| Object:This study is to explore whether IL-35 alleviates cardiac damage caused by LPS and to elucidate its possible molecular mechanisms.Method:1.A total of 24 C57BL/6 mice were firstly randomly divided into three groups:(1)Control group(n=8):Control group was pretreated with PBS and then no other treatment;(2)LPS group(n=8):LPS group was pretreated with PBS by tail vein for 7 days and then injected with LPS(10 mg/kg)for 12 hours;(3)LPS+IL-35group(n=8):LPS+IL-35 group was pretreated with pIL-35 by tail vein for 7 days and then injected with LPS(10 mg/kg)for 12 hours.Large volume rapid tail vein injection of 2 ml PBS solution which contain 100?g plasmid DNA pIL-35 pretreat mice using plasmid transfer technique.After mice were treated by LPS for 12 h,the heart function of mice was detected by ultrasound,then the mice were killed,and the tissues were taken for other tests;the serum cardiac injury markers(LDH,CK-MB)were detected by automatic biochemical analyzer;the serum inflammatory factor changes were detected by ELISA;the heart sections were taken for HE?Masson staining;and other items were detected by RT-qPCR and Western blot.2.Mice cardiac fibroblasts(CFs)cells were obtained and seeded into 6-well plates at a density of 1×10~6 cells with Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%fetal bovine serum(FBS)and 1%penicillin/streptomycin(Hyclone)under a humidified incubator of 5%CO2 at 37°C.Before experimental treatments,the cells should be grown to 90%confluency.Available CFs were firstly pretreated with IL-35 at low(1ng/ml),medium(10ng/ml),and high(50ng/ml)concentration for 1 hour,and then challenged with LPS(100ng/mL)for another 24hours.Cultured CFs were randomly assigned into five groups as follows:(1)Control group:without adding of IL-35 or LPS;(2)LPS group:with adding of LPS(100ng/mL);(3)Low-dose IL-35 pretreatment+LPS group:LPS+IL-35(1ng/ml),with adding of IL-35(1ng/ml)and LPS(100ng/mL);(4)Medium-dose IL35pretreatment+LPS group:LPS+IL-35(10ng/ml),with adding of IL-35(10ng/ml)and LPS(100ng/mL);(5)High-dose IL35 pretreatment+LPS group:LPS+IL-35(50ng/ml),with adding of IL-35(50ng/ml)and LPS(100ng/mL).After these treatments,cells were harvested for further experiments.Such as CCK8,RT-qPCR,Western blot experiments.Result:1.Western blot results showed that compared with the Control group,the expression level of IL-35 in the heart of the LPS group decreased.Compared with LPS group mice,the expression level of IL-35 in the heart of LPS+IL-35 group mice pretreated with plasmid DNA(PIL-35)was significantly increased.2.Compared with the Control group,the cardiac function of the LPS group was significantly reduced.Compared with LPS group mice,LPS+IL-35 group mice pretreated with plasmid DNA(PIL-35)significantly reduced the cardiac dysfunction caused by LPS.3.The results of HE staining,Western blot and RT-qPCR showed that compared with the Control group,the expression of cardiac inflammatory factors in the LPS group was significantly increased.Compared with LPS group mice,LPS+IL-35group mice pretreated with plasmid DNA(PIL-35)significantly reduced the cardiac inflammatory response caused by LPS.4.Western blot results showed that compared with the Control group,the apoptosis level of heart cells in the LPS group was significantly increased.Compared with LPS group mice,LPS+IL-35 group mice pretreated with plasmid DNA(PIL-35)significantly reduced LPS-induced cardiac apoptosis.5.Massion staining,Western blot and RT-qPCR showed that compared with the Control group,the level of cardiac fibrosis in the LPS group was significantly increased.Compared with LPS group mice,LPS+IL-35 group mice pretreated with plasmid DNA(PIL-35)significantly reduced the level of heart fibrosis caused by LPS.6.CCK-8 test results showed that preconditioning CFs cells with different concentrations of IL-35 could reduce the decrease of CFs cell activity induced by LPS in a concentration-dependent manner.7.Western blot and RT-qPCR showed that CFs cells in the LPS group had significantly higher levels of inflammation and fibrosis than those in the Control group.Compared with CFs cells in the LPS group,pretreatment with IL-35 at different concentrations significantly reduced the level of inflammation and fibrosis in LPS-induced CFs cells in a concentration-dependent manner.Conclusion:1.IL-35 can alleviate the heart injury induced by LPS.2.IL-35 reduces LPS-induced myocardial inflammation,which mainly inhibits inflammatory responses through the NF-?B signaling pathway.3.IL-35 can inhibit the apoptosis of myocardial cells induced by LPS.4.IL-35 can reduce myocardial fibrosis induced by LPS,which mainly through inhibited TGF-?1/Smad2/3 signaling pathway. |
PDF Full Text Request