Mechanism Of Ginsenoside Rg3 Regulating Intestinal Flora In Rats With Acute Radiation Proctitis Based On TLR4 Pathway

Objective To observe the protective effect of ginsenoside Rg3(GRg3)on acute radiation proctitis(ARP)in rats,and to explore the effect of GRg3 on intestinal flora in ARP rats.To clarify the anti-inflammatory effect of GRg3 based on TLR4/My D88/NF-κB signaling pathway.Methods Before the experiment,forty-eight SPF healthy male Wistar rats were randomly divided into 6 groups: control check group(CK),model group(MG),dexamethasone group(Dexa),low-dose GRg3 group(GL),medium-dose GRg3 group(GM),and high-dose GRg3group(GH).The ARP rat model was established by a single 22-Gy irradiation of 6 MV X-rays.After modeling,the general state of rats was observed,and the body weight and disease activity index(DAI)were evaluated.Two weeks later,all rats were sacrificed after taking fresh feces and abdominal aortic blood,and rectal tissues were collected.The distribution and function of intestinal flora were detected using 16 sr RNA high-throughput sequencing.HE staining was used to observe the pathological changes of rectal tissue in rats.ELISA was used to detect the expression of IL-1β and IL-10 in serum.RT-q PCR was used to detect the m RNA expression of TLR4,My D88,and NF-κB.The protein expression levels of TLR4,My D88 and NF-κB p65 were detected by Western blot.Results After the establishment of ARP model,except CK group,the body weight of rats in the other five groups decreased significantly(P<0.05),DAI increased significantly(P<0.05).After 2 weeks of gavage,compared with MG group,all treatment groups had a significant increase in body weight(P<0.05);The DAI of GM group,GH group and Dexa group was 0,and the mean DAI of MG group and GL group was 2 and 0.625,respectively.The species distribution of intestinal flora in GRg3 was significantly different from that in ARP rats,and the difference was the most obvious in GH group.The beneficial bacteria such as Ruminococcus,Lactobacillus,Blautia and Romboutsia were significantly increased,and the harmful bacteria such as Shigella and Alloprevotella were significantly decreased.GRg3 is also closely related to amino acid metabolism.The results of HE showed that the MG group had large area of rectal tissue ulcer,serosal layer invasion,accompanied by a large number of lymphocytes infiltration,a large number of intestinal glands were replaced by connective tissue,and the submucosa was severely edema.There were different degrees of necrotic cell debris in GL and GM groups,while only mucosal edema with a small amount of inflammatory cell infiltration was observed in Dexa and GH groups.ELISA results showed that the expression of IL-1β in all treatment groups were significantly lower than that in MG group(P<0.05),and IL-10 expression was higher than that in MG group(P<0.05);There was significant difference between Dexa group and GL and GM groups(P<0.05),but there was no significant difference with GH group(P>0.05).RT-q PCR results showed that compared with the MG group,the expression of TLR4,My D88,and NF-κB m RNA was significantly decreased in all treatment groups(P<0.05);There was no significant difference in TLR4 m RNA expression between GH group and Dexa group(P>0.05).There was no significant difference in the expression of My D88 and NF-κB m RNA between GM and GH groups(P>0.05).WB results showed that compared with MG group,the expressions of TLR4,My D88 and NF-κB p65 proteins in Dexa,GM and GH groups were significantly decreased(P<0.05).Compared with Dexa group,there was no significant difference in the expression of TLR4 protein and NF-κB p65 protein in GH group(P>0.05).The expression of My D88 protein in GH group was significantly different(P<0.05),but there was no significant difference in My D88 protein expression between GH group and CK group(P>0.05).Among the GRg3 treatment groups,only the GH group showed the most significant downward trend in gene and protein expression.Conclusions GRg3 can effectively alleviate the inflammatory response in ARP rats,and GH has the most significant effect on ARP rats.GRg3 can significantly improve the imbalance of intestinal flora in ARP rats,increase the beneficial bacteria and reduce the harmful bacteria,and the difference is most obvious in GH group.The mechanism is related to the inhibition of TLR4/My D88/NF-κB signaling pathway,which provides a new theoretical basis for GRg3 in the treatment of ARP.