Effects Of Adenovirus-mediated HSP70 On Related Stress Hormone And CKs In Rats Of Cold Stress

In order to discuss the influence of the related inflammatory cytokines upon response of HSP70 in the cold stress,and the interrelationship between HSP70,Inflammatory Cytokines and hormones.In this study,we injected the rat HSP70 recombinant adenovirus mediated expression vector(pAd-CMV-HSP70)into the vein of rat's tail,and established the rat model of HSP70 overexpression.According to the different titer of recombinant adenovirus and exposure time for vivo transfection,The optimal titer and the optimal exposure time for vivo transfection were screened by Western blotting technology.After success of the appraisal model begainning acute or chronic cold stress experiment,take the SPF Wistar rats as the study object which was 12 weeks of age and sex was male,the 270 rats were divided randomly into three groups:(1)pAd-CMV-HSP70 transfected groups;(2)pAd-CMV-Null transfected groups;(3)blank groups.The above-mentioned groups were divided into normal temperature groups,acute cold stress groups and chronic cold stress groups.The acute cold stress groups was divided into acute cold stress 3h experiment group,acute cold stress 6h experiment group,acute cold stress 12 h experiment group and acute cold stress 24 h experiment group.The chronic cold stress groups was divided into chronic cold stress 3d experiment group,chronic cold stress 6d experiment group,chronic cold stress 9d experiment group and chronic cold stress 12 d experiment group.The normal temperature of blank groups was set as control group.Each group had 10 rats.According to the optimal titer and the optimal exposure time,pAd-CMV-HSP70 and pAd-CMV-Null was transfected into rats,then to carry out the cold stress experiment.The environment in the climatic room was maintained at a temperature of 24 ± 0.05?.The cold stress temperature was maintained at 4 ± 0.05?.After the treatments,rats are anesthetised by 75mg/kg sodium pentobarbital.5mL blood was taken from the anesthesized rats by entering right ventricule from their hearts.The blood was to remove serum.The concentration of cytokines(IL-6,TNF-?,IL-2,IL-4)were detected by Luminex multiplexed microsphere-based flow assay.The concentration of stress hormone(ACTH and corticosterone)was detected by ELISA technology.The experiment results showed as follows:(1)The optimal titer and the optimal exposure time for vivo transfection were screened by Western blotting technology.Determine the 2.5×109pfu/kg was the optimal titer,and 4d was the optimal exposure time for vivo transfection.According to the optimal titer and the optimal exposure time transfected the recombinant adenovirus mediated HSP70 into rats,HSP70 was overexpressed on liver and spleen compared with control group(p<0.05).(2)The TNF-? concentration increased significantly compared with control group in serum of acute cold stress rats in blank group and pAd-CMV-Null transfected group(p<0.05).The TNF-? concentration did not find difference significantly on the duration of cold stress in pAd-CMV-HSP70 transfected groups.The TNF-? concentration did not find difference significantly in the duration of chronic cold stress in the each experimental group.The IL-6 concentration increased significantly compared with control group in different duration of cold stress in the blank group and pAd-CMV-Null transfected groups(p<0.05).In the pAd-CMV-HSP70 transfected groups,the IL-6 concentration significantly increased in chronic cold stress 3d(p<0.05),and the other duration of cold stress had no significant difference.(3)In the blank groups,the IL-2 concentration significantly increased in acute cold stress 3h,6h and 12h(p<0.05).In the pAd-CMV-HSP70 transfected groups,the level of IL-2 enhanced significantly compared with control group in the normal temperature and the duration of chronic cold stress(p<0.05).(4)Comparing the control group,the ACTH concentration was significantly higher in the acute cold stress 3h,6h,12 h in the blank groups(p<0.05),and the ACTH concentration was significantly lower in the duration of chronic cold stress in the blank groups(p<0.05).In the pAd-CMV-Null transfected groups and pAd-CMV-HSP70 transfected groups,the ACTH concentration increased significantly compared with control group in the duration of acute cold stress,pAd-CMV-HSP70 transfected group,ACTH concentration was significantly lower than blank groups and pAd-CMV-Null transfected groups(p<0.05).The trend of ACTH was similar between pAd-CMV-Null transfected groups and pAd-CMV-HSP70 transfected groups in the duration of chronic cold stress.In the blank groups,pAd-CMV-Null transfected groups and pAd-CMV-HSP70 transfected groups,the corticosterone concentration increased significantly compared with control group in the different duration of cold stress except the acute cold stress 3h and the chronic cold stress 24 h in the blank groups.The corticosterone concentration was lower in the pAd-CMV-HSP70 transfected groups compared with the blank groups and the pAd-CMV-Null transfected groups in the acute cold stress 6h,12 h,and there were significant difference between the pAd-CMV-HSP70 transfected groups and the blank groups(p<0.05).Conclusions: the results suggested that the recombinant adenovirus mediated HSP70 overexpressed on liver and spleen could improve the protection of cold stress in the rats.It mainly via the follow ways: the recombinant adenovirus mediated HSP70 could induce downregulation of inflammatory cytokines TNF-? and IL-6 to suppression the occurrence of inflammatory response caused by cold stress in the rats.And stimulate the secretion of IL-2 in the serum of cold stress rats to activate the cellular immunity.Moreover,the recombinant adenovirus mediated HSP70 could cause a decrease of ACTH and corticosterone in cold stress rats to reduce the level of the stress response.