Study On The Relationship Between Kaempferol,miR-155-5p/SOCS1 And Immune Rejection Of Corneal Transplantation

Part 1Kaempferol inhibits the activation of NL RP3 inflammasome by inducing autophagy in corneal allograft rejectionObjective:To investigate the effect of kaempferol on immune rejection after allokeratoplasty in rats and whether it is effective by activating autophagy-mediated suppression of NLRP3 inflammaomes.Methods:The rat model of penetrating corneal transplantation was established and randomly divided into the control group,the isograft group and allograft group.The cornea was collected on day 5,9 and 14 after the operation,and the mRNA levels of NLRP3 and IL-1β were detected by RT-qPCR.The allograft group,vehicle group,kaempferol group,and 3-MA+kaempferol group were established.From 3 days before surgery to 5 days after surgery for collection or observation,the following treatments were performed each day:no special treatment,intraperitoneal injection of the same amount of solvent,intraperitoneal injection of 50mg/kg kaempferol,and 10mg/kg 3-MA 12 hours before administration.The cornea was taken 5 days after the operation,and the mRNA of NLRP3,IL-1β,Beclinl,ATG5,LC3 and p62 were detected by RT-qPCR.NLRP3 and IL-1β protein were detected by WB.The model of survival analysis was observed daily by Larkin score.Results:①Compared with the control group and the isograft group,the mRNA levels of NLRP3 and IL-1β of the cornea in the allograft group were significantly increased on day 5,9 and 14,and the expression peak was on day 5.②The graft survival time of each group before and after the treatment of kaempferol showed that compared with the allograft group(12.6±1.08)days and the vehicle group(11.4±0.97)days,the graft survival time of kaempferol group was significantly extended to(27.7±1.49)days.③ The effect of kaempferol on NLRP3 inflammasome was detected on the 5th day after operation:the mRNA and protein of NLRP3 and IL1β of cornea in kaempferol group were significantly lower than those in vehicle group,and no significant changes were observed in vehicle group compared with the allograft group.④Further analysis of autophagy in the treatment of kaempferol:the survival time of the graft in the 3-MA+kae group was(10.5±0.71)days,which was significantly shorter than that in the kae group(27.7± 1.49)days.⑤Autophagy related gene detection:the mRNA levels of corneal Beclinl,LC3 and ATG5 in the kae group were significantly higher than those in the solvent control group,and the mRNA levels of p62 were significantly lower,suggesting autophagy activation.After the addition of 3-MA treatment,Beclinl,LC3 and ATG5 significantly decreased,and p62 significantly increased,suggesting autophagic inhibition.Regarding NLRP3 inflammasome:mRNA and protein expressions of NLRP3 and IL1β in the 3-MA+kae group increased significantly compared with that in the kae group.Conclusion:It is preliminarily confirmed that kaempferol can drive autophagy to mediate the inhibition of NLRP3 Inflammasome,reduce the release of pro-inflammatory cytokines such as IL-1β,and significantly delay the occurrence and development of corneal allograft rejection in rats.Part 2The role of miR-155-5p/SOCS1 in corneal allograft rejectionObjective:To investigate the role of miR-155-5p/SOCS1 in corneal allograft rejection in rats and whether TobraDex eye drops can inhibit miR-155-5p/SOCS1 pathway to delay rejection.Methods:The rat model of keratoplasty was established and randomly divided into two groups:Isograft group(B),allograft group(C),and the allograft group treatment with Tobradex(D),blank control group(A).On the 14th day after operation,the cornea was taken for pathological observation by HE staining,and miR-155-5p was detected by RT-qPCR.SOCS1 protein was detected by WB.Results:Pathology:the corneal structure was clear in group A,there was no vascular and lymphocyte infiltration,a large number of inflammatory cell infiltration and neovascularization could be seen in each layer of group C,and there were only a few inflammatory cells and neovascularization in group B and D.Compared with group A,group B and group C,the expression of corneal miR-155-5p in group RT-qPCR:was significantly increased,and the expression of corneal miR-15 5-5p in group C was significantly higher than that in group B.The expression of SOCS1 protein in WB:group was significantly higher than that in group A and B,while that in group C was significantly lower than that in group A.After treatment with TobraDex,the expression of miR-155-5p decreased and the expression of SOCS1 protein increased..Conclusion:miR-155-5p/SOCS1 is involved in the rejection after keratoplasty in rats,and the rejection in TobraDex eye drops group may be delayed by blocking this pathway.