Journal of Alternative Complementary & Integrative Medicine Category: Medicine Type: Research Article

Research on Mechanism of Shenling Baizhu San on Diabetic Obesity Based on Meta-Analysis and Network Pharmacology

Diyao Wu1, Shu Wang2, Shumao Pan2, Xiuyun Li2, Zheng Yang1, Xin Kuang1, Xingxing Gong2, Xinyou Zhang1,2 and Xiaofan Chen1*

1 College Of Traditional Chinese Medicine, Jiangxi University Of Traditional Chinese Medicine, Nanchang, Jiangxi, China
2 College Of Pharmacy, Jiangxi University Of Traditional Chinese Medicine, Nanchang, Jiangxi, China

*Corresponding Author(s):
Xiaofan Chen
College Of Traditional Chinese Medicine, Jiangxi University Of Traditional Chinese Medicine, Nanchang, Jiangxi, China
Email:xiaofanci122306@163.com

Received Date: Feb 09, 2022
Accepted Date: Mar 07, 2022
Published Date: Mar 14, 2022

Abstract

Objective: To evaluate the therapeutic effects of Shenling Baizhu San on diabetic obesity by literature meta-analysis, and analyze the mechanism of Shenling Baizhu San on diabetic obesity by combining network pharmacology and animal experiment methods. 

Methods: Relevant literatures at home and abroad were included. The BMI, glycosylated hemoglobin (HbA1c), 2h postprandial blood glucose (2hPG), fasting blood glucose (FBG), Homa-IR, waist-to-hip ratio were used as outcome indicators to evaluate the effects of Shenling Baizhu San on diabetic obesity. The related genes of diabetes obesity in GEO chip were downloaded, and the targets of Shenling Baizhu San were downloaded in TCMSP, TCMID and BATMAN-TCM. Protein interaction analysis was carried out on the intersection targets of the two, and then the core target protein was screened out based on topological analysis. The model of diabetic obese mice was established, and the mice were treated with Shenling Baizhu San after grouping. After two weeks, the body mass, blood sugar and Lee's index of mice in each group were measured. The volume changes of adipocytes in each group were analyzed by Hematoxylin-Eosin method, and the concentrations of cholesterol, triglyceride and core target protein in mice serum were measured. 

Results: A total of 9 literatures were included, the data were complete, no publication bias was found, and the evaluation was low risk. After administration of Shenling Baizhu San, the BMI, HbA1c, 2hPG, FBG, Homa-IR and waist-hip ratio of the patients were significantly improved. There were 337 genes related to diabetes obesity, 2067 targets of Shenling Baizhu San and 59 intersecting targets. There were 7 signal pathways related to the effect of Shenling Baizhu San on diabetes obesity, including Biosynthesis of unsaved fatty acids, Fatty acid metabolism and so on. The results of animal experiment showed that the blood glucose of mice treated with Shenling Baizhu San was not significantly changed, the body weight and Lee's index showed a significant downward trend, the fat cells were significantly reduced, and the contents of cholesterol and triglyceride, the concentration of APO-E, IGF-1 and PAI-1 protein showed a downward trend. 

Conclusion: Based on the results of meta-analysis and animal experiments, Shenling Baizhu San had obvious therapeutic effect on diabetic obesity, and its mechanism may be related to influencing the synthesis of unsaturated fatty acids and fatty acids, reducing the transformation of glucose and fat, correcting the imbalance of energy metabolism in the body, and promoting the decomposition of adipose tissue by up-regulating autophagy.

Keywords

Diabetes obesity; Mechanism; Meta analysis; Network pharmacology; Shenling Baizhu San

Introduction

Obesity or overweight has long been considered as an independent risk factor for various diseases, including hypertension, coronary heart disease, especially diabetes [1]. A series of active factors secreted by adipose tissue is able to induce insulin resistance, and thus result in affecting glucose metabolism [2]. Therefore, obesity combined with diabetes is extremely common in clinical practice. Insulin resistance caused by obesity will further progress to metabolic syndrome, leading to the occurrence of diabetic cardio-cerebrovascular disease (DM-CVD), which poses a serious threat to human health [3] and has become a global public health problem [4,5] . 

