Study On The Mechanism Of Shaoyao-Gancao Decotion In The Treatment Of Polycystic Ovary Syndrome Dyslipidemia Based On The Gut Microbiota-bile Acids-FXR Axis

Objective:Polycystic ovary syndrome（PCOS）is a common endocrine disorder,characterized by obesity,ovulation disorders,hyperandrogenism and other metabolic abnormalities.Shaoyao Gancao Decoction（SGD）has been widely used to improve PCOS,and has been highly recommended by successive generations of physicians to this day.However,the relationship between traditional Chinese medicine theories and modern pharmacological mechanisms of SGD in treating PCOS has not been elucidated clearly.Our previous research found that after SGD intervened in PCOS rats,the structure and diversity of the gut microbiota in the rats changed significantly.It suggested that studying the mechanism of SGD in treating PCOS from the perspective of gut microbiota is in line with the theory of microecological balance in modern medicine.However,whether the therapeutic effect of SGD on PCOS is gut microbiota-dependent,and the mechanism which the dominant gut microbiota communicates with the host is still unclear.Based on this,our paper analyzed the changes in the gut microbiota structure after SGD intervention in PCOS rats by 16S RNA sequencing,and constructed FMT rats to further clarify the key gut microbiota in response to SGD intervention.Our experiment also used UPLC-MS/MS to measure the content of bile acids,and investigate the changes in the bile acid profiles.The molecular mechanism of SGD regulating the gut microbiota-bile acid axis was further explored.Our investigation provided a scientific basis for the wider clinical application of SGD.Methods:1.Effect of SGD on PCOS ratsPCOS rats were prepared by continuous gavage with letrozole for 21 days,followed by gavage with SGD for 14 days.We observed changes in body weight and estrous cycles in the rats of each group.After the experiments,bilateral ovarian weight was obtained.Ovarian pathology was observed by staining with hematoxylin and eosin.Serum concentrations of the rats’neutral hormones（E₂ and T）were measured by ELISA,and lipid（TG,T-CHO,LDL-C,HDL-C）levels were measured by Colorimetric Assay Kits.2.Improvement of lipid metabolism abnormalities in PCOS rats by SGD depends on gut microbiota（1）Analysis of the gut microbiota profiles to examine the effect of SGD on gut microbiota abundance in PCOS rats.Fresh feces samples from normal,PCOS,and SGD rats were collected.The 16S r RNA analysis was performed to identify the predominant gut microbiota associated with SGD.KEGG pathways further clarified whether the differential gut microbiota was related to lipid metabolism.Pearson correlation was used to analyze correlations between serum biochemical indicators and gut microbiota.（2）Prepared FMT rats and verify the efficacy of SGD on PCOS depends on the involvement of gut microbiotaThe FMT experiment was started after preparing germ-free PCOS rats by gavage with ABX for 4 days.24 germ-free PCOS rats were divided into FMT-N group and FMT-S group（n=12）,then they were transplanted fresh feces from N and SGD donor rats respectively.Germ-free PCOS rats were given 1 m L of freshly prepared fecal supernatant by intragastric administration every day for 21 consecutive days.After the experiment,16S r RNA was used to analyze the Alpha and Beta diversity of the gut microbiota to verify the success of the FMT experiment.The pharmacodynamic indicators such as body weight,estrous cycles,ovarian pathological changes,serum sex hormones and blood lipid levels were investigated.3.SGD improves PCOS lipid metabolism disorders based on BAs-FXR axis.Serum total bile acids were measured using Colorimetric Assay Kits.BAs in liver,serum,and feces were determined using the UPLC-MS/MS method.Correlations between microbiota and bile acids were analyzed using the Pearson correlation and visualized by heatmaps.The 16S r RNA analysis was used to identify the predominant gut microbiota at different levels after FMT.Metabolic functions of the gut microbiota were predicted through KEGG pathways to analyze biological differences.The expressions of m RNA related to hepatic and ileum BAs synthesis and lipid metabolism were measured by RT-q PCR.Results:1.Effect of SGD on PCOS ratsAfter continuous gavage of letrozole for 21 days,there was a larger continuous increase in body weight in PCOS rats compared with normal rats（P<0.05）.Similarly,the ovarian weight in PCOS rats significantly increased（P<0.05）.The estrous cycles of PCOS rats were disrupted at the metaestrus and diestrus（M/D）phases.Ovarian cystic follicles increased significantly（P<0.001）,and corpus luteum numbers decreased markedly（P<0.05）.Serum E₂ levels were significantly