Effect Of Lycium Ruthenicum Murry Anthocyanins On Hypoxic Adaptability Of H9c2 Rat Cardiomyocytes

Improving the hypoxia adaptation of cardiomyocytes is of great significance for animals to prevent myocardial injury and protect cardiac function.Cardiomyocyte apoptosis and myocardial damage aroused by hypoxia can be effectively alleviate by anthocyanins within it’s strong antioxidant activity.Therefore,we built a hypoxic model of H9c2 rat cardiomyocytes in vitro to detect the effect of LRMA on hypoxia adaptation.Normoxia group,hypoxia group and hypoxia+LRMA group were set up.Then cell viability was detected by CCK-8 kit to select the optimal induction concentration and time of LRMA.Then LDH content and the cell morphology were observed.To detect the effect of LRMA on apoptosis induced by hypoxia of H9c2 rat cardiomyocytes,the content of ROS was explored by fluorescent enzyme labeling instrument.And then,cell apoptosis was detected by Hoechst33342/PI double staining and flow cytometry.In addition,the expression of apoptosis related factors(Bcl2,Bax,Bcl2/Bax)at m RNA and protein levels were tested by q RT-PCR and Western blot,respestively.Besides,lnc RNAs-mi RNAs-m RNAs ce RNA network related to hypoxia adaptation of H9c2 rat cardiomyocytes regulated by LRMA were built by RNA-seq and bioinformatics technology.Finally,flow cytometry was performed to defined the effect of LRMA on cell cycle,the m RNA and protein expressions of proliferation related genes(CCNT2,CDK9 and FOXP1)were tested by q RT-PCR and western blot,respectively.It was aimed to build a foundation of adaptability research for mammals in hypoxic environment,and also provide a theoretical basis to develope LRMA as a feed additive to improve the hypoxia adaptability of plateau animals.Results showed that:1.The protective effect of LRMA on H9c2 rat cardiomyocytes aroused by hypoxiaThe result of CCK-8 showed that H9c2 rat cardiomyocytes induced by 25,50 and100 μg/m L LRMA for 24,36,and 48 h could effectively improve the viability of H9c2 rat cardiomyocytes under hypoxic conditions(P<0.05).And the best way was that cells were pre-induced by 50 μg/m L LRMA for 12 h,and then aroused by LRMA under hypoxia condition for 24 h.Then,cells were treated as this way in the subsequent experiments.LDH activity in hypoxia(P<0.01)was significantly higher than that in normoxia group.Compared with hypoxia group,LDH activity was significantly decreased in hypoxia+LRMA group(P<0.05).Besides,compared with normoxia group,cell density decreased,cells shrink,cells morphology deformed and cell boundary blurred in the hypoxia group.Compared with hypoxia group,cells in hypoxia+LRMA group had higher cell density,clear cell boundary and normal cell morphology.Those results showed that H9c2 rat cardiomyocytes aroused by hypoxia could be effectively protected by LRMA.2.Effect of LRMA on apoptosis of H9c2 rat cardiomyocytes under hypoxiaThe result showed that,compared with normoxia group,the blue and red fluorescence intensity were brighter,the content of ROS and apoptosis rate was extremely increased in hypoxia group(P<0.01).Besides,the m RNA and protein expression of Bax was extremely improved while the m RNA and protein expression of Bcl2 and Bcl2/Bax were notably reduced in hypoxia group(P<0.01).In addition,compared with hypoxia group,the blue and red fluorescence intensity were darker in hypoxia+LRMA group.LRMA can effectively decreased the content of ROS and apoptosis rate under hypoxic condition(P<0.01).Meanwhile,LRMA can obviously inhibited the m RNA and protein expression of Bax(P<0.01)while increased the m RNA and protein expressions of Bcl2 and Bcl2/Bax in hypoxia(P<0.01).Those results indicated that LRMA could effectively mitigate apoptosis of H9c2 rat cardiomyocytes aroused by hypoxia.3.The hypoxia-related ce RNA network regulated by LRMAIt was found that there were 1404 lnc RNAs,20 mi RNAs and 829 m RNAs regulated by hypoxia.We obtained 1769 differentially expressed lnc RNAs,62 mi RNAs and 2517 m RNAs involved in the regulation of both LRMA and hypoxic.Then we obtained hypoxia related differentially expressed 862 lnc RNAs,7 mi RNAs and 351 m RNAs regulated by LRMA.In addition,it was found that the related differential genes regulated by LRMA were mainly enriched in the pathways related to cell proliferation and division,cell energy metabolism,inflammatory response and cell development.8lnc RNA-mi RNA relationship pairs and 33 mi RNA-m RNA relationship pairs were obtained by ce RNA network analysis,and 5 pairs of ce RNA were finally screened.The m RNA expression trend was consistent with the sequencing results.The further study showed that LRMA could effectively protect the proliferation activity of H9c2 rat cardiomyocytes under hypoxic conditions.So,it was speculated that LRMA could improve the hypoxic adaptability of H9c2 rat cardiomyocytes by promoting proliferation,which may be related to the expression of CCNT2 and FOXP1.4.Effect of LRMA on proliferation of H9c2 rat cardiomyocytes under hypoxiaThe results of previous studies showed that the hypoxic proliferation activity of H9c2 rat cardiomyocytes was protected by LRMA,which may be related to the expression of CCNT2 and FOXP1.The further study of flow cytometry showed that,compared with normoxia group,the number of H9c2 rat cardiomyocytes in G0/G1 phase was significantly increased(P<0.05),while the number of cells in S phase and G2/M phase were significantly decreased in hypoxia group(P<0.05).Besides,the m RNA and protein expression of CCNT2,CDK9 and FOXP1 were notably down regulated in hypoxia group(P<0.01).Compared with hypoxia group,the number of H9c2 rat cardiomyocytes in G0/G1 phase was significantly reduced(P<0.05),while the number of cells in S phase and G2/M phase were significantly improved in hypoxia+LRMA group(P<0.05).In addition,the m RNA and protein expression of CCNT2,CDK9 and FOXP1 were obviously up regulated in hypoxia+LRMA group(P<0.01).Those results showed that LRMA could promote the hypoxic adaptation of H9c2 rat cardiomyocytes through increasing cell proliferation.