From the perspective of Traditional Chinese Medicine (TCM), diabetic obesity is mainly due to the usual addiction to fat diet and sweetness, prolonged sitting and multiple lying, resulting in spleen deficiency and loss of transport and transformation. In modern clinical practice, numerous studies have confirmed that [6,7] traditional Chinese medicine has definite curative effect in treating diabetes obesity, especially the compound Chinese medicines for invigorating spleen and eliminating dampness, such as Shenling Baizhu San (SLBZS) and Jianpi Xiaozhi Decoction, which have been clinically proved to not only reduce the patient's weight, but also assist in controlling the development of diabetes [8,9]. Nevertheless, its specific mechanism of action has not yet been studied clearly. The present study aims to evaluate the therapeutic effect of SLBZS on diabetic obesity through literature meta-analysis, and then analyze the mechanism of SLBZS on diabetic obesity in combination with network pharmacology and animal experiments, so as to promote its application in diabetic obesity and improve the clinical cure rate and safety of diabetic obesity.

Materials And Methods

The overall flow chart of this paper is shown in figure 1.

Figure 1: Flow chart of the mechanism of SLBZS on diabetes obesity based on Meta and network pharmacology. 

Meta analysis 

Literature inclusion criteria: 

  • The randomized controlled trial (RCT) published at home and abroad
  • All the subjects were in line with the diagnostic criteria for diabetes, including Chinese Guidelines for Prevention and Treatment of Type 2 Diabetes (2017 Edition) and Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults
  • All the subjects had no other concomitant diseases, and there was no statistical difference in baseline data

Literature exclusion criteria: 

  • Documents did not meet the inclusion criteria
  • Animal, review, and repetitive publications
  • Documents containing other intervention measures and comorbidities 
  • Documents with incomplete or incorrect test information and inconsistent design 

Outcome indicators: BMI, glycosylated hemoglobin (HbA1c), 2h postprandial blood glucose (2h PG), fasting blood glucose (FBG), Homa-IR, waist-hip ratio. 

Literature retrieval strategy: With "diabetes and obesity" and "Shen ling Bai zhu San" as the subject words, the relevant literatures were searched in CNKI, VIP, WANGFANG and CBM. With "diabetes obesity" and "Shen ling Bai zhu San/powder" as the subject words, the relevant literatures were searched in PubMed and web of science databases. The time was from the construction of the database to June 2021. 

Literature screening and quality evaluation: 

  • Data extraction: Two evaluators with professional knowledge background of evidence-based medicine independently screened the literatures and excluded the literatures which obviously did not meet the inclusion criteria. After that, the two evaluators exchanged the results and checked the documents and data extracted from the documents. If there are differences in opinions between the two parties, another professional evaluation is required until the final opinions are reached.
  • Evaluation of literature quality: According to Cochrane Manual of Evidence-based Medicine, the bias risk of systematic evaluation results from was evaluated based on the aspects of random sequence generation, distribution concealment and the integrity of outcome data.
  • Statistical processing: Meta-analysis was carried out on the data extracted from the literature by using RevMan5.3 analysis software. The Ratio (OR) was taken as the relative effect index variable of counting data, and the Standardized Mean Difference (SMD) or Mean Difference (MD) was taken as the relative effect index of measuring data. The effect value and its 95% Confidence Interval (CI) were taken as the index of the reliability of the results. The heterogeneity of results was mainly judged by I2 test. If P> 0.10 and I2< 50% in the results, the heterogeneity of outcome indicators was small, and the fixed effect model should be adopted for analysis. If P< 0.10 and I2> 50% in the results, the heterogeneity of the outcome indicators was large, and the random effect model should be adopted for analysis. 

Network pharmacological analysis 

Disease targets of diabetes and obesity: The obesity gene of diabetes was downloaded from GEO chip (https://www.ncbi.nlm.nih.gov/). During this period, the GEO DateSets was selected on the left side of the search box, "Diabetic Obesity" was searched, and the GSE number, platforms and sample information were recorded. Then, data were analyzed by R language, and the related genes of diabetic obesity were obtained. 

