# The Mechanism of the Yigutang-Mediated P13K/AKT/GSK-3β Signal Pathway to Regulate Osteogenic Differentiation of Bone Marrow Stromal Stem Cells to Treat Osteoporosis

Authors: Ning Li; Yichen Gong
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

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## Abstract

Objectives. To explore the mechanism of Yigutang mediating the P13K/AKT/GSK-3β signaling pathway to regulate the osteogenic differentiation of bone marrow stromal stem cells to treat osteoporosis (OP). Methods. Sixty 12-week-old female SD rats were randomly divided into the normal group, model group, Yigutang group, and estrogen group, with 15 cases in each group. In the model group, Yigutang group, and estrogen group, the ovaries on both sides were removed to construct the model, and the bone mineral density (BMD) of the upper metaphysis of the right femur of the rats in each group was detected. The left femur of each group of rats was removed, and the load-deformation curve of the left femur of each group of rats was calculated. The number of osteoblasts was observed by H&E staining. After extracting the right femurs of rats in each group, real-time fluorescent quantitative PCR and Western blot were used to detect the expression levels of genes and proteins related to the P13K/AKT/GSK-3β signaling pathway. Results. The BMD of the upper metaphysis of the right femur in the Yigutang group and the estrogen group was significantly higher than that of the model group (P<0.05), while both Yigutang and estrogen groups had no significant difference compared with the normal group (P>0.05). In addition, there was no significant difference between the Yigutang group and the estrogen group (P>0.05). The elastic load and maximum load of the Yigutang group and the estrogen group were significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05). The number of osteoblasts in the Yigutang group and the estrogen group was significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05). The P13K gene expression in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that in the model group (P<0.05), and the AKT gene expression did not change significantly (P>0.05). The gene expression of GSK-3β was significantly lower than that of the model group (P<0.05). Compared with the normal group, the gene expression of P13K, AKT, and GSK-3β in the right femur bone tissue of the Yigutang group and the estrogen group did not change significantly (P>0.05). The protein expression of P13K and P-AKt in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that of the model group (P<0.05), and the protein expression of AKT did not change significantly (P>0.05). The protein expression of GSK-3β was significantly lower than the model group (P<0.05). Compared with the normal group, the protein expression of P13K, AKT, P-AKt, and GSK-3β in the right femoral bone tissue of the Yigutang group and the estrogen group did not change significantly (P>0.05). Conclusions. Yigutang can regulate the differentiation of bone marrow stromal stem cells into osteoblasts, which may be achieved by regulating the P13K/AKT/GSK-3β signaling pathway.


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## Body

## 1. Introduction

Osteoporosis (OP) is a systemic metabolic disease characterized by decreased bone mass and destruction of bone microstructure, which in turn leads to increased bone fragility and prone to fractures. The main affected populations are postmenopausal women and the elderly [1]. Postmenopausal osteoporosis is caused by the decrease of estrogen in postmenopausal women. The elderly osteoporosis is caused by the growth of age and the lack of hormone in the body, which affect the formation and differentiation of osteoblasts. At present, there are many drugs for treating osteoporosis, including estrogen drugs, bisphosphonates, calcitonin, and vitamin D analogs. However, these drugs have many adverse reactions [2]. Some literatures have shown that oral antiosteoporosis drugs have the risk of nausea, vomiting, gastric bleeding, and skin pruritus, which limit their clinical application.OP patients have an imbalance between osteogenesis and bone loss. This imbalance is mainly due to the close relationship between the proliferation and differentiation of osteoblasts and osteoclasts. Osteoblasts and osteoclasts are derived from bone marrow stromal stem cells. Therefore, the signal pathway of bone marrow stromal stem cells to differentiate into osteoblasts or osteoclasts is a hot research topic, such as the Wnt/β-catenin signaling pathway, PI3K/Akt signaling pathway, MAPK signal pathway, RANKL/RANK/OPG signal pathway, and so on. The Yigutang developed by Professor Yao Xinmiao of our hospital (the prescriptions are Drynaria fortunei, Epimedium, psoralen, danshen, Shengdi, Chinese yam) can significantly improve the clinical symptoms of OP patients and reduce the incidence of fractures, but so far, its mechanism of action is not clear.In this study, ovariectomized female SD rats were used as the research object to detect the BMD of the upper metaphysis of the right femur of each group of rats, remove the left femur of each group of rats, and calculate the load-deformation of the left femur of each group of rats curve. Then, the number of osteoblasts was observed by H&E staining, and the positive rate of BrdU cells was detected by BrdU immunohistochemical staining. After extracting the right femurs of rats in each group, real-time fluorescent quantitative PCR and Western blot were used to detect the expression levels of genes and proteins related to the P13K/AKT/GSK-3β signaling pathway. This study deeply explored the mechanism of Yigutang in preventing and treating OP, which laid the foundation for the clinical application of Yigutang.


