# Dynamic Protective Effect of Chinese Herbal Prescription, Yiqi Jiedu Decoction, on Testis in Mice with Acute Radiation Injury

Authors: Tian-Qi Wang; Xin Zhang; Jia Yang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

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## Abstract

Objective. A Chinese herbal prescription, Yiqi Jiedu (YQJD) decoction, is used for injuries caused by radiotherapy. This study aimed to investigate the dynamic protective effects of YQJD decoction in mice on radiation damage at different time points of the testis. Methods. The 200 male Balb/c mice were randomly divided into 5 groups (blank, model, Anduolin, low-dose YQJD, and high-dose YQJD groups). Blank and model groups were all given stomach deionized water. Anduolin group and high and low dose of Chinese herb groups were treated with Chinese material medical decoction compound for 2 weeks before being irradiated. Except for the blank group, other groups were given whole-body one-time irradiation of 5.5 Gy 60Co gamma rays. Histopathology examination of testes was conducted on the 1st, 7th, 21st, and 35th day after irradiation, respectively. Indexes of sperm motility rate, sperm count, serum levels of testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also measured correspondingly. Result. Compared to 1 day and 7 days after irradiation, most of the indexes, including testis index, sperm motility, and count, were decreased on the 21st and 35th day in all irradiated groups. However, compared with the model group, the indexes to assess sperm quality and the morphological structures of testis in low-dose and high-dose YQJD groups were significantly recovered on the 21st and 35th days. Additionally, reproductive hormones, including testosterone and estradiol, were recovered more in low-dose and high-dose YQJD groups on the 21st and 35th days. Conclusion. The YQJD decoction is able to accelerate the recovery of sperm indexes, testosterone, and estradiol levels of sex hormones in mice after irradiation and demonstrated good protective effects on the testis.


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## Body

## 1. Introduction

The 1998 World Health Organization survey showed that ionizing radiation can cause direct damage to the human reproductive system, hematopoietic system, and immune system [1]. It is a predisposing factor for infertility and teratogenesis, for it can reduce male sexual function, female endocrine disorders, and menstrual disorders. According to statistics, the current ratio of infertile people among people of childbearing age is as high as 10%, and the incidence of male infertility accounts for about 15% to 50% of infertile couples [2]. At the same time, the incidence of reproductive health problems in males exposed to radiation for a long time becomes higher [3]. Therefore, the effect of ionizing radiation on the human reproductive system is attracting more and more attention [4].In recent years, with the country’s strong support for traditional Chinese medicine, many scholars have devoted themselves to the treatment of testicular damage caused by radiation, including single Chinese medicine and its extracts [5, 6] and Chinese medicine compound prescriptions [7].A Chinese herbal prescription, Yiqi Jiedu (YQJD) decoction, was based on Chinese medicine expertise. As shown in Table1 and Figure 1, it consisted of the Chinese herbs such as Fructus Lycii, Radix Angelicae Sinensis, and Radix Astragali, which is used for syndrome induced by radiotherapy. The composition of YQJD decoction has been granted a Chinese invention patent on December 11, 2020 (No: 4144448). Recently, it has been reported that the main components of herbs in YQJD decoction have a certain antiradiation effect in animal models [8–10]. A previous study has shown that YQJD decoction attenuated irradiation-induced testis injury in mice and its potential mechanism was related to the TLR5 signaling pathway on the 7th day after irradiation [11]. Moreover, YQJD decoction could also increase SOD activity [12], reduce the content of TNF-α [13], and alleviate the radiation-induced peripheral blood cytopenia [14]. Most of the existing studies have focused on the acute protective effect of YQJD decoction against radiation within 7 days; however, little is known about its far-back protective effect on testicular tissue, especially among in a spermatogenic cycle (35 days) [15]. In order to investigate the dynamic protective effects of YQJD decoction in reducing the radiation injury on testis both immediately effect and in the long term, we observed the improving testicle structure and function and sex hormone levels at 4 different time points: the 1st, 7th, 21st, and 35th day after irradiation.Table 1
Composition of YQJD decoction.
CompositionRatio (%)Astragali Radix30Angelicae Sinensis Radix6Lycii Fructus10Panacis Quinquefolii Radix6Paeoniae Radix Alba10Crataegi Fructus7Poria12Portulacae Herba15Figure 1
HPLC fingerprint and the chemical components of YQJD extract. The chemical compounds were identified by UHPLC-Orbitrap-HRMS/MS. 1, citric acid; 2, manninotriose; 3, succinic acid; 4, protocatechuic acid; 5, gentiopicroside; 6, oxypaeoniflorin; 7, catechin hydrate; 8, gentisic acid; 9, paeoniflorin; 10, calycosin-7-O-β-D-glucoside; 11, ecgonine methyl ester; 12, calycosin; 13, albiflorin; 14, ononin; 15, hydroxygenkwanin; 16, formononetin; 17, daidzein methyl ether; 18, benzoylpaeoniflorin; 19, astragaloside IV; 20, lupenone; 21, astragaloside II; 22, isoastragaloside I; 23, ligustilide; and 24, levistilide A.


## 2. Methods and Materials

### 2.1. Drug Preparation

According to recent studies [16], Anduolin Capsules had a protective effect in mice against irradiation, so it was chosen as the positive medicine. Anduolin Capsules were provided by Fuzhou Adolla Pharmaceutical Co., Ltd., and the Chinese herbs were appraised by a professor at the Beijing University of Chinese Medicine.


### 2.2. Dose Consideration

The drug powder in the Anduolin capsule was dissolved in deionized water and fully mixed with deionized water to make an aqueous solution. The drug dose was equivalent to the human clinical dose, namely, 0.4 g/mL.The YQJD decoction pieces were purchased from Beijing Tongren Drug Store and were identified as genuine by the pharmacists of traditional Chinese medicine. The decoction was prepared by our laboratory. The decoction pieces were immersed in 10 times volume of water for 1 hour and brewed under intense fire until boiling. The boiled preparation was then brewed under mild fire for 1 hour to make the first decoction. The dregs of YQJD added to 8 times volume of boiling water were brewed for another 1 hour to make the second decoction. Finally, the two doses of decoctions were merged. After cooling the decoction, absolute ethanol was added to a concentration of 55–60%. Place it in a refrigerator at 4°C for 24 h, then go through vacuum suction and filtration, recover ethanol, and dilute the drug solution to the required concentration. Based on the recent studies [14], the doses in low-dose group were 0.4 g/mL (human clinical equivalent dose) and 0.8 g/mL (2 times equivalent dose) in high-dose group.


### 2.3. Animals and Irradiation

Two hundred Balb/c male mice, weighed 20 ± 2 g, were provided by Sibefu Experimental Animal Science and Technology Co., Ltd., animal license number SCXK (Beijing) 2011-0004. The 200 mice were divided into 4 batches of 50 mice in each batch according to the random number table. The 50 mice in each batch were randomly divided into 5 groups again according to the random number table: blank control (Ctrl,n = 10), irradiation + no pretreatment (IR, n = 10), irradiation + Anduolin Capsules pretreatment (IRA, n = 10), irradiation + YQJD decoction of low-dose group pretreatment (IRY-low, n = 10), and irradiation + YQJD decoction of high-dose group pretreatment (IRY-high, n = 10). The Ctrl group and the IR group were gavaged with deionized water, the IRA group was provided with Anduolin water solution, and the IRY-high and IRY-low groups were given high-concentration and low-concentration YQJD decoction compound, once a day, for 14 days.After 14 days of intervention, mice in all groups except the Ctrl group received one-time whole-bodyγ-ray irradiation in the Cobalt Source Laboratory of College of Chemistry and Molecular Engineering, Peking University, with the irradiation dose of 5.5 Gy and the irradiation dose rate of 1.1 Gy/min. After irradiation, no drug was given to each group, and samples were collected at 4 time points, 1, 7, 21, and 35 days after irradiation, respectively, and the experimental indexes were dynamically detected, using 3% pentobarbital sodium 1 ml/kg intraperitoneal injection anesthesia. The design of the animal experiment program is reasonable, and the experimental methods and objectives are in line with human moral and ethical standards and international practices.


### 2.4. Testis Index

The abdominal cavity was opened to isolate the mice’s bilateral testicles. Testis was weighted and removed. After being rinsed with normal saline, the surface debris was wiped with a filter paper, and the indexes of each organ were weighed with a 1/10000 balance. Organ index = organ weight (mg)/body weight (g).


