Protective Effect And Mechanism Of Karounidiol Pretreatment On Hypoxia-reoxygenated Myocardial Cells

Objective:(1)To investigate the protective effect of karounidiol(Kar)pretreatment on hypoxia-reoxygenated(H/R)injured cardiomyocytes.(2)To explore whether Kar pretreatment inhibits apoptosis through PI3K/Akt/NF-?B pathway,thereby protecting myocardial cells from H/R induced injury.(3)To further determine whether Kar pretreatment regulates autophagy through PI3K/Akt/mTOR pathway,thereby protecting myocardial cells from H/R induced injury.Method:(1)CCK-8 was used to detect the toxicity and proliferation promoting effects of Kar on H9c2(2-1)cells and AC16 cells.(2)The H9c2(2-1)cells and AC16 cells were pretreated with different concentrations of Kar on the established H/R cell model.CCK-8 was used to measure the cell survival rate and LDH was used to detect the cell mortality rate.The optimal protection concentration(10 ?mol/L)of Kar on H/R injured myocardial cells was determined according to the results.(3)Both of the H9c2(2-1)cells and AC16 cells were divided into the following groups:Normoxia group(Normoxia),hypoxia reoxygenation group(DMSO+hypoxia reoxygenation,H/R),karounidiol pretreatment group(karounidiol+H/R,Kar),PI3K inhibitor group(LY294002+Kar+H/R,LY294002),Akt inhibitor group(Perifosine+Kar+H/R,Perifosine),NF-?B inhibitor group(BAY 11-7082+Kar+H/R,BAY 11-7082),mTOR inhibitor group(Rapamycin+Kar+H/R,Rapamycin).(4)The protein expression of p-I3K,T-PI3K,p-Akt,T-Akt,p-p5,p65,Cleaved-Caspase-3,T-Caspase3,Bcl-2,Bax,p-mTOR,T-mTOR,P62,Beclin-1,LC3?/? were measured by the Western blot.(5)The adenovirus of mRFP-GFP fluorescent protein double labled LC3? autophagy probe(Ad-mRFP-GFP-LC3)was used to infect the H9c2(2-1)cells or AC16 cells to monitor the autophagy flux changes by the laser confocal microscopy.(6)TUNEL staining and DAPI counterstain were used to detect the apoptosis.Result:(1)H9c2(2-1)cells or AC16 cells were incubated with different doses of Kar(0.01?mol/L?0.1 ?mol/L?1?mol/L?10 ?mol/L L)for 24 h.Then the CCK-8 was applied to evaluate the cell proliferation or toxicity of them.Compared with the blank control or the solvent control(0.01%DMSO),all doses of Kar had no obvious cell proliferation or toxicity effect on both cell lines(all P>0.05).(2)Compared with the Normoxia group,the H/R group had significantly lower cell survival rate and significantly higher cell death rate(all P<0.01);Compared with the H/R group,the survival rates of cells in all Kar groups were significantly improved except the 0.1 ?mol/L group,and the 10 ?mol/L group had the strongest effect(P<0.01);Compared with the H/R group,the cell death rates were significantly reduced in all Kar groups(all P<0.05),and the effect was most significant in the 10?mol/L group(P<0.01).According to the above results,the optimal dosage of Kar was determined to be 10?mol/L.(3)Compared with the Normoxia group,the protein expression of the Cleaved-Caspase-3,Bax,Beclin-1,LC3?/? in H/R group were significantly higher(all P<0.05),and p-PI3K/T-PI3K,p-Akt/T-Akt,p-p65/p65,p-mTOR/T-mTOR,P62 and Bcl2 protein expression were significantly lower(all P<0.05);Compared with H/R group,the protein expression of Cleaved-Caspase-3,Bax,Beclin-1,LC3?/? in Kar group were significantly decreased(all P<0.05),and p-PI3K/T-PI3K,p-Akt/T-Akt,p-p65/p65,p-mTOR/T-mTOR,P62,Bcl-2 protein expression were significantly increased(all P<0.05);Compared with the Kar group,the protein expression of the Cleaved-caspase-3 and Bax increased(all P<0.05)and the protein expression of p-PI3K/T-PI3K,p-Akt/T-Akt,p-p65/p65 and Bcl-2 decreased(all P<0.05)in LY294002 group,Perifosine group and BAY 11-7082 group;Compared with the Kar group,the expression of Beclin-1,LC3?/? in LY294002 group,Perifosine group and Rapamycin group all increased(all P<0.05),and p-PI3K/T-PI3K,p-Akt/T-Akt,p-mTOR/T-mTOR,P62 protein expression reduced(all P<0.05).(4)The mRFP-GFP double-labeled adenovirus fluorescent protein autophagy probe(Ad-mRFP-GFP-LC3)was detected by the laser confocal microscopy.The results showed that compared with the Normoxia group,the yellow and red fluorescent puncta in the H/R group both increased significantly;Compared with the H/R group,both the yellow and red puncta in Kar group were reduced significantly;Compared with the Kar group,the yellow and red fluorescence puncta were increased in LY294002 group,Perifosine group and also Rapamycin group.(5)TUNEL and DAPI double staining was used to monitor the cell apoptosis.The Results indicated that compared with the Normoxia group,the green fluorescence intensity in H/R group was increased obviously;Compared with H/R group,the green fluorescence intensity of the Kar group was reduced obviously;Compared with the Kar group,the green fluorescence intensity of the LY294002 group,Perifosine group and the BAY 11-7082 group were all increased.Conclusion:(1)Pretreatment with the trichosanthes karounidiol has a dose-dependent protective effect on myocardial cells damaged by hypoxia-reoxygenation.(2)Activating PI3K/Akt/NP-?B pathway was involved in the the karounidiol pretreatment's anti-apoptosis mechanisms on hypoxic-reoxygenated cardiomyocyte.(3)Activating PI3 K/Akt/NF-?B pathway was involved in the karounidiol pretreatment's autophagy inhibition mechanisms on hypoxic-reoxygenated cardiomyocyte.