## 2. Materials and Methods

### 2.1. Test Drug

The water extract of Yigu decoction was prepared by the Chinese Medicine Laboratory of Zhejiang University of Traditional Chinese Medicine. The formula is composed of 10 g of psoralen, 15 g ofDrynaria fortunei, 15 g of Radix Rehmanniae, 15 g of Xianling spleen, 15 g of Chinese yam, and 30 g of Salvia miltiorrhiza, containing 2.0 g of crude drug/ml, and kept in the refrigerator for later use. Pentobarbital sodium is produced by Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.


### 2.2. Cultivation, Proliferation, and Labeling of Rat Bone Marrow Stromal Stem Cells

Ten 3-week-old male SD rats were used for the culture and proliferation of bone marrow stromal stem cells. The experimental animals were provided by the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine.


#### 2.2.1. Cultivation and Proliferation of Rat Bone Marrow Stromal Stem Cells

The traditional method of passage and adherence screening is used to isolate rat bone marrow stromal stem cells and then replace the culture medium every 3- 4 days. When the cells are mostly fused, use 0.25% pancreatin digest, aspirate the digestion solution and add 10% fetal bovine serum to the culture medium of DMEM by pipetting, and passage the cells after fusion every 4–7 days.


#### 2.2.2. BrdU Labeling of Rat Bone Marrow Stromal Stem Cells

Take the third generation of bone marrow stromal stem cells, take an appropriate amount of bone marrow stromal stem cells for cell climbing, add BrdU (final concentration of 25μmol/L) when the fusion is about 50%, and wait for 48 hours to take out. Then, it was fixed at room temperature with 10% neutral formalin, blocked at room temperature with 3% H2O2, fixed at room temperature with 2 mol/L HCl, mouse anti-BrdU primary antibody (1 : 100) in a humidified box overnight at 4°C, and added biologically. The vegetarian goat anti-mouse IgG secondary antibody was incubated in a wet box, and streptavidin-biotin-peroxidase complex (SABC) was added dropwise and incubated in a wet box. Finally, it was rinsed with PBS liquid, and after 15 minutes of paraformaldehyde fixation, immunohistochemistry and immunofluorescence staining were performed and stained and mounted for observation.


### 2.3. In Vivo Transplantation of Bone Marrow Stromal Stem Cells

After successfully labeling the third-generation bone marrow stromal stem cells with BrdU, make a cell suspension with DMEM containing 10% fetal bovine serum. The concentration of the cell suspension is3∗106/ml. After preparation, use a small amount to test the cell viability and then inject 3∗106 cells per rat through the tail vein of each rat.


### 2.4. Experimental Animals and Grouping

60 12-week-old female SD rats were provided by the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine (clean level). The rats were randomly divided into the normal group, model group, and bone-benefiting group according to the number random table method. Decoction group and estrogen group have 15 cases in each group. Regular feed feeding, free drinking water, good ventilation in the room, room temperature control at 22°C, alternating light and dark conditions every 12 hours.


### 2.5. Modeling

After intraperitoneal injection of pentobarbital sodium 35 m/kg bodyweight anesthetized, rats in the model group, Yigutang group, and estrogen group opened the abdominal cavity, removed both ovaries, and sutured layer by layer. After the ovaries were removed, 4U penicillin was injected subcutaneously into the abdominal cavity every day to prevent postoperative infection.


### 2.6. Feeding and Administration

After the operation, the 60 rats were fed in separate cages, first fed with regular feed for 12 weeks and then fed separately from the 13th week. The normal group was given no treatment. The model group was given normal saline 10 mL·kg−1 by gavage, Yigutang group was given Yigutang water extract 10 mL·kg−1 by gavage, and the estrogen group was given dissolved estradiol valerate tablets. 10 mL of distilled water was then given by gavage according to 10 mL·kg−1. The entire feeding process lasted for 8 weeks. Each rat in each group received a tail vein injection of bone marrow stromal stem cells every 4 weeks.


### 2.7. Indicator Detection

#### 2.7.1. Bone Density Measurement

Rats in each group were sacrificed by cervical dislocation, and their right femurs were taken and fixed on the BMD measuring table. After the rats were straightened, the BMD of the upper metaphysis of the right femur of each rat was measured.


