Anti-heat Stress Protection And Mechanism Of Vitamin C-Na On Myocardidial Cells By Inducing Hsps Expression

Heat stress has become one of major factors restricting the development of livestock and poultry,with the intensification of intensive farming and global warming.Heat stress can cause various tissue damage,and even death in severe cases.It was reported that sudden death due to heat stress is closely related to myocardial injury.The heart is sensitive to heat,when suffered to heat stress,and becomes one of the primary organ to be damaged.It has been a hot spot for researchers to explore the mechanism of heat stress leading to myocardial injury and to find effective ways to protect cardiomyocytes from heat stress injury.Heat shock protein(Hsp)is a group of highly conserved protective proteins which induce synthesis or synthesis increase when cells are stimulated by various stress factors,which can help peptide chains correct folding,assembly,translocation,and recovery.In addition,Hsps are also involved in various signal pathways such as apoptosis,cell cycle and immune,indicating Hsps have important endogenous protective mechanism in the body.In this paper,heat stressing H9C2 cardiomyocytes were used as an in vitro model to explore the relationship between pathological lesions and Hsps expressions in H9C2 cells under heat stress and recovery conditions.In addition,aB-crystallin was mainly selected to investigate the protection mechanism by overexpressing in improving H9C2 cells from heat damages.The molecular mechanism of heat shock factor 1(HSF1)regulating Hsps expression was also explored under heat stress and recovery conditions.Besides,vitamin C-Na(VC-Na),as a heat-stress-resistant drug,was screened and verified its effective function as well as the mechanism in relieving heat stress injury in vivo and in vitro.In order to explore the heat stressed damage,transcription levels of hsps mRNA and the expression levels of Hsps in cardiomyocytes under the heat stress and the recovery conditions,the stressed lesions,the variations of the transcription and the expression of Hsps were studied,by using of histopathological observation,RT-PCR and western blot,under the establishment of the heat stress and the recovery model of myocardial cells.The results showed that the swelling including granular degeneration and vacuolar degeneration is observed in H9C2 cells after heat stress at 42? for 0.5 h,and much more obvious swelling,severe granular degeneration and vacuolar degeneration appeared after heat stress for 2 h.After 12 h of recovery,the pathological lesions slightly weakened.However,necrosis of myocardial cells was observed after heat stress at 42? for 5 h,and no obvious recovery from cell damages was observed after 12 h following culture recovery.The transcription levels of hsp27 and hsp70 up-regulated at 0.5 h of heat stress.The transcription levels of CRYAB,hsp47,hsp60,hsp90,and hsp110 showed significant inductions at 2 h of heat stress.After 12 h following culture recovert,the transcription levels of CRYAB increased continuously,while the transcription levels of hsp27,hsp47,hsp60,hsp70,hsp90,hsp110 showed reduction tendency.The expressions of Hsp27,Hsp47,Hsp60,Hsp70,Hsp90 and Hsp110 showed induction trend,except for reduction trend of CRYAB.After 12 h of culture recovery,the expression levels of CRYAB,Hsp27,Hsp60,Hsp70 and Hsp110 increased,and the level of Hsp47 recovered to the normal level.It is indicated that Hsps play important roles in improving the heat stress resistance and damage repair.In this study,we firstly constructed the H9C2 cell line with the feature of stably expressing CRYAB,then examined by histopathological lesions,cell cycle detection,co-location of CRYAB and F-actin,apoptosis and related proteins detection to explore the function and mechanisms of CRYAB on cardiomyocytes following heat stress.In this study,three cell lines were screened out,one is for stably transfecting blank plasmid and named with control,the other two were both overexpression CRYAB protein in different degrees with names of CRYAB-5 and CRYAB-7,respectively.Our results showed that both cell lines of CRYAB-5 and CRYAB-7 could significantly reduce the granular degeneration and vacuolar degeneration caused by heat stress,the same to relieve the cell blockage in G0/G1 phase.The protective effect is positive to the expression of CRYAB protein.Immunofluorescence analysis showed CRYAB can transfer from cytoplasm to nucleus under heat stress conditions,except that,CRYAB was co-localized with F-actin,which occurred to accumulate under stress conditions.Overexpression of CRYAB could significantly reduce the aggregation of F-actin caused by heat stress.In addition,over-expressing CRYAB