Study On The Mechanism Of Shaoyao-gancao Decoction Regulating TLR4/NF-κB Signal Pathway In The Improvement Of Polycystic Ovary Syndrome Via Remodeling Gut Microbiota

Objective:The effective treatment of Shaoyao-Gancao Decoction(SGD)on polycystic ovary syndrome(PCOS)fully demonstrates the characteristics and advantages of "promoting ovulation and assisting pregnancy" in traditional Chinese medicine.However,the relationship between TCM treatment theory and modern biological mechanism of SGD were also not clear.From the point of view of gut microbiota,the scientific connotation of SGD in treating PCOS based on the theory of invigorating spleen to nourish qi accords with the holism of traditional Chinese medicine.This project intends to explore the role of gut microbiota in the therapeutic effect of SGD,and to clarify its potential molecular mechanism in depth,in order to provide a theoretical basis for the wider clinical application of SGD.Methods:1.SGD relieves symptoms of PCOS rats by improving chronic low-grade inflammationThe rats were randomly divided into normal group,PCOS group and SGD group.Except for normal rats,all rats were given letrozole(1 mg/kg/d)by gavage for 3 weeks.The body weight of rats was recorded every day,and vaginal smears were used to observe the changes of estrous cycle after Giemsa staining.The weight gain and estrous cycle disorder showed that the PCOS model was established successfully.PCOS rats were treated with SGD(25 g/kg/d)for 2 weeks.24 hours after the last administration,blood samples from abdominal aorta were taken to determine the levels of plasma sex hormones(T,E2,LH,FSH).Ovarian tissues were taken to observe the histopathological changes of ovary.The intervention effect of SGD in the treatment of PCOS was evaluated by measuring changes in body weight,estrous cycle,pathology and sex hormone content.Elisa method was used to detect the effect of SGD on plasma inflammatory cytokines(TNF-α,IL-1β,IL-6,IL-18)in PCOS rats.At the same time,RT-PCR was used to further study the effect of SGD on levels of these inflammatory factors in PCOS rat ovarian tissue.To observe the effect of SGD on chronic low-grade inflammation in PCOS rats.2.SGD antagonizes chronic low-grade inflammation in PCOS rats by regulating the structure of gut microbiota.The structure of intestinal flora was determined by 16 s rRNA sequencing,the α andβ diversity of microflora were evaluated,and the microflora with different abundance among groups were found,and the regulatory effect of SGD on the structure and abundance of gut microbiota in PCOS rats was evaluated.Elisa method was used to determine the effect of SGD on plasma lipopolysaccharide(LPS)content in PCOS rats.To analyze the regulatory effect of SGD on the Intestinal mucosal integrity of PCOS rats,RT-PCR was used to detect the effect of SGD on the gene expression of ZO-1,Occludin,Claudin-1 in the terminal ileum of PCOS rats.In order to find out the key flora that SGD plays an important role in the regulation of PCOS,spearman correlation analysis was used to compare the relationship between differential flora and related biochemical indexes in PCOS disease state,and it was found whether SGD antagonized the chronic low-grade inflammation in PCOS rats by regulating the structure of gut microbiota.3.Study on the Mechanism of SGD on PCOS Rats based on TLR4/NF-κB signal pathway.To further study the molecular mechanism of SGD improving PCOS by regulating the structure of gut microbiota,the intervention effect of SGD on TLR4/NF-κB signal pathway in PCOS rats was observed.The effects of SGD on the gene and protein levels of TLR4,PI3K,Akt and NF-κB p65 in TLR4/NF-κB signal pathway in ovarian tissue of PCOS rats were determined by RT-PCR and Western blotting,and at the same time,the inflammatory model was established by stimulating RAW264.7 macrophages with LPS in vitro.The NO production of RAW264.7 cells induced by LPS was measured by NO kit,and the effect of SGD on the survival rate of RAW264.7 cells was determined by CCK-8 method.The effect of SGD on the gene expression of TLR4/NF-κB signal pathway in RAW264.7 cells stimulated by LPS was measured by PCR.To find out whether the regulatory effect of SGD on PCOS rats is related to the regulation of TLR4/NF-κB signaling pathway.Results:1.SGD relieves symptoms of PCOS rats by improving chronic low-grade inflammationCompared with the normal group,the body weight of PCOS rats increased significantly,and the estrous cycle showed different degrees of disorder,and most of them were in diestrus and metestrus.The number of granulosa cell layers and the corpus luteum were significantly decreased,the plasma sex hormone T and LH levels were significantly increased,and E2 and FSH levels are significantly reduced(P < 0.01).The results showed that the PCOS rat model was successful,and the levels of ovarian cystic dilated follicles were significantly increased,the layers of granulosa cells and corpus luteum were decreased,and the levels of plasma sex hormones T and FSH were significantly increased.On the 35 th day after SGD intervention,the body weight of rats decreased significantly,the estrous cycle resumed,there were fewer follicles in cystic dilatation of ovary,more granulosa cells and more corpus luteum.The levels of plasma sex hormone T and LH decreased significantly,and the levels of E2 and FSH increased significantly(P < 0.01).There was no significant difference between the control group and the normal group.It is suggested that SGD can significantly improve