#### 2.7.2. Biomechanics Test

Take the left femur of each rat and use the universal electronic material testing machine to make the whole bone three-point bending test. The maximum load is 20 kg, the span at both ends is 20 mm, and the loading speed is 2 mm/min. The computer automatically traces the load-deformation curve of each group of rat femurs.


#### 2.7.3. Immunohistochemical Analysis

Take the right tibia of each rat, fix it with formalin, rinse with distilled water, decalcify, take the material and rinse with water, then dehydrate, embed in paraffin and section, give H&E stain, observe, and record the number of osteoblasts (count the number of osteoblasts in 5 fields under a 200-fold light microscope and take the average).


#### 2.7.4. Real-Time Fluorescent Quantitative PCR was Used to Detect the Expression of Genes Related to the P13K/AKT/GSK-3β Signaling Pathway

TRIzol one-step method was used to extract total RNA from the right femoral bone tissue of rats in each group, and the fluorescent quantitative PCR was performed according to the instructions on the kit. Usingβ-actin as the internal control and the normal group as the reference group, the relative expression levels of P13K, AKT, and GSK-3β in the right femoral bone tissue of each group were obtained.


#### 2.7.5. Western Blot was Used to Detect the Expression of Proteins Related to the P13K/AKT/GSK-3β Signaling Pathway

After the right femur of each group of rats was crushed, 1 g of bone tissue was taken, and tissue lysate containing RIPA was added, and protease inhibitor (PMSF) was added at the same time for protein quantification after boiling water for about 15 minutes. Take 50μg of each sample for sodium dodecyl sulfate (SDS) gel electrophoresis, transfer the membrane to polyvinylidene fluoride (PVDF) membrane by the semidry method, and seal with skim milk powder for 1 hour. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. Then, add ECL and expose, use the image processing system to scan, and calculate the absorbance value of each band, and the ratio of it to the absorbance value of the internal reference strip represents the expression of P13K, AKT, P-AKt, and GSK-3β protein in each group of femoral bone tissues.


### 2.8. Statistical Analysis

In this study, SPSS 19.0 statistical software was used for statistical analysis. Single-factor analysis of variance was used to compare multiple groups of measurement data. When the variance is homogeneous, the LSD method is used, and when the variance is uneven, Dunnett’s T3 method is used.P<0.05 is considered statistically significant.Figure 1
Normal group number of osteoblasts.Figure 2
Model group number of osteoblasts.Figure 3
Yigutang group number of osteoblasts.Figure 4
Estrogen group number of osteoblasts.Figure 5
P13k protein expression.Figure 6
AKT protein expression.Figure 7
P-AKt protein expression.Figure 8
GSK-3β protein expression.Table 1
Comparison of BMD in the upper metaphysis of right femur in rats (g/cm2, x ± s).
GroupNumberBMD in the upper metaphysis of the right femurNormal150.236 ± 0.03Model150.143 ± 0.04Yigutang150.245 ± 0.017Estrogen150.241 ± 0.02Table 2
Comparison of biomechanical indexes of the left femur in rats (x ± s).
GroupNumberElastic load (N)Elastic deflection (mm)Maximum load (N)Maximum deflection (mm)Normal15139 ± 10.20.82 ± 0.19145.0 ± 5.60.83 ± 0.19Model15107 ± 4.60.69 ± 4.6115.1 ± 4.90.71 ± 0.32Yigutang15125 ± 7.90.72 ± 3.7126.7 ± 9.20.76 ± 0.42Estrogen15126 ± 8.70.71 ± 9.2129 ± 10.10.75 ± 0.37Table 3
Comparison of the number of osteoblasts in rats (x ± s).
GroupNumberNumber of osteoblastsNormal1526.86 ± 10.2Model1512.45 ± 7.9Yigutang1521.79 ± 9.8Estrogen1520.91 ± 10.3Table 4
Comparison of P13k, AKT, and GSK-3β mRNA expressions in the right femur bone tissue of rats.
GroupNumberP13kAKTGSK-3βNormal151.00 ± 0.001.00 ± 0.000.29 ± 0.25Model150.19 ± 0.010.98 ± 0.011.79 ± 0.38Yigutang150.88 ± 0.050.93 ± 0.050.32 ± 0.52Estrogen150.82 ± 0.030.89 ± 0.090.35 ± 0.07Table 5
Comparison of the P13k, AKT, P-AKt, and GSK-3β protein expressions in right femur bone tissue of rats.
GroupNumberP13kAKTP-AKtGSK-3βNormal151.08 ± 0.121.24 ± 0.091.16 ± 0.170.59 ± 0.07Model150.27 ± 0.021.19 ± 0.130.43 ± 0.092.89 ± 0.28Yigutang150.93 ± 0.071.20 ± 0.241.07 ± 0.890.48 ± 0.09Estrogen150.94 ± 0.451.21 ± 0.741.14 ± 0.230.52 ± 0.12