Action target of SLBZS: The action targets of SLBZS were obtained from TCMSP (https://old.tcmsp-e.com/tcmsp.php), TCMID (http://119.3.41.228:8000/tcmid/) and BATMAN-TCM respectively. After removing the duplicates, the data were integrated to obtain the action targets of SLBZS. Venny diagram was drawn by JVENN net (http://jvenn.toulouse.inra.fr/app/example.html) to get the intersection, which was the action target of SLBZS in treating diabetic obesity. 

Core target of SLBZS in treating diabetic obesity: All the targets of SLBZS in treating diabetic obesity were input into String platform (https://www.string-db.org/) for protein interaction analysis, and after removing unconnected targets, the network diagram of protein interaction was obtained. Then, the network diagram was input into Cytoscape_v3.8.2, and APP--cytoNCA plug-in was selected. Degree Centrality (DC), Closeness Centrality (CC) and Betweenness Centrality (BC) were clicked and screen out the core target of SLBZS in diabetic obesity. 

Animal experiment 

Experimental mice: Sixty SD mice weighing 20–25g which were provided by Hunan Slake Jingda Experimental Animal Co., Ltd,(Certificate No.SYXK(Gan)2017-0004) were kept in SPF-grade experimental animal houses. The illumination time was from 6:00 to 18:00. All mice were housed under conditions of constant humidity. The mice were fed with standard fodder and water freely. After 7 days of adaptive feeding, the mice were randomly divided into conventional feeding group (ND) and high fat feeding group (HFD), with a ratio of 10:50. 

Drugs and reagents: High sugar and high fat mouse feed was purchased from Nanjing Shengmin animal farm (batch No. 210610); All 10 traditional Chinese medicines contained in Shenling Baizhu powder (White Lentils, Atractylodes, Poria, Licorice, Platycodon, Lotus Seed, Ginseng, Amomum, Chinese Yam, Coix Seed) were purchased from the pharmacy of the Affiliated Hospital of Jiangxi University of traditional Chinese medicine; 4% paraformaldehyde was purchased from Biosharp company; Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Item No.100092683); Xylene was purchased from Sinopharm Chemical Reagent Co., Ltd. (Item No.10023418); HE matoxylin eosin (He) staining solution was purchased from Beijing Regan Biotechnology Co., Ltd. (Item No.DH 0006); Neutral gum was purchased from Sinopharm Chemical Reagents Co., Ltd. (Item No.10004160). Elisa test boxes for TC, TG, APO-E, IGF-1 and PAI-1 were purchased from Nanjing jiancheng technology Co., Ltd. 

Apparatus: Paraffin embedding machine EG1150 (Leica); RM2016 (Leica); Mingmei MF43 microscope (Mingmei); Minmei MC50 Digital Imaging Measurement and Analysis System (Mingmei); Labsystems Finnpipette 100μl single channel pipette; Thermo 50μl 8-channel pipette; HH-4 Digital Display Constant Temperature Water Bath Pot (Guohua Electric Appliance Co., Ltd.); Electronic DG5033A microplate reader (Nanjing Huadong Electronics Group Medical Equipment Co., Ltd.); Blood glucose meter (Sannuo Intelligent Blood Glucose Meter). 

Experimental method: Establishment and Grouping of Diabetic Obesity Models: In view of the fact that diabetic obesity patients were mostly caused by high-sugar and high-fat diet [10], we adopted dietary induction method to establish the diabetic obesity model. Due to the irregular eating time of mice, it was impossible to determine the blood glucose before and after meals. The diagnostic criteria of diabetes based on fasting plasma glucose or postprandial blood glucose are not applicable to this study. Therefore, the change of blood glucose was selected as the measurement index. The establishment criteria of the model were as follows: the weight of mice in model group ≥ the weight of mice in blank group ×120%, and the blood glucose of mice after modeling ≥ the blood sugar of mice before modeling ×130%. If the criteria were met, it was judged that the model had been successfully established. 