the hormone level and ovarian morphology of PCOS rats,and has a good therapeutic effect on PCOS.Compared with the normal group,the elevated levels of pro-inflammatory factors(TNF-α,IL-1β,IL-6,IL-18)in the plasma and ovarian tissue of PCOS rats were significantly increased,indicating that PCOS rats are at a chronic low-grade inflammation state.SGD can significantly reduce the elevated levels of pro-inflammatory factors(TNF-α,IL-1β,IL-6,IL-18)in the plasma and ovarian tissue of PCOS rats,and improve chronic low-grade inflammation in PCOS rats.2.SGD antagonizes chronic low-grade inflammation in PCOS rats by regulating the structure of gut microbiota.By observing the gut microbiota of rats,it was found that the α diversity of gut microbiota of PCOS rats decreased,and the α diversity of PCOS rats increased after the intervention of SGD.β diversity showed that there were differences in species composition among normal group,PCOS group and SGD group,indicating the significance of grouping.At the same time,it was found that at the phylum level,SGD could significantly regulate the increased abundance of Firmicutes,Actinobacteria and LPS-producing bacteria(Proteobacteria)in PCOS rats,and up-regulate the decreased abundance of Bacteroidetes and Verrucomicrobia in PCOS rats.In the PCOS rat model,the ratio of Firmicutes to Bacteroidetes(F/B)was also significantly increased than that of the normal group.and decreased significantly after administration of SGD,indicating that the intestinal flora structure of PCOS rats was out of balance,and SGD could improve the intestinal flora structure of PCOS rats.At genus level,the intestinal Turicibacter of PCOS rats increased significantly,while the abundance of Butyricicoccus,Coprococcus,Akkermansia and Blautia decreased significantly(P < 0.0l),SGD could regulate the increased abundance of Turicibacter in PCOS model rats and up-regulate the decreased abundance of Bacteroides,Butyricicoccus,Coprococcus,Akkermansia and Blautia in PCOS rats.It can be concluded that SGD can improve the bacterial community structure and disease status of PCOS rats.The content of LPS in plasma of PCOS rats was significantly higher than that of normal rats(P < 0.01),SGD could significantly reduce the increased content of LPS in systemic circulation of PCOS rats(P < 0.01).At the same time,the contents of intestinal tight junction proteins(Occludin and Claudin-1)were significantly decreased in PCOS rats(P < 0.01),SGD could down-regulate the expression of intestinal tight junction proteins in PCOS rats.The results suggest that the intestinal barrier function of PCOS rats is impaired,and SGD can restore the integrity of intestinal mucosa of PCOS rats,thus reducing the amount of intestinal LPS entering systemic circulation in PCOS rats and improving chronic low-grade inflammation in PCOS rats.In addition,by comparing the correlation between the different flora and the relevant biochemical indexes in the state of PCOS disease,it was found that Firmicutes,Bacteroidetes and Proteobacteria(phylum level),Blautia,Bacteroides,Butyricicoccus,and Akkermansia(genus level)played an important role in the improvement of SGD on PCOS rats.The potential mechanism may be related to reducing the abundance of LPS-producing bacteria,improving the integrity of mucosal barrier,and then improving the state of inflammation and hormonal abnormalities.3.Study on the Mechanism of SGD on PCOS rats based on TLR4/NF-κB signal pathway.Compared with normal rats,the expression of TLR4,PI3K,Akt and NF-κB p65 genes and proteins in ovarian tissue of PCOS rats was significantly upregulated,SGD could significantly reduce the increased expression of TLR4,PI3K,Akt and NF-κB p65 genes and proteins in ovarian tissues of PCOS rats.It suggested that SGD could improve the symptoms of Chronic low-grade inflammation in PCOS rats.The molecular mechanism may be related to the inhibition of the TLR4/NF-κB inflammatory pathway.SGD significantly inhibited mRNA expression levels of key regulatory factors in the TLR4/NF-κB signaling pathway in LPS-stimulated RAW264.7 macrophages,while SGD did not affect the gene expression of this pathway in normal RAW264.7 cells.These results suggest that TLR4/NF-κB signal pathway is the potential molecular mechanism of SGD in the treatment of PCOS.Conclusion:SGD can improve the symptoms of PCOS rats and has a good intervention effect on PCOS diseases.PCOS rats have high expression of pro-inflammatory factors and present a state of chronic low-grade inflammation.SGD intervention can significantly improve chronic low-grade inflammation in PCOS rats.SGD can reverse the abundance of different bacteria in the intestine of PCOS rats(such as Firmicutes,Proteobacteria,Bacteroidetes,Akkermansia,Blautia,Bacteroides,Butyricicoccus,etc.).At the same time,the expression of tight junction protein is increased,and the content of LPS in plasma is reduced.By analyzing the correlation between these different bacteria and PCOS-related biochemical indicators,it is found that the flora intervened by SGD mainly regulates the intestinal barrier function and reduces the content of LPS in the body,thereby alleviating the inflammation in the PCOS rats.SGD can significantly inhibit the TLR4/NF-κB signaling pathway in PCOS rats and LPS-induced RAW264.7 cells.In summary,SGD can regulate the gut microbiota,improve the intestinal barrier,reduce the translocation of LPS,and then inhibit the inflammatory response mediated by TLR4/NF-κB,and improve the chronic low-grade inflammation in PCOS rats.