### 2.5. Sperm Quality Test

The scrotum was cut open, the epididymis on both sides of the testis was separated, and the epididymis was put into a small beaker containing 4 mL of normal saline. The temperature of the normal saline should be kept at 37°C for several minutes. The epididymis was cut into pieces with the ophthalmic scissors to make the sperm overflow and then mixed into sperm suspension. A drop of sperm suspension was taken with a straw and the equivalent Medium 199 (Beijing Bainowei Biotechnology Co., Ltd., Lot No: SH30253.01b) was mixed evenly and then dropped on the slide. After 1 min, it was pushed into thin slices. After a short period of natural drying in the air, it was observed under the computer reproductive assistance system (Wlyi-90000 Computer Reproductive Assistance System, Beijing Weili New Century Co., Ltd). The prepared slides were examined under a low-power microscope (Olympus BX51 microscope, Olympus Corporation, Japan) to find the parts with less overlap and clear background and then observed under a high-power microscope. The computer-assisted sperm analyzer was used for objective sperm analysis and sperm count, while the hemocytometric method was applied for counting sperms. The intact nonoverlapping sperms were examined in each mouse, and indicators such as sperm activity rate and sperm count were recorded.


### 2.6. Morphological and Structural Examination of Testis

The testis and epididymis of 6 mice in each group were placed in 10% neutral formalin for 24h [17]. Gradient ethanol dehydration protocol was applied; xylene transparent, paraffin-embedded, 4 μm paraffin slices were prepared, baked in 60°C oven, and stained with hematoxylin-eosin (H&E). The pathological changes of testis and epididymis were observed under a light microscope [18].


### 2.7. Sex Hormone Level Test

The blood from the mouse eyeball was placed in a nonanticoagulant tube. The nonanticoagulant blood of mice was centrifugated (3000 r/min), and the serum of mice was isolated. The serum levels of testosterone (Beijing Bainowei Biotechnology Co., Ltd., lot number: AB108666), estradiol (Beijing Bainowei Biotechnology Co., Ltd., lot number: AB108667), follicle-stimulating hormone (Beijing Bainowei Biotechnology Co., Ltd., lot number: CEA830Mu), and lutein (Beijing Bainowei Biotechnology Co., Ltd., lot number: CEA441Mu) were measured according to the instructions of ELISA kit.


### 2.8. Statistical Analysis

The data of each group were expressed as mean ± standard deviation (x¯ ± s) and analyzed by SPSS 21.0 statistical software. One-way ANOVA was used for measurement data among multiple groups. For pair comparison, LSD was used for homogeneity of variances, and Dunnett T3 was used for heterogeneity of variances. p<0.05 was considered statistically significant.


## 2.1. Drug Preparation

According to recent studies [16], Anduolin Capsules had a protective effect in mice against irradiation, so it was chosen as the positive medicine. Anduolin Capsules were provided by Fuzhou Adolla Pharmaceutical Co., Ltd., and the Chinese herbs were appraised by a professor at the Beijing University of Chinese Medicine.


## 2.2. Dose Consideration

The drug powder in the Anduolin capsule was dissolved in deionized water and fully mixed with deionized water to make an aqueous solution. The drug dose was equivalent to the human clinical dose, namely, 0.4 g/mL.The YQJD decoction pieces were purchased from Beijing Tongren Drug Store and were identified as genuine by the pharmacists of traditional Chinese medicine. The decoction was prepared by our laboratory. The decoction pieces were immersed in 10 times volume of water for 1 hour and brewed under intense fire until boiling. The boiled preparation was then brewed under mild fire for 1 hour to make the first decoction. The dregs of YQJD added to 8 times volume of boiling water were brewed for another 1 hour to make the second decoction. Finally, the two doses of decoctions were merged. After cooling the decoction, absolute ethanol was added to a concentration of 55–60%. Place it in a refrigerator at 4°C for 24 h, then go through vacuum suction and filtration, recover ethanol, and dilute the drug solution to the required concentration. Based on the recent studies [14], the doses in low-dose group were 0.4 g/mL (human clinical equivalent dose) and 0.8 g/mL (2 times equivalent dose) in high-dose group.


## 2.3. Animals and Irradiation

Two hundred Balb/c male mice, weighed 20 ± 2 g, were provided by Sibefu Experimental Animal Science and Technology Co., Ltd., animal license number SCXK (Beijing) 2011-0004. The 200 mice were divided into 4 batches of 50 mice in each batch according to the random number table. The 50 mice in each batch were randomly divided into 5 groups again according to the random number table: blank control (Ctrl,n = 10), irradiation + no pretreatment (IR, n = 10), irradiation + Anduolin Capsules pretreatment (IRA, n = 10), irradiation + YQJD decoction of low-dose group pretreatment (IRY-low, n = 10), and irradiation + YQJD decoction of high-dose group pretreatment (IRY-high, n = 10). The Ctrl group and the IR group were gavaged with deionized water, the IRA group was provided with Anduolin water solution, and the IRY-high and IRY-low groups were given high-concentration and low-concentration YQJD decoction compound, once a day, for 14 days.After 14 days of intervention, mice in all groups except the Ctrl group received one-time whole-bodyγ-ray irradiation in the Cobalt Source Laboratory of College of Chemistry and Molecular Engineering, Peking University, with the irradiation dose of 5.5 Gy and the irradiation dose rate of 1.1 Gy/min. After irradiation, no drug was given to each group, and samples were collected at 4 time points, 1, 7, 21, and 35 days after irradiation, respectively, and the experimental indexes were dynamically detected, using 3% pentobarbital sodium 1 ml/kg intraperitoneal injection anesthesia. The design of the animal experiment program is reasonable, and the experimental methods and objectives are in line with human moral and ethical standards and international practices.


## 2.4. Testis Index

The abdominal cavity was opened to isolate the mice’s bilateral testicles. Testis was weighted and removed. After being rinsed with normal saline, the surface debris was wiped with a filter paper, and the indexes of each organ were weighed with a 1/10000 balance. Organ index = organ weight (mg)/body weight (g).


## 2.5. Sperm Quality Test

The scrotum was cut open, the epididymis on both sides of the testis was separated, and the epididymis was put into a small beaker containing 4 mL of normal saline. The temperature of the normal saline should be kept at 37°C for several minutes. The epididymis was cut into pieces with the ophthalmic scissors to make the sperm overflow and then mixed into sperm suspension. A drop of sperm suspension was taken with a straw and the equivalent Medium 199 (Beijing Bainowei Biotechnology Co., Ltd., Lot No: SH30253.01b) was mixed evenly and then dropped on the slide. After 1 min, it was pushed into thin slices. After a short period of natural drying in the air, it was observed under the computer reproductive assistance system (Wlyi-90000 Computer Reproductive Assistance System, Beijing Weili New Century Co., Ltd). The prepared slides were examined under a low-power microscope (Olympus BX51 microscope, Olympus Corporation, Japan) to find the parts with less overlap and clear background and then observed under a high-power microscope. The computer-assisted sperm analyzer was used for objective sperm analysis and sperm count, while the hemocytometric method was applied for counting sperms. The intact nonoverlapping sperms were examined in each mouse, and indicators such as sperm activity rate and sperm count were recorded.


## 2.6. Morphological and Structural Examination of Testis

The testis and epididymis of 6 mice in each group were placed in 10% neutral formalin for 24h [17]. Gradient ethanol dehydration protocol was applied; xylene transparent, paraffin-embedded, 4 μm paraffin slices were prepared, baked in 60°C oven, and stained with hematoxylin-eosin (H&E). The pathological changes of testis and epididymis were observed under a light microscope [18].


## 2.7. Sex Hormone Level Test

The blood from the mouse eyeball was placed in a nonanticoagulant tube. The nonanticoagulant blood of mice was centrifugated (3000 r/min), and the serum of mice was isolated. The serum levels of testosterone (Beijing Bainowei Biotechnology Co., Ltd., lot number: AB108666), estradiol (Beijing Bainowei Biotechnology Co., Ltd., lot number: AB108667), follicle-stimulating hormone (Beijing Bainowei Biotechnology Co., Ltd., lot number: CEA830Mu), and lutein (Beijing Bainowei Biotechnology Co., Ltd., lot number: CEA441Mu) were measured according to the instructions of ELISA kit.


## 2.8. Statistical Analysis

The data of each group were expressed as mean ± standard deviation (x¯ ± s) and analyzed by SPSS 21.0 statistical software. One-way ANOVA was used for measurement data among multiple groups. For pair comparison, LSD was used for homogeneity of variances, and Dunnett T3 was used for heterogeneity of variances. p<0.05 was considered statistically significant.


