The Effects And Mechanism Of IL11 On Fibroblast Senescence Induced By Oxygen Glucose Deprivation

Cellular stress senescence induced by ischemia is a potential target for the occurrence and development of cardiovascular diseases.It has been found that cardiomyocyte senescence induced by myocardial ischemia is the main cause of cardiac insufficiency.Fibroblasts are important participants in tissue repair after ischemic injury.Oxygen glucose deprivation（OGD）caused by acute myocardial infarction can induce fibroblasts to differentiate into myofibroblasts,which mediate tissue repair by secreting collagen and promote the formation of scar at the injured site.However,it is found that fibroblast senescence interferes with the repair of ischemic tissue injury.On the one hand,senecent fibroblasts amplify the cascade inflammatory response and aggravate tissue damage by secreting cytokines.On the other hand,collagen deposition can be caused by maintaining self-activation,and lead to aggravate fibrosis and damage tissue function.Therefore,clarifying the mechanism of fibroblast senescence induced by ischemia can provide an effective theoretical basis for the clinical treatment of senescence related ischemic diseases,which has important clinical significance.Recent studies have shown that ischemia can induce specific high expression of interleukin 11（IL11）in fibroblast,and IL11 can induce collagen secretion and deposition by activating JAK2/STAT3 pathway in fibroblast,and participate in the process of fibrosis.It is found that IL11 is one of the important components of senescence associated secret phenotype（SASP）in senescent fibroblasts,which can promote fibrosis by promoting collagen deposition in fibroblasts.It is speculated that IL11 may be involved in stress-induced senescence of fibroblasts induced by ischemia,but its effect and mechanism on stress-induced senescence induced by ischemia remain unclear.Changes in kynurenine（KYN）metabolic pathway activity are associated with senescence and other diseases,intervention of KYN pathway can prolong the life span of Drosophila melanogaster and nematode.Indoleamine 2,3-dioxygenase 1（IDO1）is a key rate-limiting enzyme in KYN metabolic pathway.It was found that the expression of IDO1 was down regulated under hypoxia in fibroblasts,and the KYN mediated by IDO1 can play an anti-senescence and anti-fibrosis role,suggesting that IDO1 is involved in the cell senescence process of fibroblasts.It has been reported that IDO1 can be degraded by JAK2/STAT3.However,the effect of IL11 on KYN metabolism in fibroblasts has not been clarified.In conclusion,this study speculates that IL11 may influence the development of fibroblast senescence through the secretion of KYN mediated by IDO1 during ischemia-induced stress senescence.Objetives:To investigate the effect of IL11 on stress-induced senescence of NIH3T3fibroblasts and L929 fibroblasts induced by OGD in vitro;Explore the effect of IL11on kynurenine in NIH3T3 fibroblasts and L929 fibroblasts senescence model induced by OGD;Further clarify the mechanism of IL11 regulating KYN in NIH3T3fibroblasts and L929 fibroblasts senescence model induced by OGD.Methods:1.The OGD-induced stress senescence model in NIH3T3 and L929 cells was establishedNIH3T3 fibroblasts derived from mouse embryos and L929 fibroblasts derived from dermis were used as experimental objects.Sugar free serum-free medium,1%O₂,94%N₂and 5%CO₂were used as culture environment to create OGD conditions.MTT assay was used.to detect the survival rate of NIH3T3 fibroblasts and L929fibroblasts treated with OGD for 2h,4h,6h,8h and 10h.Western blot was used to detect the expression of senescence markers,such as p56,p21 and p16.According to the above experimental results,the effect of OGD on stress-induced senescence of NIH3T3 and L929 cells was clarified,and the optimal time of stress-induced senescence model induced by OGD was determined.RT-q PCR was used to detect the m RNA expression of collagen I,collagen III in NIH3T3 and L929 treated with OGD for different times.Collagen I,collagen III,α-SMA,TGFβproteins expression were detected by western blot in fibroblasts.According to the above experimental results,determine the changes of collagen expression in stress-induced senescence fibroblasts.2.To determine the effect of IL11 on stress-induced senescence and collagen expression of NIH3T3 and L929 cells induced by OGDThe effects of inhibition of IL11 on the expression of senescence markers and collagen were investigated by RT-q PCR and Western blot in NIH3T3 and L929 cell senescence models induced by OGD after inhibiting the expression of IL11 in fibroblasts by si RNA.Then,Collagen contraction experiment was used to detect the expression of collagen in NIH3T3 and L929 cell senescence model induced by OGD.After realizing the high expression of IL11 in NIH3T3 cells and L929 cells by supplementing recombinant mouse IL11,RT-q PCR and Western blot were used to detect the changes of senescence markers and collagen expression in OGD induced NIH3T3 and L929 cell senescence model.Collagen contraction experiment was used to detect the expression of collagen in NIH3T3 and L929 cell senescence model induced by OGD after exogenous administration of IL11.3.To explore the effect of IL11 on kynurenine expression in NIH3T3 and L929fibroblasts senescence induced by OGDEhrlich method was used to detect the level changes of KYN from cell supernatant,a metabolite of kynurenine,in the stress-induced