## 2.1. Test Drug

The water extract of Yigu decoction was prepared by the Chinese Medicine Laboratory of Zhejiang University of Traditional Chinese Medicine. The formula is composed of 10 g of psoralen, 15 g ofDrynaria fortunei, 15 g of Radix Rehmanniae, 15 g of Xianling spleen, 15 g of Chinese yam, and 30 g of Salvia miltiorrhiza, containing 2.0 g of crude drug/ml, and kept in the refrigerator for later use. Pentobarbital sodium is produced by Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.


## 2.2. Cultivation, Proliferation, and Labeling of Rat Bone Marrow Stromal Stem Cells

Ten 3-week-old male SD rats were used for the culture and proliferation of bone marrow stromal stem cells. The experimental animals were provided by the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine.


### 2.2.1. Cultivation and Proliferation of Rat Bone Marrow Stromal Stem Cells

The traditional method of passage and adherence screening is used to isolate rat bone marrow stromal stem cells and then replace the culture medium every 3- 4 days. When the cells are mostly fused, use 0.25% pancreatin digest, aspirate the digestion solution and add 10% fetal bovine serum to the culture medium of DMEM by pipetting, and passage the cells after fusion every 4–7 days.


### 2.2.2. BrdU Labeling of Rat Bone Marrow Stromal Stem Cells

Take the third generation of bone marrow stromal stem cells, take an appropriate amount of bone marrow stromal stem cells for cell climbing, add BrdU (final concentration of 25μmol/L) when the fusion is about 50%, and wait for 48 hours to take out. Then, it was fixed at room temperature with 10% neutral formalin, blocked at room temperature with 3% H2O2, fixed at room temperature with 2 mol/L HCl, mouse anti-BrdU primary antibody (1 : 100) in a humidified box overnight at 4°C, and added biologically. The vegetarian goat anti-mouse IgG secondary antibody was incubated in a wet box, and streptavidin-biotin-peroxidase complex (SABC) was added dropwise and incubated in a wet box. Finally, it was rinsed with PBS liquid, and after 15 minutes of paraformaldehyde fixation, immunohistochemistry and immunofluorescence staining were performed and stained and mounted for observation.


## 2.2.1. Cultivation and Proliferation of Rat Bone Marrow Stromal Stem Cells

The traditional method of passage and adherence screening is used to isolate rat bone marrow stromal stem cells and then replace the culture medium every 3- 4 days. When the cells are mostly fused, use 0.25% pancreatin digest, aspirate the digestion solution and add 10% fetal bovine serum to the culture medium of DMEM by pipetting, and passage the cells after fusion every 4–7 days.


## 2.2.2. BrdU Labeling of Rat Bone Marrow Stromal Stem Cells

Take the third generation of bone marrow stromal stem cells, take an appropriate amount of bone marrow stromal stem cells for cell climbing, add BrdU (final concentration of 25μmol/L) when the fusion is about 50%, and wait for 48 hours to take out. Then, it was fixed at room temperature with 10% neutral formalin, blocked at room temperature with 3% H2O2, fixed at room temperature with 2 mol/L HCl, mouse anti-BrdU primary antibody (1 : 100) in a humidified box overnight at 4°C, and added biologically. The vegetarian goat anti-mouse IgG secondary antibody was incubated in a wet box, and streptavidin-biotin-peroxidase complex (SABC) was added dropwise and incubated in a wet box. Finally, it was rinsed with PBS liquid, and after 15 minutes of paraformaldehyde fixation, immunohistochemistry and immunofluorescence staining were performed and stained and mounted for observation.


## 2.3. In Vivo Transplantation of Bone Marrow Stromal Stem Cells

After successfully labeling the third-generation bone marrow stromal stem cells with BrdU, make a cell suspension with DMEM containing 10% fetal bovine serum. The concentration of the cell suspension is3∗106/ml. After preparation, use a small amount to test the cell viability and then inject 3∗106 cells per rat through the tail vein of each rat.