Grouping of mice: Fifty mice in high-fat feeding group were randomly divided into model group (high-sugar and high-fat diet) and administration group (high-sugar and high-fat diet+ SLBZS) after feeding for 30 days. The administration doses of SLBZS were 0.33g/ day (SLBZS-L), 0.66g/ day (SLBZS-M) and 1.32g/ day respectively. After two weeks of intragastric treatment, mice were dissected, blood and adipose tissue were collected, and all experimental operations were in compliance with the requirements of the Ethics Committee of Jiangxi University of Traditional Chinese Medicine. 

Measurement of body weight and blood sugar: From the modeling period, the body weight of mice was measured every five days, and the blood from the tail tip vein of mice was taken at a fixed time point every five days to measure blood glucose and record the data. 

Lee's index measurement: The weight of mice was measured before dissection, the body length (length from nose tip to anus) was measured by straightening, and the Lee's index of each mouse was calculated. 

HE staining: After the mice were dissected, the abdominal adipose tissue was taken. After baking, sectioning, dewaxing, dyeing, dehydration, transparency and sealing, the abdominal adipose tissue was observed, photographed and analyzed under eyepieces 40x, 100x and 400x. 

Blood lipid analysis: The blood of mouse orbital venous plexus was collected and put into the test tube. After coagulation, the upper serum was taken. The contents of cholesterol and triglyceride in mouse serum were determined by enzyme labeling colorimetry. 

Core target analysis: According to the core target results obtained in 1.2.3, the concentration of core target protein in mouse serum was detected by ELISA kit.

Results

Results of meta-analysis 

Results of literature retrieval and inclusion: According to the above retrieval strategies, 66 articles were included in total, including 1 PubMed, 18 CNKI, 21 Wanfang, 11 VIP and 15 CBM. After removing duplicates, 26 articles were obtained. After reading the title and abstract, 14 unrelated literatures were deleted, and 2 were deleted after reading the full text. Finally, 9 literatures were obtained, including 3058 patients. The literature screening process and results were shown in figure 2.

Figure 2: Screening process of included literature. 

Basic characteristics of included literature: A total of 9 literatures, 769 patients, were included, including 386 cases in the treatment group and 383 cases in the control group. The outcome indicators mainly included BMI, glycosylated hemoglobin (HbA1c), 2h postprandial blood glucose (2hPG), fasting blood glucose (FBG), Homa-IR, waist-to-hip ratio. The basic characteristics of the included literature were shown in table 1.

Table 1: Basic characteristics of included literatures. 

Bias risk assessment of included literature: According to the biased risk assessment method of Cochrane risk assessment table, the quality of 9 included literatures was evaluated, and the evaluation results were described by "low risk", "unclear risk" and "high risk". All the literatures included in this study mentioned random grouping, among which 5 literatures [11,15-19] clearly explained the specific random method, which was assessed as low risk; 4 literatures [12-14,17] only mentioned random grouping, which was assessed as unclear risk; None of the included studies mentioned allocation concealment and blindness, which was assessed as unclear risk; Other biases were unknown, which was assessed as unclear risk; All the literatures had clear outcome indicators and complete data, no publication bias was found, and thus was assessed as unclear risk. The assessment results were shown in figure 3.

Figure 3: The evaluation results of bias risk of inclusion study. 

Analysis of glycosylated hemoglobin: Four literatures [12,14,17,18] reported the effects of SLBZS combined with western medicine on glycosylated hemoglobin, including 197 cases in the treatment group and 197 cases in the control group. Heterogeneity test showed that heterogeneity was not significant [χ2=4.51, df=3, P=0.21, I2=33%], and the fixed effect model was adopted. Meta-analysis showed that the glycosylated hemoglobin in the treatment group was lower than that in the control group, and the difference was statistically significant [SMD=-0.47, 95% CI (-0.67, -0.26), Z=4.54, P< 0.00001], as shown in figure 4.