senescence model of NIH3T3 and L929 cells induced by OGD.Western blot was used to detect the expression of collagen in NIH3T3 and L929 cell stress-induced senescence model induced by OGD after supplementing L-KYN.Ehrlich method was used to detect the level changes of KYN from cell supernatant in NIH3T3 and L929 cell senescence models induced by OGD after inhibition and supplementation of IL11.Western blot was used to detect the changes of collagen expression in NIH3T3 and L929 cell stress-induced senescence model induced by OGD.RT-q PCR,Western blot,immunofluorescence and ELISA kit were used to detect the expression of IDO1 in NIH3T3 and L929 cell senescence models induced by OGD after inhibition and supplementation of IL11.4.To explore the mechanism of IL11 regulating IDO1 in NIH3T3 and L929 cell senescence induced by OGDWestern blot was used to detect the protein expression of Akt,p-Akt,m TOR,p-m TOR,JAK2,p-JAK2,STAT3,p-STAT3,SOCS3 and IDO1 in NIH3T3 cells and L929 cells induced by OGD after inhibition and supplementation of IL11.After JAK/STAT3 inhibitor AG490 and recombinant mouse IL11 cytokine given at the same time,the protein expressions of JAK2,p-JAK2,STAT3,p-STAT3,SOCS3 and IDO1 in OGD induced fibroblast senescence model were detected by Western blot.Results:1.Establishment of stress-induced fibroblasts senescence model induced by oxygen and glucose deprivation（1）After OGD treatment for 2 h,4 h,6 h,8 h and 10 h,the survival rates of NIH3T3 and L929 cells decreased significantly at 4 h,6 h,8 h and 10 h.（2）The expressions of senescence marker proteins p53,p21 and p16 in NIH3T3and L929 cells increased significantly at 4 h,6 h,8 h and 10 h after OGD treatment.OGD for 6 hours was selected as the best time for stress-induced senescence model of NIH3T3 cells and L929 cells induced by OGD.（3）TGFβ,collagen I,collagen III and myofibroblast markersα-SMA protein expression increased in NIH3T3 cell and L929 cell senescence models induced by OGD.The m RNA and protein expressions of collagen I and collagen III increased significantly at 6 h,8 h and 10 h after OGD treatment。The above results showed that fibroblast stress-induced senescence model induced by OGD was established,and collagen expression increased in NIH3T3 cell and L929 cell senescence models induced by OGD.2.Effect of IL11 on stress-induced fibroblasts senescence and collogen expression（1）In the fibroblast senescence model induced by OGD,the expression of IL11m RNA and protein increased.（2）After inhibiting IL11,in the fibroblast senescence model induced by OGD,the expression of p53,p21,p16,collagen I and collagen III was decreased.（3）After supplement of exogenous IL11,the expression of senescence marker proteins p53,p21 and p16 were increased.The expression of collagen I and collagen III and collagen contraction were significantly increased in NIH3T3 and L929 cell senescence models induced by OGD.The above results showed that IL11 promoted fibroblast stress-induced senescence and collagen expression.3.IL11 affects the expression of kynurenine in fibroblasts stress-induced senescence（1）The level of KYN decreased in NIH3T3 and L929 cell senescence models induced by OGD.After supplementing L-KYN,the expression of collagen was decreased in NIH3T3 and L929 cell senescence models induced by OGD.（2）After inhibiting IL11,the level of KYN was up-regulated in NIH3T3 and L929 cell senescence models induced by OGD.After supplementation of IL11,the level of KYN in NIH3T3 and L929 cell senescence models induced by OGD was decreased.When IL11 and L-KYN were supplemented at the same time,the expression of collagen in NIH3T3 and L929 cell senescence model induced by OGD was decreased.（3）The expression of IDO1 was inhibited in NIH3T3 and L929 cell senescence models induced by OGD.After inhibiting IL11,IDO1 protein expression was increased in NIH3T3 and L929 cell senescence models.After supplementation of IL11,the expression of IDO1 was decreased in NIH3T3 and L929 cell senescence models induced by OGD.The above results show that IL11 can reduce the production of KYN and promote the expression of collagen in stress-induced senescent fibroblasts by inhibiting the expression of IDO1.4.The regulatory mechanism of IL11 on IDO1 expression in fibroblasts stress-induced senescence（1）After inhibiting IL11,in the fibroblast senescence model induced by OGD,JAK2/STAT3/SOCS3 and Akt/m TOR pathways were affected in NIH3T3 cells.The phosphorylation and expression of JAK2/STAT3/SOCS3 pathway protein were inhibited in L929 cells,but the phosphorylation level of Akt/m TOR pathway protein did not change.（2）After supplementation of IL11,in NIH3T3 and L929 cell senescence models induced by OGD,JAK2/STAT3/SOCS3 and Akt/m TOR pathway were activated.（3）After IL11 and JAK2/STAT3 pathway inhibitors were given at the same time,in NIH3T3 and L929 cell senescence models induced by OGD,JAK2/STAT3/SOCS3pathway was affected and IDO1 expression was down regulated.The above results showed that IL11 could inhibit IDO1 expression through JAK2/STAT3/SOCS3.Conclusions:1.IL11 can promote the increase of collagen expression in NIH3T3 and L929cell senescence models induced by OGD.2.IL11 may promote the accumulation of KYN in fibroblasts by regulating IDO1/KYN metabolic pathway,and participate in stress-induced senescent of fibroblast.3.IL11 can inhibit IDO1 expression through JAK2/STAT3/SOCS3 pathway and promote KYN mediated fibroblast senescence in the fibroblast senescence model induced by stress.