Regulatory Mechanism Of Taxifolin On Mouse Uterine Macrophages

Object: To investigate the inhibitory effect of Taxifolin(Tax)on mice uterine inflammation and the regulation mechanism of Tax on macrophages and related cytokines at inflammation sites.Methods: 25 sexually mature mice were randomly divided into control group and treatment groups.The treatment groups were divided into LPS,LPS+ low concentration Tax(LPS+Tax 25 mg/kg),LPS+ medium concentration Tax(LPS+Tax 50 mg/kg)and LPS+ high concentration Tax(LPS+Tax 100 mg/kg)treatment group.The control group and LPS treatment group were given PBS 0.4 ml,1 times per day at 9a.m.,and the control group was injected PBS 0.2 ml into the caudal vein on the fifth day,while the LPS treatment group was injected LPS 0.2 ml on the fifth day.LPS+Tax 25 mg/kg treatment group was given a gavage Tax 25 mg/kg at 9 am every day,LPS+Tax 50 mg/kg group were administered 50mg/kg Tax,LPS+Tax 100mg/kg group were administered 100mg/kg Tax.LPS+ low,medium and high Tax treatment groups were intravenous injection of LPS 0.2 ml on the fifth day.All LPS treated mice were injected until day 6.All the mice were killed by cervical dislocation on the 8th day.The uterus was taken and the shape changes were observed.The difference in the number and distribution of CD86(M1)and CD163(M2)type macrophages in the uterus were detected by immunohistochemistry.The difference expression of inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 in each experimental group were detected by ELISA.The difference expression of CXCL12 and its receptor CXCR4 mRNA levels in each experimental group were detected by q PCR method.Expressionof CXCL12/CXCR4 protein were detected by western blot.The above test results were used to infer the effect of Tax confrontation inflammatory mechanisms.Results: After LPS treatment induced inflammation in mouse uterus,immunohistochemistry showed that compared with the control group,the expression level of M1 macrophage surface marker protein CD86 was significantly increased in LPS treated mice.However,we found that when inflammatory mouse uterus treated with different concentrations of texifolin,the expression of CD86 protein decreased with the increase of the concentration of Tax.The high concentration treatment group mice uterine CD86 protein expression had no significant difference compared with the control group.The same method was used to detect the M2 macrophage surface marker protein CD163.It was found that compared with the control group,the expression of CD163 protein in mouse uterus decreased after LPS treatment,and the difference was very significant compared with the control group.With the increase of Tax concentration,the expression of CD163 protein was increased.The difference was significant between medium concentration group and the control group,but there was no significant difference between the high concentration group and the control group.The results of ELISA test showed that the expression of TNF-α in the inflammation uterus of mice induced by LPS was significantly higher than that of the control group.The expression of TNF-α was decreased with the increase of the concentration of Tax.The same method was used to detect an anti-inflammatory cytokine IL-10.The results showed that with the increase of the Tax concentration,the expression of IL-10 showed a correspondingly increasing trend.The differences expression of M2 type macrophage chemokine CXCL12 and its receptor CXCR4 mRNA and protein were detected by q PCR and western blot.The resultsshowed that both of CXCL12/CXCR4 gene and protein level were decreased after LPS was injected into the mice tail vein.Expression of CXCL12 gene and protein in the uterus of high concentration Tax-treated mice were not significantly different compared with each corresponding control group.The expression level of CXCR4 protein was increased,but the difference was still significant bentwwn high concentration Tax-treated group and control group.As the processing concentration of Tax increased,gene and protein expression levels of CXCL12/CXCR4 showed a correspondingly increasing trend.Conclusion:(1)The results of immunohistochemical and ELISA experiments showed that a certain concentration of Tax could effectively inhibit the inflammatory response of mouse uterus.(2)All experment reaults suggested that one of the mechanisms about Tax preventing mice uterine inflammation is that Tax affects the polarization direction of macrophages in inflammatory sites by regulating the expression a type two macrophage chemotactic protein CXCL12 and its receptor CXCR4.The expression of different cytokines was further affected.(3)A certain concentration of Tax inhibited the Th1 type immune response and promoted the Th2 type immune response.