## 2.4. Experimental Animals and Grouping

60 12-week-old female SD rats were provided by the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine (clean level). The rats were randomly divided into the normal group, model group, and bone-benefiting group according to the number random table method. Decoction group and estrogen group have 15 cases in each group. Regular feed feeding, free drinking water, good ventilation in the room, room temperature control at 22°C, alternating light and dark conditions every 12 hours.


## 2.5. Modeling

After intraperitoneal injection of pentobarbital sodium 35 m/kg bodyweight anesthetized, rats in the model group, Yigutang group, and estrogen group opened the abdominal cavity, removed both ovaries, and sutured layer by layer. After the ovaries were removed, 4U penicillin was injected subcutaneously into the abdominal cavity every day to prevent postoperative infection.


## 2.6. Feeding and Administration

After the operation, the 60 rats were fed in separate cages, first fed with regular feed for 12 weeks and then fed separately from the 13th week. The normal group was given no treatment. The model group was given normal saline 10 mL·kg−1 by gavage, Yigutang group was given Yigutang water extract 10 mL·kg−1 by gavage, and the estrogen group was given dissolved estradiol valerate tablets. 10 mL of distilled water was then given by gavage according to 10 mL·kg−1. The entire feeding process lasted for 8 weeks. Each rat in each group received a tail vein injection of bone marrow stromal stem cells every 4 weeks.


## 2.7. Indicator Detection

### 2.7.1. Bone Density Measurement

Rats in each group were sacrificed by cervical dislocation, and their right femurs were taken and fixed on the BMD measuring table. After the rats were straightened, the BMD of the upper metaphysis of the right femur of each rat was measured.


### 2.7.2. Biomechanics Test

Take the left femur of each rat and use the universal electronic material testing machine to make the whole bone three-point bending test. The maximum load is 20 kg, the span at both ends is 20 mm, and the loading speed is 2 mm/min. The computer automatically traces the load-deformation curve of each group of rat femurs.


### 2.7.3. Immunohistochemical Analysis

Take the right tibia of each rat, fix it with formalin, rinse with distilled water, decalcify, take the material and rinse with water, then dehydrate, embed in paraffin and section, give H&E stain, observe, and record the number of osteoblasts (count the number of osteoblasts in 5 fields under a 200-fold light microscope and take the average).


### 2.7.4. Real-Time Fluorescent Quantitative PCR was Used to Detect the Expression of Genes Related to the P13K/AKT/GSK-3β Signaling Pathway

TRIzol one-step method was used to extract total RNA from the right femoral bone tissue of rats in each group, and the fluorescent quantitative PCR was performed according to the instructions on the kit. Usingβ-actin as the internal control and the normal group as the reference group, the relative expression levels of P13K, AKT, and GSK-3β in the right femoral bone tissue of each group were obtained.


### 2.7.5. Western Blot was Used to Detect the Expression of Proteins Related to the P13K/AKT/GSK-3β Signaling Pathway

After the right femur of each group of rats was crushed, 1 g of bone tissue was taken, and tissue lysate containing RIPA was added, and protease inhibitor (PMSF) was added at the same time for protein quantification after boiling water for about 15 minutes. Take 50μg of each sample for sodium dodecyl sulfate (SDS) gel electrophoresis, transfer the membrane to polyvinylidene fluoride (PVDF) membrane by the semidry method, and seal with skim milk powder for 1 hour. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. Then, add ECL and expose, use the image processing system to scan, and calculate the absorbance value of each band, and the ratio of it to the absorbance value of the internal reference strip represents the expression of P13K, AKT, P-AKt, and GSK-3β protein in each group of femoral bone tissues.


## 2.7.1. Bone Density Measurement

Rats in each group were sacrificed by cervical dislocation, and their right femurs were taken and fixed on the BMD measuring table. After the rats were straightened, the BMD of the upper metaphysis of the right femur of each rat was measured.


## 2.7.2. Biomechanics Test

Take the left femur of each rat and use the universal electronic material testing machine to make the whole bone three-point bending test. The maximum load is 20 kg, the span at both ends is 20 mm, and the loading speed is 2 mm/min. The computer automatically traces the load-deformation curve of each group of rat femurs.


## 2.7.3. Immunohistochemical Analysis

Take the right tibia of each rat, fix it with formalin, rinse with distilled water, decalcify, take the material and rinse with water, then dehydrate, embed in paraffin and section, give H&E stain, observe, and record the number of osteoblasts (count the number of osteoblasts in 5 fields under a 200-fold light microscope and take the average).


