The Shugan Qingre Formula suppresses inflammatory responses in mouse model of plasma cell mastitis by modulating gut microbiota and the Ras-MAPK pathway
Objective To explore the anti-inflammatory effect of Shugan Qingre(soothing liver and relieving heat)For-mula on plasma cell mastitis(PCMM)in mouse model,and the therapeutic mechanism by analyzing its regulatory mechan-ism on the Ras-MAPK signaling pathway and intestinal flora.Methods A total of 60 female C57BL/6 mice were ran-domly divided into healthy control group(HC group),plasma cell mastitis model group(PCMM group)and Shugan Qin-gre prescription treatment group(PCMM+Tx group)(n=20).The PCMM model was induced by injecting 4％formalin in-to the mammary gland.The mice in the PCMM+Tx group were treated with Shugan Qingre prescription by gavage.HE staining was used to observe the pathological changes of breast tissue.ELISA was used to detect MPO,LPS and cytokine levels in serum and breast tissue.Western Blot was used to detect the expression of proteins related to the Ras-MAPK sig-naling pathway in breast tissue,and 16S rRNA gene sequencing analysis was performed.The changes in the diversity and composition of intestinal flora were observed,and correlation analysis was performed.Results The mammary gland tis-sue structure of mice in the PCMM group was severely damaged and the infiltration of inflammatory cells increased signi-ficantly.Treatment with Shugan Qingre formula significantly improved the mammary gland tissue structure and reduced the infiltration of inflammatory cells.ELISA results showed that the levels of MPO(Serum t=-2.401,Mammary gland tis-sue t=-5.120)and LPS in the serum and breast tissue of mice in the PCMM group significantly increased(P＜0.001),while those in the PCMM+Tx group significantly decreased(P＜0.001).Cytokine analysis showed that the levels of IL-1β(t=2.822)and IL-6(t=4.812)in the breast tissue of mice in the PCMM group significantly increased(P＜0.050),and those of IL-4(t=2.822)and IL-10(t=4.812)significantly decreased(P＜0.050),while the levels of IL-1β(t=3.335)and IL-6(t=4.281)significantly increased(P＜0.050).The level of cytokines in the Tx group was close to that in the HC group(P＜0.050).Western Blot results showed that the protein expressions of Ras(t=2.312),Raf-1(t=4.726)and p-MEK1/2(t=2.311)in the breast tissue of mice in the PCMM group significantly increased(P＜0.050),while those in the PCMM+Tx group significantly decreased(P＜0.050).16S rRNA gene sequencing results showed that the Chaol index(t=6.323)and Shannon index(t=6.526)of intestinal flora of mice in the PCMM group significantly reduced(P＜0.001),while those in the PCMM+Tx group was significantly restored(P＜0.001).LEfSe analysis results showed that the abundance of Proteo-bacteria in the intestines of mice in the PCMM group significantly increased(t=6.216,P＜0.001),and those of Firmicutes,Bacteroidetes,Actinobacteria and Verrucomicrobia significantly decreased(t=8.122,P＜0.001),while these bacterial groups were abundant in the PCMM+Tx group.The degree of recovery was significant(t=6.443,P＜0.001).Correlation analysis showed that Proteobacteria was significantly positively correlated with MPO(r=0.748),LPS(r=0.703),IL-1β(r=0.682),IL-6(r-0.649)(P＜0.001),and significantly negatively correlated with IL-4(r=-0.554)and IL-10(r=-0.583)(P＜0.001).Conclusion Shugan Qingre Formula can significantly improve the inflammatory state of PCMM mice,re-duce breast tissue damage,reduce pro-inflammatory cytokine levels,and increase anti-inflammatory cytokine levels by reg-ulating the Ras-MAPK signaling pathway and intestinal flora structure.
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