## 3. Results

### 3.1. Dynamic Protective Effects of YQJD Decoction on Testis Index

The testicular index was compared between different groups at the same time point:(a)
Twenty-one days after irradiation: it was significantly reduced (p<0.001) in all groups; it was higher in the Anduolin group and high-dose group than that in the model group (p<0.05).(b)
Thirty-five days after irradiation: it was significantly reduced (p<0.001) in all groups; it was much higher in the low-dose group than that in the model group (p<0.01).The testicular index of mice was compared in the same group and at different time points:(i)
Model group: compared with the values at 1 and 7 days after irradiation, testis index decreased significantly at 21 and 35 days (p<0.01; p<0.001); compared with the values at 21 days after irradiation, testis index decreased at 35 days (p<0.05).(ii)
Treatment groups (Anduolin, high-dose, and low-dose groups): compared with the values at 1 and 7 days after irradiation, testis index decreased significantly at 21 and 35 days (p<0.01; p<0.001); the testis index decreased at 35 days after irradiation compared with that at 21 days (p<0.05) (Figure 2).Figure 2
Comparison of testis index at different time points.∗p<0.05, ∗∗p<0.01¸and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05 vs. 21 days after irradiation.


### 3.2. Dynamic Protective Effects of YQJD Decoction on Sperm Motility Rate

The sperm motility rates were compared between different groups at the same time point:(a)
One day after irradiation: compared with blank group, the rates in both Anduolin group and low-dose group increased (bothp<0.05).(b)
Seven days after irradiation: compared to the values at 1 day after irradiation, the rates in model group increased (p<0.05), whereas they significantly decreased in the treatment groups (p<0.01 for Anduolin group; p<0.001 for low-dose and high-dose groups).(c)
Twenty-one days after irradiation: the rates were sharply dropped in the model group compared to those in the blank group (p<0.01); significant decreases were observed in both the Anduolin group and high-dose group (both p<0.01); the rates were decreased in the low-dose group (p<0.05). Compared with the model group, the rates in the low-dose group increased (p<0.05).(d)
Thirty-five days after irradiation: the rates in the model group decreased significantly compared to the blank group (p<0.001); they were significantly decreased in the Anduolin group and low-dose group (both p<0.01) and decreased in the high-dose group (p<0.05). Compared with the model group, the rates in the high-dose group demonstrated a significant increase, while sperm motility rates in both Anduolin and low-dose did not change significantly (p>0.05).Figure 3
Comparison of sperm motility rates at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05 vs. 21 days after irradiation.The sperm motility rates of mice were compared in the same group at different time points:(i)
Model group: compared with the rates at 1 day after irradiation, at 7 days, they showed a significant increase (p<0.05). Compared with the rates at1 and 7 days after irradiation, the decrease at 21 and 35 days after irradiation was extremely significant (all p<0.001).(ii)
Anduolin group: compared with the rates at 1 day after irradiation, they decreased (p<0.01) at 7 days and sharply decreased at 21 and 35 days (both p<0.001). Compared with the rates at 7 days after irradiation, they were not significantly decreased at 21 and 35 days (both were p>0.05), yet the motility rates increased after 35 days compared to 21 days after irradiation (p<0.05).(iii)
High-dose group: compared with day 1, the sperm motility rate on day 7 decreased significantly (p<0.001). Compared with the rates at 1 and 7 days after irradiation, the decrease was very significant at 21 and 35 days after irradiation (both p<0.001), yet the motility rates increased after 35 days compared to 21 days after irradiation (p<0.05).(iv)
Low-dose group: compared with the rates at 1 day, they were decreased 7 days after irradiation (p<0.01); compared with the values at 1 day after irradiation, the decrease of 21 and 35 days after irradiation was extremely significant (both p<0.001) (Figure 3).


### 3.3. Dynamic Protective Effects of YQJD Decoction on Sperm Count

(a)
Seven days after irradiation: compared with the values in the blank group, they were increased significantly in the model group (p<0.001); the number of sperms in each dose group was significantly lower than that of the model group (p<0.001).(b)
Twenty-one days after irradiation: compared with the values in 9the blank group, they were significantly reduced in the irradiation groups (allp<0.001). Compared with the model group, the high- and low-dose groups both recovered (p<0.05, p<0.001, respectively).(c)
Thirty-five days after irradiation: compared with the values in the blank group, they were significantly reduced in the irradiation groups (allp<0.001). Compared with the model group, both the high- and low-dose groups recovered significantly (p<0.001, p<0.01, respectively).The sperm numbers of mice in the same group were compared at different time points:(i)
Model group: compared with the count at 1 day after irradiation, the sperm count of mice at 7 days increased (p<0.05) and was significantly decreased at 21 and 35 days after irradiation (p<0.01, p<0.001). In comparison with the count at 7 days after irradiation, the sperm counts decreased significantly at 21 and 35 days (p<0.01, p<0.001).(ii)
Each dose group: compared with the counts at 1 day and 7 days after irradiation, the sperm counts of mice on 21 and 35 days were significantly reduced (bothp<0.01, p<0.001). Compared with the counts at 21 days after irradiation, they were decreased in both the Anduolin group and low-dose group (both p<0.05) and increased in the high-dose group on 35 days (p<0.05) (Figure 4).Figure 4
Comparison of sperm count at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05 vs. 21 days after irradiation.


### 3.4. Dynamic Protective Effects of YQJD Decoction on Testosterone (T)

Serum testosterone levels in mice were compared among different groups at the same time point:(a)
One day after irradiation: compared with the level in the blank group, the testosterone level of the model group increased significantly (p<0.001)/ Compared with the level in the model group, the testosterone levels of each dose group were significantly decreased (all p<0.001).(b)
Seven days after irradiation: compared with the level in the blank group, the testosterone levels decreased in the model group (p<0.05) and significantly decreased in each dose group (all p<0.001). Compared with the level in the model group, testosterone levels decreased in both the Anduolin group and low-dose group (all p<0.05).(c)
Twenty-one days after irradiation: compared with the level in the blank group, they decreased significantly in the model group (p<0.01) and decreased in both the Anduolin group and high-dose group (both p<0.05).(d)
Thirty-five days after irradiation: compared with blank group, model group, Anduolin group and low-dose group all had significantly decreased testosterone levels (allp<0.001); the levels were decreased significantly the high-dose group (p<0.01).The serum testosterone levels in mice were compared in the same group at different time points:(i)
Model group: compared with the levels at 1 day after irradiation, those at 7 days, 21 days, and 35 days were significantly decreased (allp<0.001).(ii)
Each dose group: compared with the levels at 1 day after irradiation, those in all the dose groups decreased at 7 days (allp<0.05) (Figure 5).Figure 5
Comparison of serum testosterone level at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation.


### 3.5. Dynamic Protective Effects of YQJD Decoction on Estradiol (E2)

The mouse serum estradiol levels were compared between different groups at the same time point:(a)
One day after irradiation: compared with the levels in the blank group, the serum estradiol levels increased in the model group (p<0.05). Compared with model group, Anduolin (p<0.05), high-dose (p<0.01), and low-dose (p<0.001) groups had decreased serum estradiol levels.(b)
Seven days after irradiation: compared with the blank group, the model group had increased serum estradiol levels (p<0.05).(c)
Thirty-five days after irradiation: Compared with the levels in the blank group, the levels in model group decreased (p<0.05), and compared with model group, both high and low-dose groups had increased levels (both p<0.05).The serum estradiol levels were compared in the same group at different time points:(i)
Model group: compared with the levels at 1 day and 7 days after irradiation, there was a significant decrease in 21 and 35 days (bothp<0.001).(ii)
Each dose group: compared with the levels at 1 day after irradiation, serum estradiol levels at 7 days all increased (p<0.05 for Anduolin and high-dose group; p<0.001 for low-dose group) and at 21 days and 35 days both decreased significantly (all p<0.001) (Figure 6).Figure 6
Comparison of serum estradiol level at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation.


