The Efficacy And Mechanism Study Of Astilbin On Endometritis In Rats

Background and ObjectiveEndometritis is one of the common inflammatory diseases in the female reproductive system,affecting 10%～15%of women of childbearing age.Nowadays,antibiotic resistance caused by the overuse of antibiotics in endometriotic clinical treatment leads to an unguaranteed treatment effect and reduced patient safety.Astilbin(ASB)widely exists in Chinese herbal medicines and foods,and has different biological activities,such as antioxidation,anti-inflammatory and hypoglycemic properties.Moreover,it has lower toxicity and higher safety compared with antibiotic treatment.Up to now,no study has revealed the effect of ASB on endometritis.The study aimed to investigate the effect and molecular mechanism of ASB on endometritis in rats.Method(1)Using LPS and H2O2 to induce RAW264.7 and ASB intervention,ELISA was used to detect the inflammatory factors in the supernatant;kit was used to detect the level of oxidative stress in cells;WB was used to detect the expression of inflammatory pathway proteins;RT-qPCR and WB were used to detect the expression of oxidative stress pathway and its regulated target genes.(2)The model of endometritis in rats was established,including a model group,blank control group,ASB(high,medium and low dose)treatment groups and a dexamethasone positive control group.The parameters of Hemorheology and blood routine examination were measured by an automatic whole blood analyzer;the inflammatory factors in serum were measured by ELISA;the pathological changes of uterine tissue were observed by HE staining;the expression of inflammatory factors in uterine tissue was measured by immunohistochemistry;the level of oxidative stress in uterine tissue was detected by kit;the expression of the inflammatory signaling pathway,the oxidative stress pathway and its regulated target genes were identified by WB and RT-qPCR.(3)Using LPS to induce HEECs and ASB intervention,the levels of inflammatory factors in the supernatant and oxidative stress in cells were detected by kit;the expression of inflammatory signaling pathway proteins was detected by WB,double luciferase reporter genes and immunofluorescence;the expression of oxidative stress pathway and its regulated target genes were detected by WB and RT-qPCR;the molecular mechanism of ASB was verified by the specific interference of agonists and siRNA.Results(1)ASB not only down-regulates the expression of inflammatory factors,such as IL-6,TNF-α,IL-1β,iNOS and COX-2 by inhibiting the activation of TLR4 and its downstream NF-κB and MAPK signaling pathways,but also activates Keap1/Nrf2 antioxidant signaling pathway,up-regulates the expression of antioxidant factors,such as NQO1 and HO-1,and reduces H2O2-induced cell damage and ROS production.(2)ASB reduces the swelling of the uterus,improves the pathological changes of uterus;reduces the hemorheology indexes and blood routine;not only reduces the inflammation indexes of peripheral blood,but also reduces the expression of inflammatory factors/mediators in the uterus;up-regulates PPAR-y expression,inhibits TLR4/NF-κB and IL-6R signal transduction pathway;activates Nrf2 signal pathway and up-regulates the expression of antioxidant factors,and reduces ROS and MDA levels.(3)ASB activates PPAR-γ,interferes NF-κB and STAT3 signal transduction and reduces the expression of inflammatory factors;regulates the expression of antioxidant factors and reduces the level of ROS and MDA by up-regulating Nrf2 signaling pathway;ASB has the same effect on reducing inflammatory responses as the PPAR-γ selective agonist;siRNA silencing PPAR-γ cancels the effect of ASB on reducing the inflammatory response.Conclusion(1)ASB can play both anti-inflammatory and anti-oxidative stress roles,in RAW264.7 cells.(2)ASB can reduce the inflammation and oxidative stress in endometritis,and play a therapeutical role in endometritis.(3)ASB can interfere with TLR4/NF-κB and IL-6R/AK2/STAT3 signal transduction by upregulating PPAR-y,which blocks their positive feedback signaling cascade to inhibit the inflammatory response;activate Keap1/Nrf2 signaling,and inhibit oxidative stress induced by the inflammatory response.