The Research On The Protective Effect And Mechanism Of JAK2-STAT3 Signaling Pathway Inhibitor SC99 On The Ischemia-reperfusion Injury In Rats

Objective To investigate the protective effect and its mechanism of SC99,a novel specific inhibitor targeting JAK2-STAT3 signaling pathway on cerebral isehemia-reperfusion injury in rats.Methods The present study was divided into three parts:In part 1,the rat model of middle cerebral artery occlusion and reperfusion(MCAO/R)was established.The expression of p-JAK2,JAK2,p-STAT3 and STAT3 in ischemic penumbra was detected by western blot at 3,6,12,24,48 and 7 days,respectively.In part 2,rat models of MCAO/R and microglia hypoxia and glucose deprivation/reperfusion(OGD/R)in vitro were established,respectively.In vivo,144 rats were randomly divided into six groups:Sham group,MCAO/R group,MCAO/R+Vehicle group(15?L,intracerebroventricular injection[icv]),MCAO/R+SC99-L group(5 mmol/L,15 ?L,icv),MCAO/R+SC99-M group(15 mmol/L,15?L,icv),MCAO/R+SC99-H group(25 mmol/L,15 ?L,icv),24 rats in each group.Based on the results of part 1,the time point with the highest phosphorylation of JAK2 and STAT3 after MCAO/R was selected(24h).Behavioral activities of each group were observed and neurological deficit scores were scored.Brain tissue was taken for TTC staining to calculate cerebral infarction volume.Western blot was employed to detect the expression levels of p-JAK2,JAK2,p-STAT3 and STAT3.TUNEL staining and FJB staining were used to detect apoptosis and necrosis in each group.Enzyme-linked immunosorbent assay(ELISA)was applied to detect proinflammatory and anti-inflammatory factors in supernatant and dry-wet weighing method was used to detect brain edema in each group.In vitro,primary cultured rat microglia were randomly divided into six groups:Sham group,OGD/R group,OGD/R+Vehicle group(2 ?L),OGD/R+SC99-L group(5?mmol,2 ?L),OGD/R+SC99-M group(10?mmol,2 ?L),OGD/R+SC99-H group(20 ?mmnol,2?L),and the supernatant was extracted 24 hours later for ELISA.Immunofluorescence was used to detect the activation of p-JAK2 and p-STAT3 in microglia.In part 3,based on the results of experiment part 2,the middle dose was chosen as the best dose in vivo(1.5 mmol/L,15?L,icv)and in vitro(10?mmol,2?L).In vivo,48 rats were randomly divided into four groups:Sham group,MCAO/R group,MCAO/R+Vehicle group(15?L,icv),MCAO/R+SC99-M group(1.5 mmol/L,15?L,icv).24 hours after MCAO/R,western blot was used to detect the levels of pro-inflammatory and anti-inflammatory factors,and immunofluorescence was employed to detect the effect of SC99 on microglia polarization.In vitro,primary cultured rat microglia were randomly divided into Sham group,OGD/R group,OGD/R+Vehicle group(2?L),OGD/R+SC99-M group(10?mmol,2?L).Immunofluorescence was performed to detect the effect of SC99 on microglia polarization at 24h after OGD/R.Results1.Part 1:In the acute phase of brain I/R,the levels of total JAK2 and STAT3 did not change significantly at all time points(P>0.05).The levels of p-JAK2 and p-STAT3 increased at 3h after reperfusion and reached the peak at 24 hours(P<0.001),then decreased gradually.2.Part 2:In vivo,western blot results showed that the levels of p-JAK2 and p-STAT3 in ischemic penumbra brain tissue in MCAO/R group increased significantly.Three different doses of SC99 all inhibited it.SC99-M and SC99-H groups had the strongest inhibitory effect and there was no significant difference between the two groups(P>0.05).Correspondingly,different doses of SC99 can reduce the infarct volume,improve neurological deficit,alleviate brain edema,and reduce neuronal apoptosis and necrosis after MCAO/R.SC99-M and SC99-H groups have the strongest protective effect and there is no significant difference between the two groups(P>0.05).In vitro,different doses of SC99 could decrease the levels of p-JAK2 and p-STAT3 in microglia and promote the translocation of p-STAT3 from cytoplasm to nucleus.SC99-M and SC99-H had the strongest effect and there was no significant difference between the two groups(P>0.05).3.Part 3:Immunofluorescence showed that SC99 promoted microglia polarization to anti-inflammatory M2 phenotype(CD206/CDllb positive cells)and inhibited microglia polarization to pro-inflammatory Ml phenotype(CD16/CDllb positive cells)after MCAO/R in vivo.Western blot showed that SC99 promoted the production of M2 microglia-related anti-inflammatory factors(IL-4 and IL-10)and inhibited the production of M1 microglia-related inflammatory factors(TNF-a,IL-1? and iNOS)after MCAO/R.In vitro,SC99 inhibited microglia polarization to pro-inflammatory M1 phenotype and promoted microglia polarization to anti-inflammatory M2 phenotype after OGD/R,thus playing a protective role.Conclusions1.MCAO/R can induce the phosphorylation of JAK2/STAT3 signaling pathway in ischemic penumbra,and the phosphorylation degree is the strongest in 24 hours after reperfusion.2.SC99 can inhibit the phosphorylation of JAK2/STAT3 signaling pathway induced by MCOA/R or OGD/R and has protective effect.3.SC99 can inhibit the polarization of microglia to pro-inflammatory M1 phenotype and promote the polarization of microglia to anti-inflammatory M2 phenotype to play an effective protective role after MCOA/R or OGD/R.