Effect Of Hydrogen Sulfide (H2S) On ER Stress-mediated Autophagy In Cardiomyocytes In The Myocardial Injury In Sepsis

Objective: To investigate the effect of exogenous hydrogen sulfide(NaSH)on ER stress-mediated autophagy in rats with septic cardiomyopathy.Methods: Cell model: H9C2 cardiomyocyte sepsis model was induced with LPS(5 u g/ml),NaSH(exogenous H2 S donor),PAG(endogenous H2 S inhibitor)were cultured with cells,with concentration of NaSH(50 u mol/L)and PAG(200 u mol/L).Cells were randomly divided into seven groups,control(Control),sepsis(LPS),control and NaSH co-treatment(Control + NaSH),sepsis co-treatment(LPS +NaSH),control and PAG(Control + PAG),sepsis co-treatment with PAG(LPS + PAG),and sepsis and NaHS and PAG(LPS + NaSH + PAG).Cell viability was measured using the CCK-8 kit,and the ER stress proteins perk,EIF2,IREI-,CHOP,autophagy protein LC3-,p62 were detected by western-blot.Comparisons between groups were analyzed by one-way ANOVA with P <0.05.Animal model: Taking 48 SD rats from seven to eight weeks old,Using cecal ligation puncture(CLP),Rats were randomly divided into six groups,False surgery group(sham),sepsis group(CLP),sodium sulfur hydride treatment of sepsis group(CLP + NaSH);The Sepsis Group(CLP),Sepsis and the ER stress inhibitor 4-phenyl butyric acid(CLP + 4-PBA),Sepsis and the ER stress inducer tunicamycin(CLP + TM)in each group of 8 patients,Comparison of plasma H2 S content between control and sepsis rats,Rat left heart function was observed by echocardiography,Heart muscle tissue morphological changes were observed by HE staining,The mitochondrial structure,the number of autophagosomes,The level of apoptosis in myocardial tissue was assessed by tunel staining.The expression of ER stress protein perk,EIF2,IREI-I,CHOP,LC3-,h-,p62,CSE,3-MST,and the morphology of rat myocardium was observed by hematoxylin eosin(H & E)staining.Immunophluorescence colocalization observed the degree of LC3-binding to p62 to evaluate the level of autophagic flux.The comparison between groups was analyzed by one-way ANOVA,P <0.05,hydrogen sulfide and myocardial function index(LVEF,CKMB,LDH),P<0.05,and | r |> 0.6.Results: In vitro model:(1)in H9C2 cell line sepsis model,cell viability in LPS decreased(P <0.05),inhibited endogenous H2S(LPS)(P <0.05),LPS(P <0.05),and NaHS(Control + NaHS)or PAG(Control+ PAG)had no significant effect on cell viability(P> 0.05).(2)Compared with the Control,The LPS group,the ER stress markers p-perk,p-e IF2,chop,The level of autophagy was all increased(P <0.05);In contrast to the LPS,In the LPS + PAG group,The ER stress markers p-perk,p-e IF2,chop,Higher levels of autophagy were even more pronounced(P <0.05),After the administration of NaHS in the LPS group,Both ER stress and autophagy proteins were downregulated(P <0.05),Adding NaHS(Control + NaHS)in control group or PAG(Control + PAG)in control group had no significant effect on ER stress and autophagic protein(P> 0.05)In vivo model:(1)when compared to the Sham group,Rat plasma hydrogen sulfide levels were significantly reduced in the CLP group,Echocardiogram showed a left ventricular volume expansion,Ejection fraction(LVEF)was decreased,The LVEF levels were increased after CLP + NaSH(P <0.05),And we have found that,Hydrogen sulfide levels were associated linearly with the ejection fraction,To explore the reasons for the reduction of hydrogen sulfide levels in rats,This study further examined two key endogenous enzymes for hydrogen-sulfide synthesis(CSE,3-MST)highly expressed in myocardial tissue,The results showed decreased decrease in CSE and 3-MST(P <0.05)closely associated with the cardiovascular system in the CLP group.(2)From the pathological histological staining,myocardial tissue damage was obvious in CLP group,muscle fiber rupture,cell edema,disordered arrangement,myocardial injury released CK-MB and LDH,and plasma CK-MB and LDH were significantly higher than Sham group(P <0.05),CK-MB levels and LDH decreased(P <0.05).In tunel staining for myocardial apoptosis,myocardial apoptosis increased,and myocardial apoptosis decreased after CLP + NaSH(P <0.05).(3)Observe the ER stress on mitochondria and autophagosomes,it can be seen that the Sham group mitochondria short rod,cristae structure is complete,and CLP group visible mitochondria is vacuole,cristae structure partially dissolved,mitochondrial morphology is irregular,and a large number of autophagosomes,CLP + NaSH group reduction,mitochondrial morphology recovery.(4)ER stress marker proteins and autophagy markers were detected,It was found that the ER stress initiation proteins p-perk,p-e IF2,IRE1,and chop were upregulated in the CLP(P <0.05),The autophagy marker LC3 was also upregulated(P <0.05),The expression of p62 was decreased(P <0.05),The opposite trend of p62 and autophagy levels,Given that the enhanced levels of autophagy,After CLP + 4-PBA(ER stress inhibitor),The level of autophagy was down-regulated along with the ER stress marker proteins(p-perk,p-e IF2,IRE1,and chop),After the administration of CLP + TM(an ER stress agonist),The autophagy protein LC3 is also upregulated along with the ER stress marker proteins(p-perk,p-e IF2,IRE1,and chop).(5)In the LPS-induced H9C2 myocardial capillary sepsis model,ER stress and autophagy levels were significantly higher in LPS group than control group(P <0.05),and in LPS + NaSH group,ER stress markers p-perk,p-e IF2,IRE1,chop expression decreased(P <0.05)and autophagy levels also decreased(P<0.05).After detecting the interaction between LC3 and P62 in vitro membrane type,we can see that the cell viability decreased(P <0.05),the fluorescence intensity of p62 and LC3 increased significantly,and the autophagy level increased significantly.After LPS + NaSH,the autophagy level also decreased(P <0.05),and the cell viability increased significantly(P <0.05).Conclusion: Exogenous hydrogen sulfide(NaSH)can inhibit ER stress and mediate autophagy to reduce septic myocardial injury.