### 3.6. Dynamic Protective Effects of YQJD Decoction on Follicle-Stimulating Hormone (FSH)

(a)
One day after irradiation: compared with the blank group and model group, low-dose group had increased FSH levels (allp<0.05).(b)
Seven days after irradiation: compared with the blank group, the model group and high-dose group had significantly increased levels (bothp<0.01), and both the Anduolin group and low-dose group had increased levels (both p<0.05).(c)
Thirty-five days after irradiation: compared with the blank group, Anduolin group had increased (p<0.05) and high- and low-dose groups had significantly increased levels (both p<0.001). Compared with model group, the high-dose group had increased (p<0.05) and the increase in low-dose group had extremely significant levels (p<0.001).Serum FSH levels in mice were compared in the same group at different time points:(i)
Model group: compared with the levels at 1 day after irradiation, those at 7 days and 35 days increased (p<0.01, p<0.05). Compared with the levels at 7 days, those at 21 days after irradiation dropped sharply (p<0.001). Compared with the levels at 21 days, those at 35 days after irradiation increased significantly (p<0.01).(ii)
Anduolin group: compared with 1 day after irradiation, the increase was significant at 7 and 35 days (p<0.001, p<0.01). Compared with 7 days, 21 days decreased sharply (p<0.001); Compared with the levels at 21 days, serum FSH levels increased significantly at 35 days after irradiation (p<0.001).(iii)
High-dose group: compared with the levels at 1 day after irradiation, serum FSH levels increased significantly at both 7 days and 35 days (bothp<0.001). Compared with the levels at 7 days, serum FSH levels decreased significantly at 21 days after irradiation (p<0.001). Comparing with the levels at 21 days, serum FSH levels increased significantly 35 days after irradiation (p<0.001).(iv)
Low-dose group: Compared with the levels at 1 day after irradiation, serum FSH levels at 7 days and 35 days increased significantly (p<0.001, p<0.01). Compared with with the levels at 7 days after irradiation, serum FSH levels at 21 days dropped sharply (p<0.001), while at 35 days, they increased significantly (p<0.01). Compared with the levels at 21 days after irradiation, at 35 days, they increased significantly (p<0.001) (Figure 7).Figure 7
Comparison of FSH levels at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05, ▲▲p<0.01, and ▲▲▲p<0.001 vs. 21 days after irradiation.


### 3.7. Dynamic Protective Effects of YQJD Decoction on Luteinizing Hormone (LH)

(a)
One day after irradiation: compared with the level in the blank group, LH levels increased in both model group and high-dose group (bothp<0.05) and increased significantly in the Anduolin group and low-dose group (both p<0.01).(b)
Seven days after irradiation: compared with the level in the model group, LH decreased in the low-dose group (p<0.05).(c)
Thirty-five days after irradiation: compared with model group, high-dose group decreased (p<0.05).Serum LH levels of mice are compared in the same group at different time points:(i)
Model group: compared with the level at 1 day after irradiation, LH level decreased at 7 days (p<0.05). Compared with the level at 7 days, LH level increased at 21 days (p<0.05). Compared with the level at 21 days after irradiation, LH level decreased at 35 days (p<0.05).(ii)
Anduolin group and high-dose group: compared with the level at 1 day after irradiation, LH levels decreased significantly at both 7 and 35 days (bothp<0.001). Compared with the level at 7 days after irradiation, LH levels increased significantly at 21 days (all p<0.001). Compared with the level at 21 days after irradiation, LH levels all decreased significantly at 35 days (all p<0.001).(iii)
Low-dose group: compared with the level at 1 day after irradiation, LH levels decreased significantly at both 7 days and 35 days (p<0.001, p<0.01). Compared with the level at 7 days after irradiation, LH levels increased significantly at 21 days (p<0.001). Compared with the level at 21 days after irradiation, LH levels decreased significantly 35 days (p<0.01) (Figure 8).Figure 8
Comparison of LH levels at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05, ▲▲p<0.01, and ▲▲▲p<0.001 vs. 21 days after irradiation.


### 3.8. Dynamic Protective Effects of YQJD Decoction on Morphological Structure of Testis in Mice at Different Time Points

#### 3.8.1. Morphological Structure of Testes in Each Group One Day after Irradiation

(i) Blank Group. The lumen of the seminiferous tubules is of regular shape. There were no pathological changes.(ii) Model Group. The structure of the testis is still clear, there are no vacuoles in the seminiferous tubules, the number of spermatogenic cells in the lumen has not been reduced, and the lumen expansion is not obvious. There is no cell shedding and the seminiferous tubule epithelial cells have not thinned. The cytoplasm was plump, and the number of sperms was not significantly different from the normal group.(iii) Anduolin Group. The structure of the testis is still clear, yet the dividing layer of seminiferous tubule epithelial cells has not become thinner. The cytoplasm is full, and more sperm can be seen in the lumen.(iv) High-Dose Group. The structure of the testis is clear, the lumen shape of the seminiferous tubules is relatively regular, the arrangement of spermatogenic cells at all levels is more orderly, and the epithelial cells of the seminiferous tubules at all levels have not fallen off.(v) Low-Dose Group. The structure of the testis is clear, the lumen of the seminiferous tubules is regular in shape, the basement membrane of the tube wall is intact, the spermatogenic cells at all levels are arranged neatly, and there are no vacuoles in the lumen of the seminiferous tubules (Figure 9(a)).Figure 9
(a) The morphology of testis of each group of mice 1 day after irradiation (H&E x400). (b) The morphology of testis of each group of mice 7 days after irradiation (H&E x400). (c) The morphology of testis of each group of mice 21 days after irradiation (H&E x400). (d) The morphology of testis of each group of mice 35 days after irradiation (H&E x400).
(a)(b)(c)(d)


#### 3.8.2. Morphological Structure of Testes in Each Group 7 Days after Irradiation

(i) Blank Group. The testis structure is clear, the cells in each division layer in the seminiferous tubules are complete, the spermatogenic cells at all levels are arranged regularly and tightly, and the lumen is full of mature sperm without obvious pathological changes.(ii) Model Group. The structure of the testis is relatively clear, vacuoles appear in the seminiferous tubules, the number of spermatogenic cells in the lumen is reduced, the epithelial cells of the seminiferous tubules are thinned, and the epithelial cells of the seminiferous tubules are slightly shed. The number of sperm is reduced, the lumen is slightly enlarged, and the cytoplasm is fuller.(iii) Anduolin Group. The testis structure is still clear, and a small number of vacuoles appear in the seminiferous tubule epithelium. Compared with the model group, the local seminiferous tubule epithelial cell level is reduced, but the reduction is not obvious, and the cytoplasm is fuller.(iv) High-Dose Group. The structure of the testis is still clear, a small number of vacuoles appear in the seminiferous tubule epithelial cells, which is less that in than the model group, a small number of seminiferous tubule epithelial cells at all levels fall off, the level is slightly reduced, and more can be seen in the lumen sperm.(v) Low-Dose Group. The testis structure is still clear, a small number of vacuoles appear in the seminiferous tubules, which is slightly improved compared with the model group, the interstitial cell edema is not obvious, and more sperm can be seen in the lumen (Figure 9(b)).


#### 3.8.3. Morphological Structure of Testes in Each Group 21 Days after Irradiation

(i) Blank Group. The testis structure is clear, the split layer cells in the seminiferous tubules are intact, the spermatogenic cells at all levels are tightly arranged, and a large number of sperms and mesenchymal cells can be seen in the lumen, without obvious pathological changes.(ii) Model Group. The structure of the testis is relatively fuzzy, there are more vacuoles in the seminiferous tubules, the distance between the spermatogenic tubules becomes sparse, the epithelial cells of the seminiferous tubules become thinner, and the number of division layers is thinned. The shedding is more pronounced, and the cytoplasm is not full.(iii) Anduolin Group. The testis structure is fuzzy, and there are some vacuoles in the seminiferous tubule epithelium. Compared with the model group, the thinning of the local seminiferous tubule epithelial cells is slowed down, and the cytoplasm is not full.(iv) High-Dose Group. The structure of the testis is fuzzy, there are more vacuoles in the seminiferous tubules, the lumen movement in the vacuoles is slightly improved compared with the model group, the epithelial cells of the seminiferous tubules at all levels fall off, and the lumen deformation is more obvious. The number of primary spermatids decreases.(v) Low-Dose Group. The structure of the testis is fuzzy, and there are more vacuoles in the seminiferous tubules, which is better than the model group, but the stromal cell edema is more obvious (Figure 9(c)).