Figure 4: The forest plot comparing of glycosylated hemoglobin of 2 groups of patients

Analysis of 2h postprandial blood glucose: Six literatures [14-19] reported the effects of SLBZS combined with western medicine on 2h postprandial blood glucose, including 258 cases in the treatment group and 255 cases in the control group. Heterogeneity test showed that heterogeneity was not significant [ χ 2 = 3.82, DF = 5, P = 0.58, I2 = 0%], and the fixed effect model was adopted. Meta-analysis showed that the 2h postprandial blood glucose in the treatment group was lower than that in the control group, and the difference was statistically significant [SMD = -0.73, 95% CI (- 0.91, - 0.55), z = 8.00, P < 0.00001], as shown in figure 5.

Figure 5: The forest plot comparing of 2h postprandial blood glucose of 2 groups of patients.

Analysis of fasting blood glucose: Six literatures [14-19] reported the effects of SLBZS combined with western medicine on fasting blood glucose, including 258 cases in the treatment group and 255 cases in the control group. Heterogeneity test showed that heterogeneity was significant [χ2=16?df=5?P=0.007?I2=69%], and the random effect model was adopted. Meta-analysis showed that the fasting blood glucose in the treatment group was lower than that in the control group, and the difference was statistically significant [SMD=-0.55, 95% CI(-0.87, -0.22), Z=3.31, P= 0.0009], as shown in figure 6.

Figure 6: The forest plot comparing of fasting blood glucose of 2 groups of patients.

Analysis of Homa-IR: Two literatures [13,16] reported the effect of SLBZS combined with western medicine on Homa-IR, including 89 cases in the treatment group and 90 cases in the control group. Heterogeneity test showed that heterogeneity was not significant [χ2=1.02, df=1, P=0.31, I2=2%], and the fixed effect model was adopted. Meta-analysis showed that Homa-IR of patients in treatment group was lower than that in control group, and the difference was statistically significant [SMD=-1.05, 95% CI(-1.37, -0.74), Z=6.58, P< 0.00001], as shown in figure 7.

Figure 7: The forest plot comparing of Homa-IR of 2 groups of patients

Analysis of waist-hip ratio: Two literatures [17,18] reported the effect of SLBZS combined with western medicine on waist-hip ratio, including 100 cases in the treatment group and 100 cases in the control group. Heterogeneity test showed that heterogeneity was not significant [χ2=0.52, df=1, P=0.47, I2=0%], and the fixed effect model was adopted. Meta-analysis showed that Homa-IR of patients in treatment group was lower than that in control group, and the difference was statistically significant [MD=-0.17, 95% CI(-0.24, -0.1), Z=5, P< 0.00001], as shown in figure 8.

Figure 8: The forest plot comparing of waist-hip ratio of 2 groups of patients

Analysis of BMI: Four literatures [11,17-19] reported the effects of SLBZS combined with western medicine on BMI, including 175 cases in the treatment group and 171 cases in the control group. Heterogeneity test showed that heterogeneity was not significant [χ2=3.94, df=3, P=0.27, I2=24%], and the fixed effect model was adopted. Meta-analysis showed that the BMI of patients in the treatment group was lower than that in the control group, and the difference was statistically significant [MD=-0.68, 95% CI(-1.05, -0.31), Z=3.58, P= 0.0003], as shown in figure 9.

Figure 9: The forest plot comparing of BMI of 2 groups of patients.

Analysis of GLP-1: Two literatures [11,16] reported the effect of SLBZS combined with western medicine on GLP-1, including 89 cases in the treatment group and 90 cases in the control group. Heterogeneity test showed that heterogeneity was not significant [χ2=0.62, df=1, P=0.43, I2=0%], and the fixed effect model was adopted. Meta-analysis showed that GLP-1 in the treatment group was higher than that in the control group, and the difference was statistically significant [MD = 5.08,95% ci (4.62,5.54), Z=21.54, P< 0.00001], as shown in figure 10.

Figure 10: The forest plot comparing of GLP-1 of 2 groups of patients 

Results of Network Pharmacology 

Related targets of diabetic obesity: A total of 337 genes related to diabetic obesity were downloaded from GEO chip, as shown in Attached table 1. 

