| Sepsis is a life-threatening organ dysfunction caused by the uncontrolled infection response of living organism.In China,sepsis caused mortality rate in the ICU and hospital were 28.7% and 33.5%,respectively.Although the mortality rate of sepsis has slow decreased with the improvement of medical service quality,the incidence rate has slightly increased,and the number of patients with sepsis who are treated again in clinics has increased.Cardiac dysfunction caused by sepsis is the most common complication,with an incidence of 40%~60%.The early mortality and the incidence of cardiovascular disease after discharge were independently caused by myocardial damage resulting from sepsis in most patients.The mortality rate of patients with sepsis is 41.7% within 1 year after discharge,and as well as is 33.4% over 1 year after discharge,myocardial damage caused by sepsis is the independent factor.At present in china,the per capita hospitalization cost of patients with severe sepsis is $11,390 ± $11,455,the average daily cost per person is $502± $401,and the high mortality and high cost are increasingly payed more attention by people.Myocardial mitochondrial dysfunction caused by sepsis-induced myocardial mitochondrial damage is a key factor in myocardial damage and cardiac dysfunction.Mitochondrial autophagy has been shown to be an effective way to improve mitochondrial damage.However,there are few reports on targeting regulation of mitochondrial autophagy to improve septic myocardial mitochondrial damage.Min is one kind of antibiotic,which is more easily concentrated in ischemic cardiomyocytes than normal cardiomyocytes,and plays a role in improving myocardial injury caused by myocardial ischemia-reperfusion.Min has been shown to be a potent autophagy inducer in multiple cells with multiple pathological conditions.Whether min induces myocardial mitochondrial autophagy to improve mitochondrial damage,which rescues myocardial injury and cardiac dysfunction caused by sepsis has not been reported.Mammalian target of rapamycin(m TOR)has been proved to be one of the key molecules regulating autophagy.m TOR plays an important role in cell physiology and coordinates cell growth under different environmental conditions.Study show that Min regulated Akt/m TOR to inhibit the growth of ovarian tumors.In the heart,m TOR is widely expressed in myocardial tissue.However,whether Akt/m TOR signaling is involved in the protective effect of Min on myocardial injury and cardiac dysfunction induced by sepsis through inducing myocardial mitochondrial autophagy is not clear.In summary,this study investigated the role of Min-induced myocardial mitochondrial autophagy in myocardial mitochondrial injury induced by sepsis and the role of Akt / m TOR signaling pathway in this process respectively from animal and cell level,which provides new targets for the diagnosis and treatment of sepsis myocardial injury and cardiac dysfunction,and provides proofs of basic research for the clinical translational research of Min in the treatment of sepsis cardiac dysfunction.?Objective? 1.The appropriate dosage of Min Min for treating mice with sepsis was investigated after constructing a septic animal model induced by CLP.2.The CLP-induced mice were performed to explore whether Min Min induced septic myocardial mitochondrial autophagy and this effect on sepsis-induced myocardial mitochondrion injury were investigated.3.The effects of Min inducing myocardial autophagy on cardiac dysfunction and the survival rates in CLP-induced mice were investigated.4.Constructing LPS induced primary cardiomyocytes sepsis was performed and the appropriate dosage of Min Min for treating the primary cardiomyocytes with sepsis was investigated.5.In vitro experiments was performed to explore whether Min-induced septic cardiomyocytes autophagy and this effect on LPS-induced cardiomyocytes injury was investigated.6.To explore whether Min targeted regulation of Akt/m TOR pathway on autophagy in LPS-induced primary cardiomyocytes was performed.?Methods? 