## 2.7.4. Real-Time Fluorescent Quantitative PCR was Used to Detect the Expression of Genes Related to the P13K/AKT/GSK-3β Signaling Pathway

TRIzol one-step method was used to extract total RNA from the right femoral bone tissue of rats in each group, and the fluorescent quantitative PCR was performed according to the instructions on the kit. Usingβ-actin as the internal control and the normal group as the reference group, the relative expression levels of P13K, AKT, and GSK-3β in the right femoral bone tissue of each group were obtained.


## 2.7.5. Western Blot was Used to Detect the Expression of Proteins Related to the P13K/AKT/GSK-3β Signaling Pathway

After the right femur of each group of rats was crushed, 1 g of bone tissue was taken, and tissue lysate containing RIPA was added, and protease inhibitor (PMSF) was added at the same time for protein quantification after boiling water for about 15 minutes. Take 50μg of each sample for sodium dodecyl sulfate (SDS) gel electrophoresis, transfer the membrane to polyvinylidene fluoride (PVDF) membrane by the semidry method, and seal with skim milk powder for 1 hour. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. Then, add ECL and expose, use the image processing system to scan, and calculate the absorbance value of each band, and the ratio of it to the absorbance value of the internal reference strip represents the expression of P13K, AKT, P-AKt, and GSK-3β protein in each group of femoral bone tissues.


## 2.8. Statistical Analysis

In this study, SPSS 19.0 statistical software was used for statistical analysis. Single-factor analysis of variance was used to compare multiple groups of measurement data. When the variance is homogeneous, the LSD method is used, and when the variance is uneven, Dunnett’s T3 method is used.P<0.05 is considered statistically significant.Figure 1
Normal group number of osteoblasts.Figure 2
Model group number of osteoblasts.Figure 3
Yigutang group number of osteoblasts.Figure 4
Estrogen group number of osteoblasts.Figure 5
P13k protein expression.Figure 6
AKT protein expression.Figure 7
P-AKt protein expression.Figure 8
GSK-3β protein expression.Table 1
Comparison of BMD in the upper metaphysis of right femur in rats (g/cm2, x ± s).
GroupNumberBMD in the upper metaphysis of the right femurNormal150.236 ± 0.03Model150.143 ± 0.04Yigutang150.245 ± 0.017Estrogen150.241 ± 0.02Table 2
Comparison of biomechanical indexes of the left femur in rats (x ± s).
GroupNumberElastic load (N)Elastic deflection (mm)Maximum load (N)Maximum deflection (mm)Normal15139 ± 10.20.82 ± 0.19145.0 ± 5.60.83 ± 0.19Model15107 ± 4.60.69 ± 4.6115.1 ± 4.90.71 ± 0.32Yigutang15125 ± 7.90.72 ± 3.7126.7 ± 9.20.76 ± 0.42Estrogen15126 ± 8.70.71 ± 9.2129 ± 10.10.75 ± 0.37Table 3
Comparison of the number of osteoblasts in rats (x ± s).
GroupNumberNumber of osteoblastsNormal1526.86 ± 10.2Model1512.45 ± 7.9Yigutang1521.79 ± 9.8Estrogen1520.91 ± 10.3Table 4
Comparison of P13k, AKT, and GSK-3β mRNA expressions in the right femur bone tissue of rats.
GroupNumberP13kAKTGSK-3βNormal151.00 ± 0.001.00 ± 0.000.29 ± 0.25Model150.19 ± 0.010.98 ± 0.011.79 ± 0.38Yigutang150.88 ± 0.050.93 ± 0.050.32 ± 0.52Estrogen150.82 ± 0.030.89 ± 0.090.35 ± 0.07Table 5
Comparison of the P13k, AKT, P-AKt, and GSK-3β protein expressions in right femur bone tissue of rats.
GroupNumberP13kAKTP-AKtGSK-3βNormal151.08 ± 0.121.24 ± 0.091.16 ± 0.170.59 ± 0.07Model150.27 ± 0.021.19 ± 0.130.43 ± 0.092.89 ± 0.28Yigutang150.93 ± 0.071.20 ± 0.241.07 ± 0.890.48 ± 0.09Estrogen150.94 ± 0.451.21 ± 0.741.14 ± 0.230.52 ± 0.12