#### 3.8.4. Morphological Structure of Testes in Each Group 35 Days after Irradiation

(i) Blank Group. The structure of the testis is clear, the cells of each division layer in the seminiferous tubules are complete, a large number of sperm and mesenchymal cells can be seen in the lumen, and there is no obvious pathological change.(ii) Model Group. The structure of the testis is fuzzy, the split layers in each seminiferous tubule are fuzzy, a large number of vacuoles appear in the seminiferous tubules, cell degeneration is obvious, and the number of split layers decreases more severely 21 days after the comparison. The epithelial cells of the seminiferous tubules become thinner. The plasma is not full, the lumen is significantly dilated, and the number of sperms in the lumen is very small, which is significantly different from the blank group, and the cell shedding is significant.(iii) Anduolin Group. Testis structure is fuzzy, local seminiferous tubule epithelial cells are thinned obviously, some vacuoles appear on seminiferous tubule epithelium, the lumen is obviously dilated, a small number of sperms can be seen in the lumen, and the cells fall off significantly, which have slightly improved compared with the model group.(iv) High-Dose Group. Testicular structure is blurred, more vacuoles appear in the seminiferous tubules, the lumen movement in the vacuoles is slightly improved compared with the model group, the epithelial cells of the seminiferous tubules at all levels are shed more obviously, and the number of layers is particularly reduced. The plasma is not full.(v) Low-Dose Group. The structure of the testis is blurred, and more vacuoles appear in the seminiferous tubules, which are slightly improved compared with the model group. The interstitial cell edema is more obvious, the lumen is more obvious, and a small number of sperms can be seen in the lumen (Figure 9(d)).


## 3.1. Dynamic Protective Effects of YQJD Decoction on Testis Index

The testicular index was compared between different groups at the same time point:(a)
Twenty-one days after irradiation: it was significantly reduced (p<0.001) in all groups; it was higher in the Anduolin group and high-dose group than that in the model group (p<0.05).(b)
Thirty-five days after irradiation: it was significantly reduced (p<0.001) in all groups; it was much higher in the low-dose group than that in the model group (p<0.01).The testicular index of mice was compared in the same group and at different time points:(i)
Model group: compared with the values at 1 and 7 days after irradiation, testis index decreased significantly at 21 and 35 days (p<0.01; p<0.001); compared with the values at 21 days after irradiation, testis index decreased at 35 days (p<0.05).(ii)
Treatment groups (Anduolin, high-dose, and low-dose groups): compared with the values at 1 and 7 days after irradiation, testis index decreased significantly at 21 and 35 days (p<0.01; p<0.001); the testis index decreased at 35 days after irradiation compared with that at 21 days (p<0.05) (Figure 2).Figure 2
Comparison of testis index at different time points.∗p<0.05, ∗∗p<0.01¸and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05 vs. 21 days after irradiation.


## 3.2. Dynamic Protective Effects of YQJD Decoction on Sperm Motility Rate

The sperm motility rates were compared between different groups at the same time point:(a)
One day after irradiation: compared with blank group, the rates in both Anduolin group and low-dose group increased (bothp<0.05).(b)
Seven days after irradiation: compared to the values at 1 day after irradiation, the rates in model group increased (p<0.05), whereas they significantly decreased in the treatment groups (p<0.01 for Anduolin group; p<0.001 for low-dose and high-dose groups).(c)
Twenty-one days after irradiation: the rates were sharply dropped in the model group compared to those in the blank group (p<0.01); significant decreases were observed in both the Anduolin group and high-dose group (both p<0.01); the rates were decreased in the low-dose group (p<0.05). Compared with the model group, the rates in the low-dose group increased (p<0.05).(d)
Thirty-five days after irradiation: the rates in the model group decreased significantly compared to the blank group (p<0.001); they were significantly decreased in the Anduolin group and low-dose group (both p<0.01) and decreased in the high-dose group (p<0.05). Compared with the model group, the rates in the high-dose group demonstrated a significant increase, while sperm motility rates in both Anduolin and low-dose did not change significantly (p>0.05).Figure 3
Comparison of sperm motility rates at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05 vs. 21 days after irradiation.The sperm motility rates of mice were compared in the same group at different time points:(i)
Model group: compared with the rates at 1 day after irradiation, at 7 days, they showed a significant increase (p<0.05). Compared with the rates at1 and 7 days after irradiation, the decrease at 21 and 35 days after irradiation was extremely significant (all p<0.001).(ii)
Anduolin group: compared with the rates at 1 day after irradiation, they decreased (p<0.01) at 7 days and sharply decreased at 21 and 35 days (both p<0.001). Compared with the rates at 7 days after irradiation, they were not significantly decreased at 21 and 35 days (both were p>0.05), yet the motility rates increased after 35 days compared to 21 days after irradiation (p<0.05).(iii)
High-dose group: compared with day 1, the sperm motility rate on day 7 decreased significantly (p<0.001). Compared with the rates at 1 and 7 days after irradiation, the decrease was very significant at 21 and 35 days after irradiation (both p<0.001), yet the motility rates increased after 35 days compared to 21 days after irradiation (p<0.05).(iv)
Low-dose group: compared with the rates at 1 day, they were decreased 7 days after irradiation (p<0.01); compared with the values at 1 day after irradiation, the decrease of 21 and 35 days after irradiation was extremely significant (both p<0.001) (Figure 3).


## 3.3. Dynamic Protective Effects of YQJD Decoction on Sperm Count

(a)
Seven days after irradiation: compared with the values in the blank group, they were increased significantly in the model group (p<0.001); the number of sperms in each dose group was significantly lower than that of the model group (p<0.001).(b)
Twenty-one days after irradiation: compared with the values in 9the blank group, they were significantly reduced in the irradiation groups (allp<0.001). Compared with the model group, the high- and low-dose groups both recovered (p<0.05, p<0.001, respectively).(c)
Thirty-five days after irradiation: compared with the values in the blank group, they were significantly reduced in the irradiation groups (allp<0.001). Compared with the model group, both the high- and low-dose groups recovered significantly (p<0.001, p<0.01, respectively).The sperm numbers of mice in the same group were compared at different time points:(i)
Model group: compared with the count at 1 day after irradiation, the sperm count of mice at 7 days increased (p<0.05) and was significantly decreased at 21 and 35 days after irradiation (p<0.01, p<0.001). In comparison with the count at 7 days after irradiation, the sperm counts decreased significantly at 21 and 35 days (p<0.01, p<0.001).(ii)
Each dose group: compared with the counts at 1 day and 7 days after irradiation, the sperm counts of mice on 21 and 35 days were significantly reduced (bothp<0.01, p<0.001). Compared with the counts at 21 days after irradiation, they were decreased in both the Anduolin group and low-dose group (both p<0.05) and increased in the high-dose group on 35 days (p<0.05) (Figure 4).Figure 4
Comparison of sperm count at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05 vs. 21 days after irradiation.


## 3.4. Dynamic Protective Effects of YQJD Decoction on Testosterone (T)

Serum testosterone levels in mice were compared among different groups at the same time point:(a)
One day after irradiation: compared with the level in the blank group, the testosterone level of the model group increased significantly (p<0.001)/ Compared with the level in the model group, the testosterone levels of each dose group were significantly decreased (all p<0.001).(b)
Seven days after irradiation: compared with the level in the blank group, the testosterone levels decreased in the model group (p<0.05) and significantly decreased in each dose group (all p<0.001). Compared with the level in the model group, testosterone levels decreased in both the Anduolin group and low-dose group (all p<0.05).(c)
Twenty-one days after irradiation: compared with the level in the blank group, they decreased significantly in the model group (p<0.01) and decreased in both the Anduolin group and high-dose group (both p<0.05).(d)
Thirty-five days after irradiation: compared with blank group, model group, Anduolin group and low-dose group all had significantly decreased testosterone levels (allp<0.001); the levels were decreased significantly the high-dose group (p<0.01).The serum testosterone levels in mice were compared in the same group at different time points:(i)
Model group: compared with the levels at 1 day after irradiation, those at 7 days, 21 days, and 35 days were significantly decreased (allp<0.001).(ii)
Each dose group: compared with the levels at 1 day after irradiation, those in all the dose groups decreased at 7 days (allp<0.05) (Figure 5).Figure 5
Comparison of serum testosterone level at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation.


## 3.5. Dynamic Protective Effects of YQJD Decoction on Estradiol (E2)

The mouse serum estradiol levels were compared between different groups at the same time point:(a)
One day after irradiation: compared with the levels in the blank group, the serum estradiol levels increased in the model group (p<0.05). Compared with model group, Anduolin (p<0.05), high-dose (p<0.01), and low-dose (p<0.001) groups had decreased serum estradiol levels.(b)
Seven days after irradiation: compared with the blank group, the model group had increased serum estradiol levels (p<0.05).(c)
Thirty-five days after irradiation: Compared with the levels in the blank group, the levels in model group decreased (p<0.05), and compared with model group, both high and low-dose groups had increased levels (both p<0.05).The serum estradiol levels were compared in the same group at different time points:(i)
Model group: compared with the levels at 1 day and 7 days after irradiation, there was a significant decrease in 21 and 35 days (bothp<0.001).(ii)
Each dose group: compared with the levels at 1 day after irradiation, serum estradiol levels at 7 days all increased (p<0.05 for Anduolin and high-dose group; p<0.001 for low-dose group) and at 21 days and 35 days both decreased significantly (all p<0.001) (Figure 6).Figure 6
Comparison of serum estradiol level at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation.