protein can significantly reduce apoptosis induced by heat stress,which may play the anti-apoptotic function by reducing the expression of cleaved-caspase.In conclusion,overexpression of CRYAB could significantly increase the heat resistance of H9C2 cells,and its protective effect is positively correlated with the overexpression degree of CRYAB.However,the detailed mechanism of CRYAB function still needs further study.To investigate the regulatory mechanism of HSF1 under heat stress and recovery conditions in H9C2 cells by detecting the changes of HSF1 in transcription and expression level,in the distribution of cytoplasm and nucleus,and in trimerization.In addition,the effect of HSF1 interference on Hsps transcription in H9C2 cells following heat stress and recovery was also explored in this study.The results showed that HSF1 mainly distributed in the cytoplasm of H9C2 cells under normal conditions.When exposed to heat stress,the transcription level and protein expression of HSF1were significantly reduced in a time-dependent manner of heat stress,the distribution of HSF1 was transferred from cytoplasm to nucleus,and phosphorylation and trimerization occurred.Recovery for 12 h after 2 h heat stress,the transcription level and protein expression of HSF1 increase significantly,the distribution of HSF1 shifts from nucleus to cytoplasm,and the phosphorylation and trimerization disappeared.HSF1 interference resulted in increased transcription of hsp47 and hsp60 in H9C2 cells under normal conditions,but had no significant effect on other hsps.Except that,HSF1 interference significantly reduced the inductions in transcriptional levels of CRYAB,hsp27,hsp70,hsp90 and hsp110 induced by heat stress and heat stress recovery conditions.We explored the ability of vitamin C alone and in combination with equimolar sodium bicarbonate(Vitamin C-Na)to stimulate endogenous antioxidants and heat shock proteins(HSPs)to relieve heat stress in H9C2 cells.The stress damages and apoptosis of myocardial cells,and the parameter of LDH,MDA,SOD,ROS and the expression of Hsps were studied in this study after the control,vitamin C(20 ?g/mL vitamin C)and vitamin C-Na(20 ?g/mL vitamin C-Na)groups were heat-stressed for 0,1,3 or 5 h.The results showed that the granular and vacuolar degeneration,and damage to nuclei such as karyopyknosis and mitochondria were clearly reduced in treatment groups,as were apoptosis,lactate dehydrogenase activity and ROS and malondialdehyde levels,while superoxide dismutase activity was increased.Additionally,the levels of CRYAB,hsp27,hsp60 and hsp70 mRNA were upregulated at 3 h(P<0.01),and protein levels of CRYAB were increased at 0 h(P<0.05)and 1 h(P<0.01),and the levels of Hsp70 increased at 3 and 5 h of heat stress(p<0.01).The results indicated that the pretreatment with vitamin C or vitamin C-Na may protect H9C2 cells against heat damage by enhancing the antioxidant ability and upregulating CRYAB and Hsp70.In order to explore the function of VC and VC-Na in relieving heat stress injury in chicken cardiomyocytes,150 Isaac brown chicken(30 day old)were selected and were randomLy divided into three groups such as the control group(normal drinking water),VC group(50 ?g/mL VC in drinking water)and VC-Na group(50 ?g/mL VC-Na in drinking water).After 7 days of administration feeding,the selected chicken were suffered heat stress for 0 h,1 h,3 h,5 h,and 10 h,respectively,and then the serum and heart tissues were collected immediately at the time point heat stress.The protection of VC and VC-Na supplement on relieving chicken myocardial cells injury following heat was studied by using of detecting the levels of LDH,CK and CK-MB in serum and by using of histopathological lesion,by using of the detection of MDA,SOD,total antioxidant capacity,and the expression of CRYAB and Hsp70 in the myocardial cells.The results showed that the supplement of 50?g/mL VC or VC-Na can significantly reduce the levels of LDH,CK,CK-MB and MDA in serum,as well as can reduce the heat stress-induced granular and vacuolar degeneration,myocardial fiber breakage and cell necrosis,indicating effective resistance to heat stress damage.In addition,the supplement of 50?g/mL VC or VC-Na can significantly increase the total antioxidant capacity in serum.Meanwhile,the supplement of VC-Na also can induce the expression of CRYAB and Hsp70 in the myocardial cells under normal and heat stress conditions,the supplement of VC can induce the expression of CRYAB under normal and heat stress conditions and induce Hsp70 under normal condition.It is indicated that the supplement of VC or VC-Na may protect chicken myocardium cells against heat injury by enhancing antioxidant capacity and inducing expression of CRYAB and Hsp70.