reduced（P<0.05）,T levels were significantly increased（P<0.01）,and serum levels of TG,T-CHO,and LDL-C were significantly increased（P<0.05）.These results indicated that PCOS rat models were prepared successfully.After 14 days of SGD treatment,body weight and ovarian weight of PCOS rats reduced significantly（P<0.001）,and estrous cycles returned to the normal distribution gradually.Cystic follicles reduced significantly（P<0.001）,corpus luteum increased significantly（P<0.05）,and granulocyte arrangements were regular and multilayered.The serum levels of T decreased（P<0.05）while E₂ levels increased（P<0.001）,and serum TG,T-CHO,and LDL-C decreased significantly（P<0.05）.These results suggested that SGD significantly improved the symptoms of PCOS,especially dyslipidemia.2.Improvement of lipid metabolism abnormalities in PCOS rats by SGD depends on gut microbiota16S rRNA sequencing showed that SGD significantly increased Alpha diversity in PCOS rats（P<0.01）,and Beta diversity showed significant differences in taxa composition among the three groups.At the phylum level,SGD intervention significantly increased the relative abundance of Verrucomicrobia and Bacteroidetes（P<0.001）,and significantly decreased the relative abundance of Actinobacteria and Firmicutes（P<0.001）.At the genus level,SGD intervention significantly decreased the relative abundance of Turicibacter（P<0.001）and significantly increased the relative abundance of Akkermansia,Bacteroides,Blautia,Coprococcus,Oscillospira and Ruminococcus（P<0.05）.Analysis of KEGG pathways revealed that differential microbiota efficiently enriched several metabolic-related pathways including lipid metabolism.These results suggested that SGD intervention could improve lipid disturbance by improving the gut microbiota structure in PCOS rats.Following FMT experiments,Alpha diversity was increased significantly in the FMT-S rats（P<0.05）,and Beta diversity showed a significant separation trend between FMT-N and FMT-S rats.Body weight and ovarian weight in the FMT-S rats decreased significantly compared with that in FMT-N rats（P<0.01）.The estrous cycles in the FMT-S rats were basically restored to the normal level.The ovarian functions were restored,few follicles were observed,and corpus luteum was significantly increased in FMT-S rats（P<0.05）.Serum T levels showed a downward trend,and serum TG,T-CHO,LDL-C,and HDL-C levels returned to the normal level.These results suggested that effects of SGD on PCOS,especially dyslipidemia,depended on gut microbiota.3.SGD improves PCOS lipid metabolism disorders based on BAs-FXR axis.SGD administration also altered the BAs profiles in PCOS rats characterized by elevating the ratio of primary/secondary BAs.Further studies showed a significant positive correlation between serum UDCA and anti-obesity probiotic Akkermansia（P<0.001）.After SGD intervention,liver FXR and its downstream target gene SHP m RNA expressions were significantly reduced（P<0.05）,and CYP7A1 and CYP27A1m RNA expressions were significantly upregulated（P<0.05）.These results suggested that SGD may inhibit liver FXR signaling pathway,and activate BAs synthesis pathway to reduce cholesterol levels.Furthermore,SGD significantly reduced the expressions of SREBP-1C and SREBP-2 which related to cholesterol synthesis（P<0.01）,upregulated the expressions of LDLR and SR-B1 which related to cholesterol reverse transport（P<0.05）,and increased the expression of ABCG8 which related to cholesterol excretion（P<0.01）.Our results indicated that SGD could inhibit cholesterol biosynthesis and promote reverse transportation and excretion of cholesterol under the control of FXR.Verrucomicrobia and Akkermansia had become the dominant gut bacteria after FMT treatment.Enriched KEGG pathways in FMT-S group were shown to accord well with that in SGD group,and“Lipid Metabolism”pathway was the most enriched in FMT-S rats.FXR and SHP expressions were significantly reduced（P<0.05）,and CYP7A1 expression was significantly upregulated（P<0.05）compared to FMT-N rats.Compared with FMT-N group,the expression levels of SREBP-1C and HMGCR which were involved in cholesterol biosynthesis significantly decreased in FMT-S group（P<0.05）.Moreover,in the cholesterol transport pathway,the levels of LDLR and PCSK9were markedly down-regulated in FMT-S group（P<0.05）.These results suggested that SGD improved dyslipidemia in PCOS rats based on the BAs-FXR axis.Conclusion:SGD significantly ameliorates symptoms in PCOS rats,especially reduces lipid levels.The mechanism may be related to the remodeling of gut microbiota,inhibition of FXR/SHP signaling and alteration of bile acids metabolism.This study has the potential to provide new theoretical evidences for future therapy of PCOS.