## 3.6. Dynamic Protective Effects of YQJD Decoction on Follicle-Stimulating Hormone (FSH)

(a)
One day after irradiation: compared with the blank group and model group, low-dose group had increased FSH levels (allp<0.05).(b)
Seven days after irradiation: compared with the blank group, the model group and high-dose group had significantly increased levels (bothp<0.01), and both the Anduolin group and low-dose group had increased levels (both p<0.05).(c)
Thirty-five days after irradiation: compared with the blank group, Anduolin group had increased (p<0.05) and high- and low-dose groups had significantly increased levels (both p<0.001). Compared with model group, the high-dose group had increased (p<0.05) and the increase in low-dose group had extremely significant levels (p<0.001).Serum FSH levels in mice were compared in the same group at different time points:(i)
Model group: compared with the levels at 1 day after irradiation, those at 7 days and 35 days increased (p<0.01, p<0.05). Compared with the levels at 7 days, those at 21 days after irradiation dropped sharply (p<0.001). Compared with the levels at 21 days, those at 35 days after irradiation increased significantly (p<0.01).(ii)
Anduolin group: compared with 1 day after irradiation, the increase was significant at 7 and 35 days (p<0.001, p<0.01). Compared with 7 days, 21 days decreased sharply (p<0.001); Compared with the levels at 21 days, serum FSH levels increased significantly at 35 days after irradiation (p<0.001).(iii)
High-dose group: compared with the levels at 1 day after irradiation, serum FSH levels increased significantly at both 7 days and 35 days (bothp<0.001). Compared with the levels at 7 days, serum FSH levels decreased significantly at 21 days after irradiation (p<0.001). Comparing with the levels at 21 days, serum FSH levels increased significantly 35 days after irradiation (p<0.001).(iv)
Low-dose group: Compared with the levels at 1 day after irradiation, serum FSH levels at 7 days and 35 days increased significantly (p<0.001, p<0.01). Compared with with the levels at 7 days after irradiation, serum FSH levels at 21 days dropped sharply (p<0.001), while at 35 days, they increased significantly (p<0.01). Compared with the levels at 21 days after irradiation, at 35 days, they increased significantly (p<0.001) (Figure 7).Figure 7
Comparison of FSH levels at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05, ▲▲p<0.01, and ▲▲▲p<0.001 vs. 21 days after irradiation.


## 3.7. Dynamic Protective Effects of YQJD Decoction on Luteinizing Hormone (LH)

(a)
One day after irradiation: compared with the level in the blank group, LH levels increased in both model group and high-dose group (bothp<0.05) and increased significantly in the Anduolin group and low-dose group (both p<0.01).(b)
Seven days after irradiation: compared with the level in the model group, LH decreased in the low-dose group (p<0.05).(c)
Thirty-five days after irradiation: compared with model group, high-dose group decreased (p<0.05).Serum LH levels of mice are compared in the same group at different time points:(i)
Model group: compared with the level at 1 day after irradiation, LH level decreased at 7 days (p<0.05). Compared with the level at 7 days, LH level increased at 21 days (p<0.05). Compared with the level at 21 days after irradiation, LH level decreased at 35 days (p<0.05).(ii)
Anduolin group and high-dose group: compared with the level at 1 day after irradiation, LH levels decreased significantly at both 7 and 35 days (bothp<0.001). Compared with the level at 7 days after irradiation, LH levels increased significantly at 21 days (all p<0.001). Compared with the level at 21 days after irradiation, LH levels all decreased significantly at 35 days (all p<0.001).(iii)
Low-dose group: compared with the level at 1 day after irradiation, LH levels decreased significantly at both 7 days and 35 days (p<0.001, p<0.01). Compared with the level at 7 days after irradiation, LH levels increased significantly at 21 days (p<0.001). Compared with the level at 21 days after irradiation, LH levels decreased significantly 35 days (p<0.01) (Figure 8).Figure 8
Comparison of LH levels at different time points.∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001 vs. Ctrl; #p<0.05 and ##p<0.01 vs. IR; Δp<0.05, ΔΔp<0.01, and ΔΔΔp<0.001 vs. 1 day after irradiation; ⋄p<0.05, ⋄⋄p<0.01, and ⋄⋄⋄p<0.001 vs. 7 days after irradiation; ▲p<0.05, ▲▲p<0.01, and ▲▲▲p<0.001 vs. 21 days after irradiation.


## 3.8. Dynamic Protective Effects of YQJD Decoction on Morphological Structure of Testis in Mice at Different Time Points

### 3.8.1. Morphological Structure of Testes in Each Group One Day after Irradiation

(i) Blank Group. The lumen of the seminiferous tubules is of regular shape. There were no pathological changes.(ii) Model Group. The structure of the testis is still clear, there are no vacuoles in the seminiferous tubules, the number of spermatogenic cells in the lumen has not been reduced, and the lumen expansion is not obvious. There is no cell shedding and the seminiferous tubule epithelial cells have not thinned. The cytoplasm was plump, and the number of sperms was not significantly different from the normal group.(iii) Anduolin Group. The structure of the testis is still clear, yet the dividing layer of seminiferous tubule epithelial cells has not become thinner. The cytoplasm is full, and more sperm can be seen in the lumen.(iv) High-Dose Group. The structure of the testis is clear, the lumen shape of the seminiferous tubules is relatively regular, the arrangement of spermatogenic cells at all levels is more orderly, and the epithelial cells of the seminiferous tubules at all levels have not fallen off.(v) Low-Dose Group. The structure of the testis is clear, the lumen of the seminiferous tubules is regular in shape, the basement membrane of the tube wall is intact, the spermatogenic cells at all levels are arranged neatly, and there are no vacuoles in the lumen of the seminiferous tubules (Figure 9(a)).Figure 9
(a) The morphology of testis of each group of mice 1 day after irradiation (H&E x400). (b) The morphology of testis of each group of mice 7 days after irradiation (H&E x400). (c) The morphology of testis of each group of mice 21 days after irradiation (H&E x400). (d) The morphology of testis of each group of mice 35 days after irradiation (H&E x400).
(a)(b)(c)(d)


### 3.8.2. Morphological Structure of Testes in Each Group 7 Days after Irradiation

(i) Blank Group. The testis structure is clear, the cells in each division layer in the seminiferous tubules are complete, the spermatogenic cells at all levels are arranged regularly and tightly, and the lumen is full of mature sperm without obvious pathological changes.(ii) Model Group. The structure of the testis is relatively clear, vacuoles appear in the seminiferous tubules, the number of spermatogenic cells in the lumen is reduced, the epithelial cells of the seminiferous tubules are thinned, and the epithelial cells of the seminiferous tubules are slightly shed. The number of sperm is reduced, the lumen is slightly enlarged, and the cytoplasm is fuller.(iii) Anduolin Group. The testis structure is still clear, and a small number of vacuoles appear in the seminiferous tubule epithelium. Compared with the model group, the local seminiferous tubule epithelial cell level is reduced, but the reduction is not obvious, and the cytoplasm is fuller.(iv) High-Dose Group. The structure of the testis is still clear, a small number of vacuoles appear in the seminiferous tubule epithelial cells, which is less that in than the model group, a small number of seminiferous tubule epithelial cells at all levels fall off, the level is slightly reduced, and more can be seen in the lumen sperm.(v) Low-Dose Group. The testis structure is still clear, a small number of vacuoles appear in the seminiferous tubules, which is slightly improved compared with the model group, the interstitial cell edema is not obvious, and more sperm can be seen in the lumen (Figure 9(b)).


### 3.8.3. Morphological Structure of Testes in Each Group 21 Days after Irradiation

(i) Blank Group. The testis structure is clear, the split layer cells in the seminiferous tubules are intact, the spermatogenic cells at all levels are tightly arranged, and a large number of sperms and mesenchymal cells can be seen in the lumen, without obvious pathological changes.(ii) Model Group. The structure of the testis is relatively fuzzy, there are more vacuoles in the seminiferous tubules, the distance between the spermatogenic tubules becomes sparse, the epithelial cells of the seminiferous tubules become thinner, and the number of division layers is thinned. The shedding is more pronounced, and the cytoplasm is not full.(iii) Anduolin Group. The testis structure is fuzzy, and there are some vacuoles in the seminiferous tubule epithelium. Compared with the model group, the thinning of the local seminiferous tubule epithelial cells is slowed down, and the cytoplasm is not full.(iv) High-Dose Group. The structure of the testis is fuzzy, there are more vacuoles in the seminiferous tubules, the lumen movement in the vacuoles is slightly improved compared with the model group, the epithelial cells of the seminiferous tubules at all levels fall off, and the lumen deformation is more obvious. The number of primary spermatids decreases.(v) Low-Dose Group. The structure of the testis is fuzzy, and there are more vacuoles in the seminiferous tubules, which is better than the model group, but the stromal cell edema is more obvious (Figure 9(c)).