Action target of SLBZS on diabetic obesity: A total of 2067 targets of Shenling Baizhu San (SLBZS) were obtained from TCMSP, TCMID and BATMAN-TCM. See Attached table 2 for details. Then, the Venny diagram was drawn to obtain the intersection of the target genes of diabetic obesity gene and SLBZS, namely, the target of Shen Ling Baizhu San acting on diabetic obesity, as shown in figure 11. In addition, there were 59 intersection targets, as shown in table 2. 

Gene

Gene

Gene

Gene

Gene

Gene

SERPINE1

FADS1

THBS1

VIP

ASS1

GSTO1

UBE2B

CKB

DGAT2

IGF1

MMAB

TP53I3

SLC25A1

SERPINB7

ADAM17

SPARC

PDGFRL

ADAP2

ACADSB

PTGER2

UCHL1

ALDH1B1

NAPEPLD

TET1

ACSS2

S100A8

COPS5

APOE

PODXL

TNFAIP3

CHRNA7

RET

HEXIM1

TYMS

AOC3

LYZ

AHCY

SCD

PNRC2

ENPP3

RFWD3

FASN

KCNA1

FADS2

SULF2

GFER

GAS6

DNAJB9

SPX

SLC16A12

CFTR

ACACA

SLC16A10

CDKN1A

EPHX2

RRM2

GSTT1

CRYZ

CLSPN

 

Table 2: The target gene for diabetes obesity gene and SLBZS.

Figure 11: The venn diagram of the target gene for diabetes obesity gene and SLBZS table 2. The target gene for diabetes obesity gene and SLBZS. 

Analysis of the core targets of SLBZS Powder on diabetic obesity: The above 59 targets were input into String platform (https://www.string-db.org/) for protein interaction analysis, and the network diagram of protein interaction was obtained, as shown in figure 12. Then, the network diagram was input into Cytoscape_v3.8.2, and three core targets of SLBZS on diabetic obesity were screened, as shown in figure 13.

Figure 12: PPI network of Diabetic obesity related targets of SLBZS.

Figure 13: 3 core targets were screened by Cytoscape. 

Through the gene enrichment analysis function in String, 7 KEGG signal pathways related to the above 59 targets were found. See table 3 for details. Through the KEGG signal pathway analysis platform (https://www.genome.jp/kegg/pathway.html), it was found that SLBZS affects the production of fat by affecting the biosynthesis of unsaturated fatty acids (hsa01040) and fatty acid metabolism (hsa01212); reduce the conversion of sugar and fat by affecting the metabolic process of pyruvate (hsa00620); corrects the imbalance of energy metabolism by activating AMPK pathway (hsa04152) and metabolic pathway (hsa01100); up-regulates autophagy to promote the decomposition of adipose tissue by activating p53 pathway (hsa04115). The relationship between pyrimidine metabolic pathway (hsa00240) and the development of obesity remains unclear, however, the literature [20] compared the differences in metabolic pathways of adipose tissue around the tumor of obese mice and on the opposite side of the tumor of obese mice. It was found that many differential pathways were involved in carcinogenesis, such as purine and pyrimidine metabolism, which indicated that obesity might be potentially associated with the occurrence of some cancers. 

pathway

description

hsa01040

Biosynthesis of unsaturated fatty acids

 

 

hsa01212

Fatty acid metabolism

 

 

hsa04115

p53 signaling pathway

 

 

hsa00620

Pyruvate metabolism

 

 

hsa00240

Pyrimidine metabolism

 

 

hsa04152

AMPK signaling pathway

 

 

hsa01100

Metabolic pathways

Table 3: Related pathways of SLBZS in treating diabetic obesity.