1.A CLP-induced mouse model of sepsis was constructed,and sham operation was used as the control model of sepsis.2.Construction of a primary cardiomyocyte sepsis model induced by LPS.After isolation of primary cardiomyocytes,different doses of LPS(0.1 ug/ml,1 ug/ml,10 ug/ml)were administered to induce a cell sepsis model,which was verified by assessment of apoptosis index and LDH level.To explore the optimal dose of LPS-induced cell sepsis was performed,experiment was randomly performed the following 4 groups: A.Contorl group: Contorl;B.0.1 ?g /ml LPS intervention group: LPS-0.1;C.1 ?g /ml LPS intervention group: LPS-1;D.10 ?g /ml LPS intervention group: LPS-10.3.Different doses of Min(25 mg/kg,50 mg/kg,100 mg/kg)were intraperitoneally injected for three consecutive times at 1 h,25 h and 49 h after CLP.The effects of Min on 7-d survival rate and serum c Tn I in septic mice were observed.The control group was admistrated by NS.To explore the optimal therapeutic dose of Min for treating sepsis was performed.The experiment was randomly performed the following 5 groups: A.Contorl group: Sham+NS;B.Model gruop: CLP+NS;C.25 mg/kg Min treament group: CLP+Min-25;D.50 mg/kg Min intervention group: CLP+Min-50;E.100 mg/kg Min treament group: CLP+Min-100.4.Different doses of autophagy inhibitor 3MA(30 mg/kg,60 mg/kg)were intraperitoneally injected 30 min before Min treatment,and the effects of 3MA on the 7-d survival rate and serum c Tn I in septic mice by Min treatment was investigated.To explore the optimal dose of 3MA for reversing the therapeutic effects of Min on sepsis was performed.The experiment was randomly performed the following 5 groups: A.Contorl group: Sham+NS;B.Model gruop: CLP+NS;C.Min treatment group: CLP+Min;D.30 mg/kg 3MA intervention group: CLP+3 MA-30+Min;E.30 mg/kg 3MA intervention group: CLP+3 MA-60+Min.5.Treatment of different doses of Min(8 ?g/ml,80 ?g/ml)were performed after 1 h LPS induction,the effects of Min on LPS-induced cardiomyocytes injury was observed through by assessment of apoptosis index and LDH level.The control group was admistrated by PBS.To explore the optimal therapeutic dose of Min for treating cell sepsis was performed.The experiment was randomly performed the following 4 groups: A.Contorl group: Contorl;B.Model gruop: LPS+PBS;C.8 ?g/ml Min treatment group: LPS+Min-8;D.80 ?g/ml Min treatment group: LPS+Min-80.6.Treatment of different doses of 3MA(10 ?m,40 ?m)were performed before 30 min Min treatment,the effects of 3MA on Min treatment to LPS-induced cardiomyocytes injury through by assessment of apoptosis index and LDH level.To explore the optimal dose of 3MA reversing the therapeutic effect of Min on cell sepsis was performed.The experiment was randomly performed the following 5 groups: A.Contorl group: Contorl;B.Model gruop: LPS+PBS;C.Min treatment group: LPS+Min;D.10 ?m 3MA intervention group: LPS+3MA-10+Min;E.40 ?m 3MA intervention group: LPS+3MA-40+Min.7.To explore the effects of Min induced mitochondrial autophagy on mitochondrial injury,myocardial injury,cardiac dysfunction and the survival in sepsis mice,the experiment was randomly divided into the following 5 groups: A.Contorl group: Sham+NS;B.Model gruop: CLP+NS;C.Min treatment group: CLP+Min;D.3MA intervention group: CLP+3MA+Min;E.3MA intervention control group: CLP+3MA.8.The effects of Min on LPS-induced autophagy,mitochondrial damage and cardiomyocyte damage in primary cardiomyocyte and the related mechanism research were explored.The experiment was randomly performed the following 5 groups: A.Contorl group: Contorl;B.Model gruop: LPS+ PBS;C.Min treatment group: LPS+Min;D.3MA intervention group: LPS+3MA+Min;E.3MA intervention control group: LPS+3MA.9.The number of mitochondrial autophagosomes and autophagosomes in the septic myocardial tissue and septic primary cardiomyocytes were assessed by transmission electron microscope,and the levels of ATP,CS activity and Mn-SOD activity were detected by ELISA to evaluate the mitochondrial function.10.Tunel staining was performed to evaluate the apoptosis of myocardium or cardiomyocytes induced by sepsis.11.Assessment of c Tn I and CK-MB by ELISA were performed to evaluate the injury of myocardium induced by sepsis.12.Assessment of LDH by ELISA was performed to evaluate cardiomyocytes damage.13.Western blotting was used to detect the expression levels of Akt,p-Akt,m TOR,pm TOR,raptor,rictor,LC3A/B and p62 protein expression in the myocardium or cardiomyocytes.14.Ultrasound was used to evaluate the cardiac function of sepsis mice.?Results? 