### 3.8.4. Morphological Structure of Testes in Each Group 35 Days after Irradiation

(i) Blank Group. The structure of the testis is clear, the cells of each division layer in the seminiferous tubules are complete, a large number of sperm and mesenchymal cells can be seen in the lumen, and there is no obvious pathological change.(ii) Model Group. The structure of the testis is fuzzy, the split layers in each seminiferous tubule are fuzzy, a large number of vacuoles appear in the seminiferous tubules, cell degeneration is obvious, and the number of split layers decreases more severely 21 days after the comparison. The epithelial cells of the seminiferous tubules become thinner. The plasma is not full, the lumen is significantly dilated, and the number of sperms in the lumen is very small, which is significantly different from the blank group, and the cell shedding is significant.(iii) Anduolin Group. Testis structure is fuzzy, local seminiferous tubule epithelial cells are thinned obviously, some vacuoles appear on seminiferous tubule epithelium, the lumen is obviously dilated, a small number of sperms can be seen in the lumen, and the cells fall off significantly, which have slightly improved compared with the model group.(iv) High-Dose Group. Testicular structure is blurred, more vacuoles appear in the seminiferous tubules, the lumen movement in the vacuoles is slightly improved compared with the model group, the epithelial cells of the seminiferous tubules at all levels are shed more obviously, and the number of layers is particularly reduced. The plasma is not full.(v) Low-Dose Group. The structure of the testis is blurred, and more vacuoles appear in the seminiferous tubules, which are slightly improved compared with the model group. The interstitial cell edema is more obvious, the lumen is more obvious, and a small number of sperms can be seen in the lumen (Figure 9(d)).


## 3.8.1. Morphological Structure of Testes in Each Group One Day after Irradiation

(i) Blank Group. The lumen of the seminiferous tubules is of regular shape. There were no pathological changes.(ii) Model Group. The structure of the testis is still clear, there are no vacuoles in the seminiferous tubules, the number of spermatogenic cells in the lumen has not been reduced, and the lumen expansion is not obvious. There is no cell shedding and the seminiferous tubule epithelial cells have not thinned. The cytoplasm was plump, and the number of sperms was not significantly different from the normal group.(iii) Anduolin Group. The structure of the testis is still clear, yet the dividing layer of seminiferous tubule epithelial cells has not become thinner. The cytoplasm is full, and more sperm can be seen in the lumen.(iv) High-Dose Group. The structure of the testis is clear, the lumen shape of the seminiferous tubules is relatively regular, the arrangement of spermatogenic cells at all levels is more orderly, and the epithelial cells of the seminiferous tubules at all levels have not fallen off.(v) Low-Dose Group. The structure of the testis is clear, the lumen of the seminiferous tubules is regular in shape, the basement membrane of the tube wall is intact, the spermatogenic cells at all levels are arranged neatly, and there are no vacuoles in the lumen of the seminiferous tubules (Figure 9(a)).Figure 9
(a) The morphology of testis of each group of mice 1 day after irradiation (H&E x400). (b) The morphology of testis of each group of mice 7 days after irradiation (H&E x400). (c) The morphology of testis of each group of mice 21 days after irradiation (H&E x400). (d) The morphology of testis of each group of mice 35 days after irradiation (H&E x400).
(a)(b)(c)(d)


## 3.8.2. Morphological Structure of Testes in Each Group 7 Days after Irradiation

(i) Blank Group. The testis structure is clear, the cells in each division layer in the seminiferous tubules are complete, the spermatogenic cells at all levels are arranged regularly and tightly, and the lumen is full of mature sperm without obvious pathological changes.(ii) Model Group. The structure of the testis is relatively clear, vacuoles appear in the seminiferous tubules, the number of spermatogenic cells in the lumen is reduced, the epithelial cells of the seminiferous tubules are thinned, and the epithelial cells of the seminiferous tubules are slightly shed. The number of sperm is reduced, the lumen is slightly enlarged, and the cytoplasm is fuller.(iii) Anduolin Group. The testis structure is still clear, and a small number of vacuoles appear in the seminiferous tubule epithelium. Compared with the model group, the local seminiferous tubule epithelial cell level is reduced, but the reduction is not obvious, and the cytoplasm is fuller.(iv) High-Dose Group. The structure of the testis is still clear, a small number of vacuoles appear in the seminiferous tubule epithelial cells, which is less that in than the model group, a small number of seminiferous tubule epithelial cells at all levels fall off, the level is slightly reduced, and more can be seen in the lumen sperm.(v) Low-Dose Group. The testis structure is still clear, a small number of vacuoles appear in the seminiferous tubules, which is slightly improved compared with the model group, the interstitial cell edema is not obvious, and more sperm can be seen in the lumen (Figure 9(b)).


## 3.8.3. Morphological Structure of Testes in Each Group 21 Days after Irradiation

(i) Blank Group. The testis structure is clear, the split layer cells in the seminiferous tubules are intact, the spermatogenic cells at all levels are tightly arranged, and a large number of sperms and mesenchymal cells can be seen in the lumen, without obvious pathological changes.(ii) Model Group. The structure of the testis is relatively fuzzy, there are more vacuoles in the seminiferous tubules, the distance between the spermatogenic tubules becomes sparse, the epithelial cells of the seminiferous tubules become thinner, and the number of division layers is thinned. The shedding is more pronounced, and the cytoplasm is not full.(iii) Anduolin Group. The testis structure is fuzzy, and there are some vacuoles in the seminiferous tubule epithelium. Compared with the model group, the thinning of the local seminiferous tubule epithelial cells is slowed down, and the cytoplasm is not full.(iv) High-Dose Group. The structure of the testis is fuzzy, there are more vacuoles in the seminiferous tubules, the lumen movement in the vacuoles is slightly improved compared with the model group, the epithelial cells of the seminiferous tubules at all levels fall off, and the lumen deformation is more obvious. The number of primary spermatids decreases.(v) Low-Dose Group. The structure of the testis is fuzzy, and there are more vacuoles in the seminiferous tubules, which is better than the model group, but the stromal cell edema is more obvious (Figure 9(c)).


## 3.8.4. Morphological Structure of Testes in Each Group 35 Days after Irradiation

(i) Blank Group. The structure of the testis is clear, the cells of each division layer in the seminiferous tubules are complete, a large number of sperm and mesenchymal cells can be seen in the lumen, and there is no obvious pathological change.(ii) Model Group. The structure of the testis is fuzzy, the split layers in each seminiferous tubule are fuzzy, a large number of vacuoles appear in the seminiferous tubules, cell degeneration is obvious, and the number of split layers decreases more severely 21 days after the comparison. The epithelial cells of the seminiferous tubules become thinner. The plasma is not full, the lumen is significantly dilated, and the number of sperms in the lumen is very small, which is significantly different from the blank group, and the cell shedding is significant.(iii) Anduolin Group. Testis structure is fuzzy, local seminiferous tubule epithelial cells are thinned obviously, some vacuoles appear on seminiferous tubule epithelium, the lumen is obviously dilated, a small number of sperms can be seen in the lumen, and the cells fall off significantly, which have slightly improved compared with the model group.(iv) High-Dose Group. Testicular structure is blurred, more vacuoles appear in the seminiferous tubules, the lumen movement in the vacuoles is slightly improved compared with the model group, the epithelial cells of the seminiferous tubules at all levels are shed more obviously, and the number of layers is particularly reduced. The plasma is not full.(v) Low-Dose Group. The structure of the testis is blurred, and more vacuoles appear in the seminiferous tubules, which are slightly improved compared with the model group. The interstitial cell edema is more obvious, the lumen is more obvious, and a small number of sperms can be seen in the lumen (Figure 9(d)).


