Effects Of Honokiol On Cardiomyopathy In Cell

BackgroundMyocardial ischemia-reperfusion injury is a common cardiovascular disease.It is predicted that nearly 300 million people currently suffer from cardiovascular diseases in China,and its mortality rate ranks first in the proportion of death causes of major diseases,and increases year by year.Patients suffering from ischemic diseases are suffering tremendously,and their families are under enormous burden.Existing methods for ischemic diseases such as major thrombolysis for myocardial infarction and coronary artery bypass surgery,but after these methods are used to restore blood flow to the ischemic site,they will still face myocardial ischemia reperfusion injury.Accumulation of fine reactive oxygen species,oxidative stress,and outbreak of cellular inflammation during myocardial ischemia-reperfusion injury will cause more serious damage to cells.When the inflammation in myocardial ischemia-reperfusion injury can not be control in time,it will cause myocardial fibrosis and affect the heart function.This study established an in vitro model of myocardial ischemia-reperfusion by glucose oxygen deprivation and reoxygenation of the cardiomyocyte H9c2 cell line,observed the effect of honokiol on hypoxia-reoxygenation-induced inflammation of H9c2 cells,as well as the subsequent fibrosis on myocardial fibroblast The impact of this is expected to provide new options for clinical drug screening for ischemia-reperfusion injury and its prognosis.and discussed its mechanism.In order to provide new options for clinical drug screening for ischemia-reperfusion injury and its prognosis.ObjectiveTo investigate the effects and potential mechanisms of Honokiol on myocardical inflammation in H9c2 cell induced by hypoxia-reoxygenation.Discussthe the inhibitory effect of honokiol on myocardial fibrosis induced by TGF-β1.Methods and resultsPart ⅠEffect of honokiol on hypoxia-reoxygenationinduced injury in H9c2 cardiomyocyte cellsMethods:The model of hypoxia-reoxygenation on rat myocardial cell line H9c2 was established by hypoxia(H)/reoxygenation(R).The effect of different concentrations of honokiol on the cell viability after H/R injury was determined using a cell counting kit(MTT)assay.Western blot was used to detect the changes of MCPIP1 expression and the expression of inflammation-related proteins at different time points 1 h,2 h,3 h,and 6 h after I/R.Silenced MCPIP1 by transfecting siRNA in advance in H9c2 cells after 15 h of hypoxia and 2 h of reoxygenation.MTT was used to detect changes in cell survival rate of each group.Results:(1)Compared to control group,the cell survival rate of each Hypoxia/Reoxygen group had decreased significantly(P<0.01);(2)Compared to control group,western blot results showed that the model group protein NF-κB phosphorylation was increase(P<0.05);meanwhile,the immunofluorescence results showed that the nuclear translocation of NF-κB increased in H9c2 cells after hypoxia-reoxygenation;(3)Western blot results showed that compared to control group,the expression of MCPIP1 protein in H9c2 cells after H15/R0h,H15/R1h,H15/R2h,H15/R3h,H15/R6h was increased(P<0.05).(4)Compared to control group,different concentrations of Honokiol had increased the cell survival rate after hypoxia-reoxygenation treatment(P<0.01).(5)Western blot results showed that compared with the model group,the expression of MCPIP1 protein was increased(P<0.05).p-NF-kB,p-JNK protein was reduced after Honokiol treated(P<0.05).(6)The MTT results showed that the effect of honokiol on the survival rate(P<0.01)of H9c2 cells after hypoxia-reoxygenation was counteracted by silencing MCPIP1.Part ⅡEffect of honokiol on TGF-β 1-induced migration in cardiac fibroblastsMethods:Primary cardiac fibroblasts were isolated and cultured.Myocardial fibroblast-specific protein Vimentin was fluorescently labeled,and immunofluorescence was used to detect the purity of cardiac fibroblasts.Wound-healing assays were adopted to detect the effect of Honokiol on rat myocardial fibroblasts migration induced by TGF-β1.Cell counting kit(MTT)assay was used to detect the activity and proliferation of myocardial fibroblasts.Immunofluorescence was used to detect the changes of fibronectin.Western blot was used to detect the expression of FN,COL-1.Results:(1)The myocardial fibroblasts were characterized as flat and irregular polygonal cells,and they account for about 0.945 of the cells isolated by differential attachment;(2)At the range of 0-20μmol·L-1 of Honokiol,there was no obvious cytotoxicity to fibroblasts,and fibroblasts can be induced to proliferate by TGF-β1 is in 10μM(P<0.05);(3)Wound-healing experiment showed that compared with the model group,Honokiol inhibited the migration of fibroblasts with concentration-dependent manner(P<0.01);(4)The results of immunofluorescence showed that compared with the control group,the fluorescence intensity of FN was increased by TGF-β1;Compared with the TGF-β1 group,the fluorescence intensity of FN was inhibited by Honokiol;(5)Western blot results showed that compared with the model group,the expression of ECM-related proteins FN,COL-1 and MMP-9 were significantly down-regulated after honokiol treated(P<0.01).Conclusion1.Honokiol can increase the survival rate of H9c2 cells after hypoxia-reoxygenation.The mechanism may be the protective effect of MCPIP1-mediated inhibition of inflammation.2.Treatment with Honokiol can reduce the migration of myocardial fibroblasts cells induced by TGF-β1,and its mechanism may be related to the inhibition of FN protein deposition.