## 3. Results

(1)
The BMD of the upper metaphysis of the right femur in the Yigutang group and the estrogen group was significantly higher than that of the model group (P<0.05), while both Yigutang and estrogen groups had no significant difference compared with the normal group (P>0.05). In addition, there was no significant difference between the Yigutang group and the estrogen group (P>0.05) (as given in Table 1).(2)
The elastic load and maximum load of the Yigutang group and the estrogen group were significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05) (as given in Table 2).(3)
The number of osteoblasts in the Yigutang group and the estrogen group was significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05) (as given in Table 3) (Figures1–4).(4)
The P13K gene expression in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that in the model group (P<0.05), and the AKT gene expression did not change significantly (P>0.05). The gene expression of GSK-3β was significantly lower than that of the model group (P<0.05). Compared with the normal group, the gene expression of P13K, AKT, and GSK-3β in the right femur bone tissue of the Yigutang group and the estrogen group did not change significantly (P>0.05) (as given in Table 4).(5)
The protein expression of P13K and P-AKt in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that of the model group (P<0.05), and the protein expression of AKT did not change significantly (P>0.05). The protein expression of GSK-3β was significantly lower than the model group (P<0.05). Compared with the normal group, the protein expression of P13K, AKT, P-AKt, and GSK-3β in the right femoral bone tissue of the Yigutang group and the estrogen group did not change significantly (P>0.05) (as given in Table 5) (Figures 5–8).