Results of animal experiments 

Establishment of diabetic obesity mouse model: The body weight and Lee's index of mice in high glucose and high fat diet group (HFD group) were significantly different from those in routine diet group (RD group) (P < 0.01), as shown in table 4. After the modeling period, the body mass of HFD group was greater than or equal to the body mass of RD group mice ×120%, as shown in figure 14. After modeling, the blood glucose of mice in HFD group was greater than or equal to the blood glucose of mice before modeling ×130%, as shown in Figure 15. Therefore, the diabetic obesity mouse model was successfully established.

Figure 14: Weight change trend of RD and HFD mice during modeling.

Figure 15: Scatter diagram of blood glucose in HFD group before and after modeling.

Results of blood glucose, body weight, body length and Lee's index of mice: The measurement results showed that compared with the HFD group, the weight of mice treated with SLBZS decreased significantly, while the decrease of blood glucose was not significant enough, as shown in figures 16-17. After two weeks of intragastric administration of SLBZS, there was an extremely significant difference in body weight between obese mice and HFD group (P < 0.01). In addition, there was a significant difference in Lee's index between low-dose administration group and high-dose administration group (P < 0.05), but there was no significant difference in body length between groups, see table 4 for details. The results showed that SLBZS has a significant effect on reducing the body mass and Lee’s index of diabetic obesity mice, and is positively correlated with the dose. The effect of SLBZS on the blood glucose of mice needs further verification.

Figure 16: Change of body weight in mice of each group.

Figure 17: Change of blood glucose in mice of each group. 

Group

Body weight?g?

Body length?cm?

Lee's index

 

 

 

 

RD

43.08±1.54a

10.6±0.19

3.3±0.072a

HFD

53.12±3.1

10.75±0.46

3.5±0.11

SLBZS-L

47.35±2.03a

10.95±0.47

3.3±0.11b

SLBZS-M

45.18±2.91a

10.61±0.42

3.36±0.12

SLBZS-H

47.4±1.52a

10.98±0.36

3.3±0.074a

Table 4: Comparison of weight, body length and Lee's index of mice in each group.

Results of HE staining analysis: HE stained photographs of adipose tissue of mice in each group were observed, as shown in figure 18. Compared with the blank group, the adipose volume of the model group was significantly larger than that of the blank group. Compared with the model group, the adipose volume of in the low dose group decreased, while the adipose volume of in the middle and high dose groups decreased significantly. Therefore, SLBZS had a significant effect on reducing the adipose volume of diabetic obesity mice.

 Figure 18: HE staining photos of adipose tissue of mice in each group under 400X microscope. 

Results of blood lipid analysis: The content of cholesterol (TC) and triglyceride (TG) in serum was measured by microplate colorimetry, and the calculation formula was as follows:

The results showed that there were significant differences in cholesterol and triglyceride between HFD mice and RD mice, which indicated that TG and TC of mice fed with high sugar and high fat were significantly increased. However, the TC and TG of mice treated with SLBZS showed a downward trend, and the higher the dose, the more significant the decrease was. Compared with HFD mice, the difference between TC and TG in middle and high dose groups was extremely significant, while that in low dose group was not significant, as shown in table 5 for details. 

 

RD

HFD

SLBZS-L

SLBZS-M

SLBZS-H

 

?mmol/L?

?mmol/L?

?mmol/L?

?mmol/L?

?mmol/L?

TG

0.615±0.083a

1.216±0.146

1.072±0.065

0.817±0.0472a

0.808±0.085a

TC

2.287±0.356a

3.317±0.46

3.260±0.46

2.60±0.27b

2.405±0.196a

Table 5: Concentration of cholesterol and triglyceride in mice of each group (X S ,n=6).

(Note: P values are compared with HFD group, a indicates P < 0.01, b indicates P < 0.05) 

Results of core target analysis: Elisa results showed that compared with RD mice, the concentrations of APO-E, IGF-1 and PAI-1 in serum of HFD group were significantly different, which indicated that the levels of APO-E, IGF-1 and PAI-1 in serum of mice fed with high sugar and high fat were significantly increased. The mice treated with SLBZS showed significantly down-regulated levels of APO-E, IGF-1 and PAI-1 in serum, and the higher the dose, the more significant the decrease was. Compared with HFD mice, the differences of APO-E, IGF-1 and PAI-1 in middle and high dose groups were significant, while those in low dose groups were not significant, as shown in table 6. 