1.Min with 50 mg/kg was intraperitoneal injected significantly improved the survival rate,alleviated myocardial injury by reducing c Tn I,improved cardiac dysfunction by increasing left ventricle EF and FS in CLP-induced sepsis mice 3 d after CLP,and Min significantly improved the 7-d survival of mice with sepsis about 40%.2.Min inducing myocardial mitochondrial autophagy significantly increased the number of myocardial mitochondrial autophagosome,the content of ATP,CS activity and significantly decreased Mn-SOD activity in myocardium to improve septic mice myocardial injury with reducing the level of c Tn I and CK-MB,and exerted antiapoptotic in myocardial cell with significantly reducing apoptosis index and the expression of caspase 3.However,administration of the autophagy inhibitor 3MA(60 mg/kg)reversed the effects of Min induced myocardial mitochondrial autophagy to improve myocardial mitochondrial and myocardial injury in sepsis mice.3.LPS 5 ?g/ml significantly induced primary cardiomyocyte apoptosis(apoptotic index about 35%)and increased LDH release levels,which was determined as the optimal dose of LPS to construct a septic cell model.80 ?g/ml Min induced autophagy in primary cardiomyocytes by significantly increasing the number of autophagosomes,and significantly improved the ATP content and CS enzyme activity in primary cardiomyocytes caused by LPS.Thus,Min exerted anti-apoptotic effect in LPS-induced primary cardiomyocytes by reducing apoptosis index,caspase 3 expression and LDH release through by Min induced autophagy improving mitochondrial damage.However,administration of the autophagy inhibitor 3MA(40 ?m)reversed the anti-apoptotic effects of Min through by inducing autophagy on LPS-induced primary cardiomyocytes.4.Min significantly up-regulated p-Akt,rictor,LC3?/ LC3?expression,and inhibited p-m TOR,ripator,p62 and caspase 3 proteins expression.Administration of the autophagy inhibitor 3MA reversed the regulating effects of Min on these protein molecules,revealing that Min targeted the Akt/m TOR signaling pathway to upregulated autophagy in LPS-induced primary cardiomyocytes.5.Min improved cardiac function in septic mice through by significantly increased left ventricular EF and FS,and decreased LVESV 3 d after CLP,thus significantly increasing the 7-d survival rate in CLP-induced mice.However,administration of the autophagy inhibitor 3MA reversed these effects of Min,which revealed that Min inducing autophagy improved cardiac dysfunction and survival in sepsis.?Conclusion? Our study showed that Min induced myocardial mitochondrial autophagy to exert reducing mitochondrial damage,inhibiting myocardial apoptosis,increasing sepsis mice cardiac function and 7-d survival rate in CLP induced-sepsis.In LPS induced-sepsis,Min induced cardiomyocytes autophagy to exert reducing mitochondrial damage and inhibiting cardiomyocytes apoptosis.And the study revealed that Min up-regulated LC3II/LC3 I levels and decreased p62/GAPDH levels in LPS-induced cardiomyocytes through by activating Akt to inhibite m TORC1 and up-regulating m TORC2 with activating Akt to inhibite m TORC1 again.These results revealed that Min may induce myocardial or myocardial mitochondrial autophagy to improve sepsis-induced cardiac dysfunction by targeting Akt/m TOR mechanism,providing a new idea for the diagnosis and treatment of sepsis. |
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