## 4. Discussion

OP is a metabolic disease characterized by a decrease in bone mass and destruction of bone microstructure, which leads to increased fragility of bone and prone to fracture. OP belongs to the category of “gubi” and “guwei” in traditional Chinese medicine, and its pathogenesis is related to the kidney. Most opinions believe that the occurrence of osteoporosis is closely related to the kidney, spleen, and blood stasis [3]. Kidney deficiency is fundamental, and weakness of the spleen and stomach is an important factor in its onset. Blood stasis is the key to the disease, and the theory of traditional Chinese medicine believes that the kidney stores sperm and produces sperm, which is the congenital foundation. The spleen is the source that controls the transport of “Qi” and blood, which is the acquired foundation. At present, the traditional Chinese medicine treatment for the primary OP is mainly based on the method of invigorating the kidney and promoting blood circulation. Professor Yao Xinmiao has been committed to clinical research on osteoporosis. Based on the classic theory of Chinese medicine, the classic prescription Yigutang for nourishing the kidney and promoting blood circulation was developed. The prescription is composed of psoralen, Rhizoma Drynariae, Chinese yam, Shengdi, and danshen. In the prescription, psoralen and Rhizoma Drynariae are the main therapeutic drugs, which can nourish the liver and kidney and strengthen the bones and muscles. Chinese yam invigorates the spleen, “Shengdi” nourishes the kidney and yin, and “danshen” has the effect of promoting blood circulation and removing blood stasis. Clinical studies have found that Yigutang can effectively increase the lumbar bone density of elderly patients with osteoporosis, reduce pain, inhibit bone resorption, and improve the clinical symptoms of patients [4]. Animal experiments show that Yigutang can significantly increase the serum hormone level of ovariectomized rats, increase bone density, can improve the biomechanical properties of rats, and promote the activity of osteoblasts to achieve the purpose of prevention and treatment of osteoporosis [5].Bone remodeling runs through the whole process of bone metabolism, the imbalance of bone formation and bone resorption can lead to osteoporosis [6], and bone formation and bone resorption are closely linked to form bone remodeling. This process is mainly completed by the joint participation of osteoblasts and osteoclasts [7]. Osteoblasts promote bone formation, osteoclasts lead to bone resorption, and the balance between osteoblasts and osteoclasts maintains normal bone density; the formation mechanism of osteoporosis is currently mainly caused by the negative balance of bone remodeling. In the process of bone remodeling, bone resorption is greater than bone formation, resulting in osteoporosis [8]. The speed of bone formation cannot catch up with the speed of bone resorption, which breaks the balance of bone remodeling. At this time, the activity of osteoclasts is greater than that of osteoblasts. Osteoblasts and osteoclasts originate from bone marrow stromal stem cells (BMSCs). Therefore, inducing BMSCs to transform into osteoblasts has become the focus of current research [9]. Studies have shown that multiple signaling pathways [10] can affect the transformation of BMSCs into osteoblasts, such as the classic Wnt signaling pathway [11], TGF-β/BMP-2 signaling pathway [12], and P13K/AKT/GSK-3β signal pathways. Classical Wnt signaling pathways [13] include the classical cellular pathway (Wnt/β-catenin) and nonclassical cellular pathways (Wnt-planner cell polarity pathway and Wnt calcium pathway). The latest experimental studies have shown that Yigu decoction can regulate Wnt, BMP2, β-catenin, Runx2, OSX, and other factors through two classic Wnt signaling pathways to induce osteoblast proliferation and differentiation and further promotes bone formation and prevents OP [14]. In addition to the Wnt/β-catenin signaling pathway, the PI3K/AKT/GSK-3β signaling pathway has always been the focus of researchers studying cell proliferation and apoptosis [15]. PI3K is an intracellular phosphatidylinositol kinase consisting of various catalytic subunits. The extracellular signal is combined with the activated Ras protein receptor, which activates P13K, P13K phosphorylates PIP2, and converts PIP2 to PIP3. PIP3 combines with the PH structure of AKt, so that AKt is recruited to the cell membrane, and AKT is the first phosphorylation of 308 enzyme amino acid [16], thereby activating AKT (p-AKt). GSK-3β is the downstream factor of this signaling pathway and the substrate on which AKT acts. AKT can phosphorylate the 9th serine of GSK-3β and reduce the phosphorylation level of GSK-3β, which can induce cell apoptosis and thus play a regulatory role in cell proliferation and differentiation [17].In this study, 60 female SD rats were divided into groups, the BMD of the upper metaphysis of the right femur of each group was detected, the left femur of each group was removed, and the load-deformation curve of the left femur of each group of rats was calculated. The number of osteoblasts was observed by H&E staining, and the positive rate of BrdU cells was detected by BrdU immunohistochemical staining. After extracting the right femur of each group of rats, real-time fluorescent quantitative PCR and Western blot were used to detect the expression levels of related genes and proteins in the P13K/AKT/GSK-3β signaling pathway. The results showed that the BMD of the upper metaphysis of the right femur in the Yigutang group and the estrogen group was significantly higher than that of the model group (P<0.05), while both Yigutang and estrogen groups had no significant difference compared with the normal group (P>0.05). In addition, there was no significant difference between the Yigutang group and the estrogen group (P>0.05). The elastic load and maximum load of the Yigutang group and the estrogen group were significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05). The number of osteoblasts in the Yigutang group and the estrogen group was significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05). The positive rates of BrdU cells in the Yigutang group and the estrogen group were significantly higher than the model group (P<0.05), but both were lower than the normal group (P<0.05). There was no significant difference between the Yigutang group and the estrogen group (P>0.05). The P13K gene expression in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that in the model group (P<0.05), and the AKT gene expression did not change significantly (P>0.05). The gene expression of GSK-3β was significantly lower than that of the model group (P<0.05). Compared with the normal group, the gene expression of P13K, AKT, and GSK-3β in the right femur bone tissue of the Yigutang group and the estrogen group did not change significantly (P>0.05). The protein expression of P13K and P-AKt in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that of the model group (P<0.05), and the protein expression of AKT did not change significantly (P>0.05). The protein expression of GSK-3β was significantly lower than the model group (P<0.05). Compared with the normal group, the protein expression of P13K, AKT, P-AKt, and GSK-3β in the right femoral bone tissue of the Yigutang group and the estrogen group did not change significantly (P>0.05). These indicate that Yigutang can regulate the differentiation of bone marrow stromal stem cells into osteoblasts. This effect may be achieved by regulating the P13K/AKT/GSK-3β signaling pathway. The mechanism of action may be that Yigu decoction upregulates the expression level of P13K and activates AKt phosphorylation at the same time. GSK-3β is a downstream factor of this signaling pathway and the first AKT substrate found in the body. Phosphorylation of AKt can also phosphorylate GSK-3β, downregulate the expression level of GSK-3β, and promote alkaline phosphatase (ALP) activity, Ca2+ deposition, the expression level of OCN and Runx2, and proliferation of osteoblasts. In this way, the purpose of preventing and treating osteoporosis is achieved, but its specific mechanism of action needs to be further studied.


## 5. Conclusion

Yigutang can not only prevent and treat osteoporosis through the classic Wnt/β-catenin signaling pathway but also achieve the purpose of preventing and curing osteoporosis through the P13K/AKT/GSK-3β signaling pathway. This project explores the mechanism of Yigu decoction in promoting osteogenic differentiation of bone marrow stromal stem cells from the molecular gene level, which lays the foundation for the modernization and objective research of Chinese medicine. In addition, this study also provides new ideas and methods for the treatment of osteoporosis in the future.



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*Source: 6699781-2021-06-18.xml*