Anti-Heat Stress Protection And Mechanism Of Co-Enzyme Q10 On Chicken Myocardial Cells By Inducing HSP70 Expression And Autophagy

Large scale or intensive farming and long distance transportation have inevitably appeared with the development of poultry breeding industry,which makes poultry more susceptible to heat stress.The growth rate,feed utilization efficiency and egg production of poultry could be reduced by heat stress,even the sudden death during high intensity of heat stress within a short time.As one of the most important organs,heart is much more sensitive to heat stress,and is easily resulted in heart failure and myocardial infarction because of heat stress.However,the body has the defense responsibility to heat stress by synthesizing a large number of heat shock proteins(HSP)for protecting himself.HSP70 is one of the members in heat shock protein family,it could bind to abnormal proteins and help them recover to normal condition or clear them directly,it could also perform anti-apoptotic function during heat stress.Heat shock factor(HSF)is a kind of factor which could bind to the heat shock element of heat shock proteins and trigger the synthesis of heat shock proteins via multimerization.Especially for poultry spices,HSF1 and HSF3 are considered the most important members among heat shock factor family.Co-enzyme Q10,is abundant in the heart which possess anti-oxidative function,has been used as an auxiliary drug for the treatment of heart diseases in human medicine.However,it is still unclear whether the co-enzyme Q10 could protect the heart of poultry under stress condition.Therefore.based on the establishment of in vitro and in vivo heat stress models,the protective function of the co-enzyme Q10 in the myocardial cells in vitro and in vivo during heat stress was studied in this paper.Because heat shock protein could protect cells from heat stress as an important molecular chaperones,the relationship between the synthesis and the mechanism of HSP70 induced by co-enzyme Q10,the relationship between the levels and the protective function of autophagy induced by co-enzyme Q10 during heat stress were also studied in this paper.1.Co-enzyme Q10 protected chicken primary myocardial cells from stress damages via inducing HSP70 expressionAfter established the cell stress model using chicken primary myocardial cells in vitro,Comparative analyses between cells in vitro pretreated with or without co-enzyme Q10 before heat stress were studied duration the different time point of heat stress(0h?5 h)by using of histopathological observation,detection of oxidative damage related enzymes and heart damage related enzymes,and flow cytometry.The results showed that the levels of CK-MB,LDH and MDA in myocardial cells pretreated with co-enzyme Q10 significantly decreased during heat stress,and the levels of T-SOD and T-AOC were upregulated during heat stress,compare to the cells with or without treatment of co-enzyme Q10.The results of the histopathological and the ultra-structural observation showed that the lesions caused by heat stress were alleviated by co-enzyme Q10.According to the results of flow cytometry and detection of cleaved caspase-3,cell apoptosis was significantly reduced when the myocardial cells were pretreated with co-enzyme Q10.It indicated that the co-enzyme Q10 pretreatment can protect the chicken primary myocardial cells from the stress damages induced by heat stress in vitro.We investigated the relationship between the protective function of co-enzyme Q10 and induction of HSP70 expression by using of western blot,detection of damage-related enzymes,pathological and cellular immunofluorescence,detection of apoptosis,RT-PCR.The results showed that co-enzyme Q10 significantly upregulated the expression of HSP70 in the chicken primary myocardial cells during heat stress compared with control group.The co-enzyme Q10 could increase the HSP70 signal intensity during heat stress and the stronger positive HSP70 signals were observed in nucleus after 5 h of heat stress.The induction of HSP70 and protective function of co-enzyme Q10 on chicken primary myocardial cells during heat stress were significantly weakened after treated with the specific inhibitor of HSP70,quercetin.According to the results of RT-PCR,western blot and immunofluorescence,the expressions of HSF1 and HSF3 were upregulated obviously after pretreated with co-enzyme Q10,and the nuclear translocation of HSF1 was also triggered by co-enzyme Q10.HSF1 binding activity to the heat shock element of HSP70 in chicken primary myocardial cells was upregulated after pretreated with co-enzyme Q10 but not HSF3.After HSF1 and HSF3 gene were silenced by siRNA respectively in chicken primary myocardial cells,the HSP70 expression induced by co-enzyme was obviously suppressed while only HSF1 gene was silenced.The above results indicated that co-enzyme Q10 can promote the expression of HSP70 and protect the chicken myocardial cells from stress damages via inducing the binding activity of HSP70 and HSF1 in vitro.To investigate the MAPK pathway is responsible for the regulation on HSF1 and HSP70 induced by co-enzyme Q10,the influence of co-enzyme Q10 on the MAPK pathway which is the upstream of HSF1 in the chicken primary myocardial cells were studied