Key Target Gene

RD

HFD

SLBZS-L

SLBZS-M

SLBZS-H

APO-E?μg/ml?

42.45±3.343a

62.62±5.604

64.49±6.593

52.65±5.66b

47.24±4.038a

IGF-1?ng/ml?

38.39±2.763a

63.93±4.803

63.55±5.575

55.81±4.734b

42.95±1.925a

PAI-1(?ng/ml)

15.37±1.182a

26.35±4.135

24.21±3.239b

20.70±2.0a

19.10±2.802a

Table 6: Protein concentrations of Apo-E, IGF-1 and PAI-1 in serum of mice in each group (X S ,n=6). 

(Note: P values are compared with HFD group, a indicates P < 0.01, b indicates P < 0.05)

Discussion

SLBZS, first recorded in the book "Taiping Huimin Heju prescription" of the Song Dynasty, has been an important prescription for the treatment of spleen and stomach deficiency and difficult diet since ancient times. The administration of SLBZS is capable of dispelling the pathological products formed by spleen's unhealthy movement, and making the patient's qi and blood smooth, viscera soft and obesity disappear [21]. Traditional Chinese medicine has a long history of treating phlegm-dampness obesity with SLBZS, however, there is still a lack of systematic and quantitative scientific data to comprehensively evaluate its therapeutic effect. Meta-analysis verified that SLBZS could significantly reduce BMI index, HbA1c, 2 hPG, FBG, Homa-IR and waist-hip ratio of diabetic obese patients. Based on the above-mentioned results, we established an animal experiment to verify the therapeutic effect of SLBZS on diabetic obesity mice. The results showed that the adipose cells of mice treated with SLBZS were significantly reduced, and the body weight, Lee's index, triglyceride and cholesterol of mice were significantly decreased, which confirmed that SLBZS had a definite therapeutic effect on obesity. However, the influence of experimental results on blood glucose of mice remained clear, which might be related to short modeling time, insufficient damage of islet β cells and unstable fluctuation of blood glucose. The results of the present study were able to provide strong evidence for the clinical application of SLBZS in the treatment of diabetic obesity. 

In addition, to further analyze the mechanism of SLBZS in the treatment of diabetic obesity, we predicted its related signal pathways and core targets through network pharmacology. The results showed that the mechanism of SLBZS in treating diabetic obesity may be related to influencing the synthesis of unsaturated fatty acids and fatty acids, reducing the transformation of sugar and fat, correcting the imbalance of energy metabolism, and promoting the decomposition of adipose tissue by up-regulating autophagy. The animal experimental results also illustrated that SLBZS had significant regulatory effects on three core targets, which provided a useful reference for us to further reveal the mechanism of SLBZS in treating diabetic obesity.

Competing interest statement

There is no conflict of interest.

Foundation

National Natural Science Foundation (Grant No. 81660727);Natural Science Foundation of Jiangxi Province (Grant No. 20192BAB205095);1050 Young Talents Project of Jiangxi University of Traditional Chinese Medicine (Grant No. 5141900102);Science and technology planning project of Jiangxi Administration of traditional Chinese Medicine(Grant No.2020A0320);Startup fund for doctoral research of Jiangxi University of traditional Chinese Medicine(Grant No.2020BSZR017)

Author’s Contribution Statement

Diyao Wu took charge of guiding the experiments and paper writing. Shumao Pan,Xiuyun Li,Shu Wang wrote the manuscript and finished data mining research.Zheng Yang,Xin Kuang and Xingxing Gong completed the experimental design and execution.Xiaofan Chen and Xinyou Zhang made the design research and provided fund support.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Citation: Wu D, Wang S, Pan S, Li X, Yang Z, et al. (2022) Research on Mechanism of Shenling Baizhu San on Diabetic Obesity Based on Meta-Analysis and Network Pharmacology. J Altern Complement Integr Med 8: 234.

Copyright: © 2022  Diyao Wu, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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