## 4. Discussion

The decline in male fertility is a major concern during the past decades, on which this study is focused. It is suggested that direct or indirect exposure to ionizing radiation as the main environmental factor plays a dominant role in the observed decline [19]. Ionizing radiation usually affects male fertility from the following aspects [20]:①
Affecting the occurrence of sperm and the quality of semen and reducing the rate of sperm motility②
Acting on the epithelium of the seminiferous tubules of the testis, directly killing the spermatogenic cells, and resulting in poor spermatogenesis and reduced sperm count③
Causing mutations in the chromosomes of spermatogenic cells and producing malformation or miscarriage④
Acting on interstitial cells to reduce androgen production and sexual function, which indirectly leads to infertilityIn terms of the sensitivity of germ cells to ionizing radiation, spermatogonial stem cells have the highest radiation sensitivity. As germ cells continue to mature and develop, their sensitivity gradually declines [21]. Radiation has the most serious genetic harm to sperm damage; therefore, the harm of ionizing radiation to human reproduction and genetics cannot be ignored. From the current point of view, there are many indicators for evaluating sperm function, including the number of sperm, and sperm motility rate.Sperm motility rate is an important parameter for evaluating male fertility [22]. It refers to the ratio of forward and nonforward motility sperm to the total number of sperms. In mammals, the sperm motility rate mainly depends on the flagella of the sperm swing [23]. Compared with the sperm motility at 1 day after irradiation, a decrease of sperm motility was observed in all treatment groups, assuming that a large radiation dose in this study has caused irreversible damage to the sperm. Therefore, on the 21st and 35th day after irradiation, the sperm motility rate of each irradiation group further decreased to varying degrees, and the sperm motility rate was significantly lower than that of the blank group, especially in the model group that had dropped to 32.3% of the blank group. This may be due to the late stage of irradiation. Free radicals produced by ionizing radiation reduce sperm motility through peroxidation damage to sperm cell membrane and changing the fluidity of cell membrane [24], which in turn causes serious damage to forward and nonforward motility sperm and the flagella of the sperm tail. The phenomenon of oligospermia is worse [25]. Therefore, it can be seen that the sperm motility rate of each irradiation group is gradually reduced from the 7th day to the 35th day after irradiation, after the fourth batch of sacrifice. The Chinese herbal prescription Yiqi Jiedu decoction group showed a better tendency to improve sperm motility from 21 to 35 days after irradiation. At 21 and 35 days, the high-dose group was 30.9% and 115.4% higher than the model group, respectively. This shows that the Chinese medicine compound may increase the testicle’s ability to produce sperm. In the later stage of irradiation, it can reduce the peroxidation damage of free radicals to the sperm cell membrane to a certain extent and prevent the change of cell membrane fluidity, thereby increasing the sperm motility rate and promoting the recovery of sperm quality, thereby improving the reproductive ability of male animals.It can be seen from this study that the number of sperms in each irradiated group is higher than that in the blank group one day after irradiation. This may be due to the fact that the mature sperms are not sensitive to radiation after one day of acute radiation, and the sperm numbers of mice are not changed significantly. Seven days after irradiation, the sperm counts in each irradiation group were higher than those in the blank group. The increasing trend in the model group was particularly obvious, 43% higher than that in the blank group. This may be due to the short time after sacrifice and the strong radiation resistance of testis’s support cells, stromal cells, sperm cells, and sperm after meiosis. There may still be functionally active secondary spermatocytes and sperm cells that continue to respond within one week after irradiation. Intensive differentiation still has the ability to produce sperm [26], so the sperm count is still maintained at a high level. On the 21st and 35th days after irradiation, the sperm count of each irradiation group showed a gradual decline, especially among the model group. The sperm count of mice decreased to 8.6% on the 35th day after irradiation of that in the blank group, which indicates that the radiation directly damaged the spermatogenic cells in the later stage of irradiation, resulting in a decrease in sperm count and also induced apoptosis of the spermatogenic cells, although the cycle of production sperm is 35 days [27]. At this time, the spermatogonia have begun to recover and new spermatogenic cells have appeared; however, it may be that the radiation dose of 5.5 Gy is much higher than the irreversible damage dose of sperm, namely, 2.5 Gy [28]. At this time, a large number of spermatogonial cells were killed, and there were not enough spermatogonial cells to produce sperm so that the number of sperms decreased. Therefore, it has a serious impact on sperm count in the later stage of irradiation [29]. However, the high-dose and low-dose groups of Chinese herbal YQJD decoction slowed down the decrease in sperm count of mice on the 21st and 35th day after irradiation, and the sperm count was 510% and 163% higher than that of the model group, respectively. This suggests that the YQJD decoction may inhibit the apoptosis of spermatogenic cells or promote the recovery of spermatogenic cells in the late stage of irradiation, thereby promoting the recovery of spermatogenic function of testicular spermatogenic cells in a spermatogenic cycle (35 days). The number of sperms further shows that the traditional Chinese medicine compound has a positive effect on the recovery of sperm quality.The dynamic balance between testosterone and estradiol is essential for body development and reproductive function. On the 7th day after irradiation, all irradiated groups demonstrated decreased level of testosterone, suggesting that radiation resulted in damage to interstitial cells of the testis. Testosterone was also significantly decreased in YQJD groups compared with that in the blank group, illuminating that the protective effect of YQJD decoction in the early phase after irradiation was not obvious. However, on the 21th and 35th day, both low-dose and high-dose YQJD groups showed a higher level of testosterone and estradiol compared with the model group. It is speculated that YQJD decoction can regulate the endocrine microenvironment of testis, gradually promoting the recovery of gonadal function in the long term.In this study, the LH and FSH of each irradiation group showed an alternating rise and fall trend at the 4 time points of 1 day, 7 days, 21 days, and 35 days after irradiation. This is due to the pulsed secretion of GnRH, and the release of gonadotropin has a discontinuous peak [30]. In this study’s 4 time points, FSH and LH levels in the model group were higher than those in the blank group, which may be due to ionizing radiation damage and negative feedback effect on the hypothalamus-pituitary gonadal axis [31]. FSH and LH secretion exacerbated the serum levels, especially at 21 and 35 days after irradiation. The serum FSH level of each irradiation group was significantly higher than that of the blank group because the hypothalamus is a site with higher radiosensitivity, and the secretion of inhibin in the blood is continuously reduced, which makes it affect the glands. The negative feedback of the pituitary gland is weakened, leading to an increase in FSH levels.According to the morphological results, it is suggested that the traditional Chinese medicine compound can increase the testosterone content in the serum by promoting the recovery of damaged spermatogenic cells and seminiferous tubules and affect the regulation of the hypothalamus-pituitary-gonad axis [32].Lastly, the proper dosage of YQJD decoction for protecting testis from irradiation needs to be modified, considering that a low dose was superior to a high dose with regard to some parameters, such as recovery of sperm motility and count on the 21st day. There was no linear relationship between dosage and response to some extent; further work should be carried out to determine the dose-effect curve, thus helping select the appropriate dose with maximum effect and minimum toxicity.Unlike the previously published sources entitled “A Chinese herbal prescription Yiqi Jiedu decoction attenuates irradiation induced testis injury in mice” [11], the innovation of this article is mainly reflected in the following aspects. Firstly, different from the previously published articles, the frequency of irradiation is different. In this article, the irradiation is carried out at four different time points (1st, 7th, 21st, and 35th day after irradiation, respectively), while in the previously published article, there is only one irradiation time point, only on 7 days of irradiation. Secondly, the total dose of exposure and the dose rate of exposure varied. In the previous article, mice in IR, IRA, and IRY groups were irradiated by whole-body exposure to 2 Gy 60Co γ-rays (the radiation dose rate: 1 Gy/min). However, in our article, the protocol followed was whole-body exposure to 5.5 Gy 60Co γ-rays (radiation dose rate: 1.1 Gy/min); the irradiation dose in this article is much higher than that in the previously published study. Thirdly, the main purposes of the two articles are quite different. This study was designed to investigate the dynamic protective effects of YQJD decoction on radiation damage of testis in mice at different time points, finding that compared with the sperm quality at 1st and 7th day after irradiation, Chinese herbal prescription Yiqi Jiedu decoction was shown to inhibit radiation-induced sperm quality on the 21st and 35th day, indicating that it has a long-term effect on the reproductive system. However, in the previously published article, they mainly focused on exploring its potential mechanisms; thus, they found that the potential mechanism was related to the TLR5 signaling pathway.


## 5. Conclusion

In conclusion, studies on different time points of whole-body irradiation on testis show that radiation induces a decrease in sperm count and sperm motility of Balb/c mice on long-term effect. However, the Chinese herbal prescription Yiqi Jiedu decoction could inhibit radiation-induced sperm quality on both 21st and 35th days, which has a direct bearing on the reproductive system.



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*Source: 6644093-2021-06-02.xml*