Polygonatum Sibiricum Polysaccharide Protects Against Myocardial Ischemic/Reperfusion Injury Through Inhibition Of TLR4

Objective In this study,we designed PSP on myocardial ischemia reperfusion injury experiment,in order to confirm whether it has myocardial protective effect,and the relationship with TLR4/NF-?B and apoptosis,pro-elaboration of PSP in ischemia reperfusion injury in the protection mechanism.Methods Part?,we have discussed the Polygonatum sibiricum polysaccharide is able to against myocardial ischemia-reperfusion in the overall level.In the whole experiment,rats were randomly assigned to different groups.Treatment groups were administered Polygonatum sibiricum polysaccharide,intragastric administration for 7 consecutive days,doses of(225,450,900,IG)mg·kg-1.Left anterior descending(LAD)coronaries were occluded for half an hour and then reperfused for two hours in each I/R,including drugs groups.ECG was used to detect ST changes before and after myocardial ischemia,and the ST segment elevation as a marker of ischemia success.And we detected datas,including left ventricular developed pressure(LVEDP),the maximum rate of left ventricular pressure rise(+dp/dt)and the maximum rate of left ventricular pressure decreased(-dp/dt)changes.Enzyme troponin and cardiac tissue tumor necrosis factor(TNF-?),interleukin-6(IL-6)were detected.We assessed tissue expression of Toll-like receptor 4(TLR4),NF-?B,Bax,Bcl-2.And we detected myocardial apoptosis.Part?,in the cell experiments.We studyed a preliminary exploration the Polygonatum sibiricum polysaccharide which was able to against myocardial ischemia-reperfusion.We used the hypoxia chambers to copy hypoxia reoxygenation model,hypoxia21,reoxygenation 6h,and found that hypoxia 21 h,reoxygenation 6h can reverse the injury.The H9c2 cells were divided as follows:(1)Control group,there was no any treatment in the cultural course of H9c2 cells.(2)Model group,the medium was replaced with glucose-free DMEM,and the cells were transferred into a triple gas incubator and cultured 21 hours.Then the medium was replaced normal culture medium,the cells were exposed to the normal culture condition for another 6 hours.(3)PSP group,the treatment was similar to that of the model group,but dealed with the final concentration of 1.0mg·ml-1PSP in the cell culture fluid in 12 hours before hypoxi H/Reoxygenation.(4)PSP group,the treatment was similar to that treatment group,but dealed with the final concentration of 1.5mg·ml-1 in the cell culture fluid 12 hours before hypoxi H/Reoxygenation.(5)PSP group,the treatment was similar to that of PSP group,but dealed with the final concentration of 2.0mg·ml-1 in the cell culture fluid12 hours before hypoxi H/Reoxygenation.Morphology change of H9c2 cells were observed under the inverted microscope.The viability of H9c2 cell was assayed by MTT.The activity of TNF-?,IL-6 and Caspase-3 in H9c2 cell were measured by ELISA assay.The protein expression of apoptosis was detected by using western blotting.Part?,in order to the further exploration of the effects of PSP on the apoptosis of myocardial ischemia-reperfusion rats and the inhibition of TLR4 pathway,which could be the mechanism of action of PSP on MIRI.Through the establish H9c2 cells model of hypoxia-reoxygenation injury,the H9c2 cells were divided as follows control group,model group,TLR4 inhibitor(TAK-242,1.0mg·ml-1)group,PSP(1.5mg·ml-1)group,PSP combined with TLR4 inhibition group.The morphological changes of apoptosis rate was determined by flow cytometric analysis.The m RNA levels of TLR4 and My D88 was detected by Real-time RT-PCR.The protein expression of I?B?,NF-?B,Bax and Bcl-2 were detected by using western blotting.Results1.Experimental results show that the Animals in sham group shows normal sinus cardiac rhythms.ST segment elevated after LAD ligated for 30 min,which suggests that the I/R model was constructed successfully.Ventricular arrhythmias was found after LAD was reperfused.Compared with the I/R group,PSP administration group LVEDP is increased significantly,especially TNF-? and IL-6 has a significant decrease.The expression of TLR4,NF-? B,Bax is decreased,and the expression of Bcl-2 is increased.At the same time,the apoptosis is decreased significantly.In order to further study,TLR4 agonist,inhibitors(TAK-242)is used alone or in combination with PSP serum to observe PSP how to protect myocardial ischemia-reperfusion injury in rats.The results suggest that PSP can against apoptosis which caused by myocardial ischemia-reperfusion by inhibiting TLR4 pathway.2.The results suggest that PSP could improve the morphology of H9c2 cells,increase the survival rate of myocardial cells,decrease the content of TNF-?,IL-6 and Caspase-3 in the cytosol,and inhibit the expression of apotosis.The results also indicate that PSP can against inflammatory injury which caused by hypoxia,and have a protective effect on hypoxia-reoxygenation H9c2 cells.3.PSP could effectively improve the morphology of apoptotic cells induced by hypoxi H/Reoxygenation,decrease the rate of apoptosis.By RT-PCR and Western blot assay TLR4,My D88 expression found PSP could reduce the expression of TLR4 and My D88 in hypoxia-reoxygenation H9c2 cells.And TLR4 inhibitor could increase the protective effect of PSP on hypoxi H/Reoxygenation injury.Then by western blot assay expression found that the regulation of PSP on apoptosis of hypoxi H/Reoxygenation may be related to the inhibition of TLR4 pathway.Conclusion In summary,PSP can protect myocardial ischemi H/Reperfusion injury in rats,TLR4 may be one of the key targets of its protective role,and its mechanism may be mediated by TLR4/NF-?B signaling pathway during ischemi H/Reperfusion,following inhibit the release of TNF-?,IL-6 and Caspase-3,reduce the apoptosis response.