by using of western blot and the detection of cell apoptosis in this paper.The results showed that no significant change of total MAPK proteins(P38MAPK,JNK,ERK)was detected in the experimental groups except for total JNK1,and no obvious expression of JNK1 was induced by co-enzyme Q10 compared to HS group.According to the detection of the phosphorylation levels of the tested proteins,expression of p-P38MAPK,p-JNK and p-ERK1 were significantly upregulated by the pretreatment of co-enzyme Q10,and the induction of HSP70 expression induced by co-enzyme Q10 were obviously suppressed in the myocardial cells during heat stress after using SB203580 and SP600125 to inhibit the phosphorylation of P38MAPK and JNK respectively.Meanwhile the cellular apoptosis was also upregulated because of the addition of the inhibitor.As the important upstream proteins of JNK and P38MAPK,the phosphorylation levels of p-MEK3/6 and p-MEK4,but not p-MEK7,in the myocardial cells during heat stress were significantly upregulated by the pre-treatment of co-enzyme Q10.The phosphorylation levels of p-PKC? and p-PKC?1 was also elevated because of the addition of co-enzyme Q10.However,the expression of HSP70 induced by co-enzyme Q10 during heat stress because of the addition of PKC phosphorylation inhibitor,GF109203X.The above results indicated that co-enzyme Q10 can upregulate the expression of HSP70 during heat stress to protect chicken primary myocardial cells from heat stress damages via PKC-MEK3/4/6-P38MAPK/JNK pathways.2.Co-enzyme Q10 protected chicken primary myocardial cells from stress damages via inducing autophagyThe anti-stress damage induced by the autophagy in the primary chicken myocardial cells after co-enzyme Q10 pretreatment and its molecular mechanism were studied in this paper by using of the cell apoptosis detection,RT-PCR,western blot,ROS detection,electron microscopy observation,cellular immunofluorescent and MDC staining.The results showed that the cellular viability was increased and the cell apoptosis was decreased during heat stress after treated with different concentrations of co-enzyme Q10 before exposure to heat stress.Treatment with 20 ?M co-enzyme Q10 upregulated autophagy-associated genes during heat stress.The expression of LC3-? was highest increased in the myocardial cells pre-treated with 20 ?M co-enzyme Q10.Pretreatment with co-enzyme Q10 decreased reactive oxygen species(ROS)levels during heat stress.The number of autophagosomes was significantly increased by the dosage of 20 ?M co-enzyme Q10 treatment,as demonstrated by electron microscopy or monodansylcadaverine(MDC)fluorescence.SQSTM1 accumulation was diminished by co-enzyme Q10 treatment during heat stress which was contrary to LC3?,the number of LC3II puncta in chicken primary myocardial cells was increased.Treatment with 20 ?M co-enzyme Q10 also decreased the phosphorylation level of PI3K/Akt/mTOR pathway.The above results indicated that the pretreatment with 20 ?M co-enzyme Q10 can protect primary chicken myocardial cells by upregulating autophagy and suppressing the PI3K/Akt/mTOR pathway in the chicken myocardial cells in vivo during heat stress.3.Co-enzyme Q10 protected chicken hearts from stress damages via inducing HSP70 expressionTo further explore the possibility of using co-enzyme Q10 in poultry production,the protective function and HSP70 expression induced by co-enzyme Q10 in chicken heart in vivo was studied,by using ELISA detection,detection of damage related enzymes,pathological observation,western blot and ChIP assays.The results showed that co-enzyme Q10 exogenously added prior heat stress(1 mg/kg)was fully absorbed by the tested chickens and the level of co-enzyme Q10 in serum maintained at high levels.The levels of heart damage-associated enzymes in the serum revealed that the treatment with co-enzyme Q10 decreased the activity levels of CK-MB,CK,and LDH compared with only heat stressed chicken group.Oxidative damages were also alleviated by co-enzyme Q10 pretreatment according to the levels of SOD,MDA,and T-AOC in the serum of the tested chickens compared with only heat stressed chicken group during heat stress.Pathologically,the chicken hearts suffered serious damages including hemorrhage,granular degeneration,karyopyknosis,and cardiac muscle fiber disorder during heat stress,however,the extent of the heart damages was reduced by co-enzyme Q10 pretreatment.The addition of co-enzyme Q10 could upregulate the expression of HSP70 during heat stress compared with only heat stressed chicken group according to detection of protein expression.The addition of co-enzyme Q10 significantly increased the gene transcription level of HSF1 during heat stress,and HSF3 only at 5 h of heat stress.The transcription of HSF2 and HSF4 was not influenced by heat stress.Co-enzyme Q10 could only accelerate the trimerization of HSF1 as well binding activities to HSP70 HSE according to native page and ChIP assays.These findings suggested that co-enzyme Q10 can protect chicken hearts in vivo from heat stress by inducing HSF1 binding activity and HSP70 expression.The results we got in this part was consistent to the results of in vitro which further proved that co-enzyme Q10 could be used in poultry production practice to help resist heat stress related damage.