Influence Of Adenovirus-mediated HSP70on Release Of Multiple Cytokines And Stress Hormones, And Expression Of Their Receptors In Cold Stressed Rats

Cold stress is normal stressor in cold region which is generally considered to suppress theimmune system, and cause increasing of morbidity and mortality in livestock. It is huge losses onstockbreeding in the cold region. Therefore, it has great significance to study the mechanism oflow immunity which could pay a potential role in prevention on animal cold stress. Researchshows that the heat shock protein70(HSP70) could play a role for repair function in damagedcells, and it is strong correlation between the HSP70and the secretion of immune-related factors.Therefore, in the present study, take the SPF Wistar rats as the study object which was12weeks of age. The recombinant adenovirus mediated HSP70was conducted by the recombinantDNA technology. High-titer stocks of recombinant adenovirus mediated HSP70were purified byCsCl density centrifugation. According to the different titer of recombinant adenovirus andexposure time for vivo transfection, recombinant adenovirus mediated HSP70(pAd-CMV-HSP70)was injected via the tail vein for vivo transfection. The optimal titer and the optimal exposuretime for vivo transfection were screened by qRT-PCR and western blotting technology.The270rats were divided randomly into blank groups, pAd-CMV-HSP70transfected groupsand pAd-CMV-Null transfected groups. The above-mentioned groups were divided into normaltemperature groups, acute cold stress groups and chronic cold stress groups. The acute cold stressgroups was divided into acute cold stress6h experiment group, acute cold stress12h experimentgroup and acute cold stress24h experiment group. The chronic cold stress groups was dividedinto chronic cold stress6d experiment group, chronic cold stress9d experiment group andchronic cold stress12d experiment group. The normal temperature of blank groups was set ascontrol group. Each group had10rats. According to the optimal titer and the optimal exposuretime, pAd-CMV-HSP70and pAd-CMV-Null was transfected into rats, then to carry out the coldstress experiment. The cold stress experiment was carried on intelligent climate room. Theenvironment in the climatic room was maintained at a temperature of24±0.05℃, relativehumidity45±0.1%. The cold stress temperature was maintained at4±0.05℃. After the treatments, rats are anesthetized by75mg/kg sodium pentobarbital.6~8mL blood was taken fromthe anesthetized rats by entering right ventricule from their hearts. The blood was to remove serumand lymphocyte. The concentration of cytokines (IL-1β, IL-6, TNF-α, IL-2, IFN-γ, IL-4, IL-10and IP-10) were detected by Luminex multiplexed microsphere-based flow assay. Theconcentration of stress hormone (ACTH and corticosterone) was detected by ELISA technology.The relative expressed levels of HSP70, ACTHR and GR was determined by qRT-PCR andwestern blotting technology in the blood lymphocytes of rats. The results were showed asfollowing:1Construction of recombinant adenovirus mediated HSP70on wistar rats and virus preparationThe recombinant adenovirus vector was constructed successfully by the recombinant DNAtechnology. High-titer stocks of recombinant adenovirus were obtained. western blotting resultsshowed that the expression levels of HSP70increased significantly after recombinant adenovirusmediated HSP70transfected into BRL cells, and the HSP70could have good immunogenicity.2The expression levels of HSP70in different tissue of rats after transfected withpAd-CMV-HSP70The optimal titer and the optimal exposure time for vivo transfection were screened byqRT-PCR and western blotting technology. Determine the2.5×109pfu/kg was the optimal titer,and4d was the optimal exposure time for vivo transfection. According to the optimal titer and theoptimal exposure time transfected the recombinant adenovirus mediated HSP70into rats, HSP70was over-expressed on liver and spleen compared with control group(p＜0.01).3Influence of adenovirus-mediated HSP70on release of pro-inflammatory cytokines (IL-1β,IL-6and TNF-α) in serum of cold stress ratsThe TNF-α concentration increased significantly compared with control group in blankgroups and pAd-CMV-Null transfected groups in the duration of acute cold stress(p＜0.05), andthe TNF-α concentration did not find significant difference on the duration of cold stress inpAd-CMV-HSP70transfected groups (p＞0.05). The IL-6concentration increased significantlycompared with control group in different duration of cold stress in the blank groups andpAd-CMV-Null transfected groups (p＜0.05). In the pAd-CMV-HSP70transfected groups, theIL-6concentration significantly increased in acute cold stress6h (p＜0.05), and the other durationof cold stress had no significant difference. In the blank groups, the IL-1β concentration did notfind significant difference in the different duration of cold stress (p＞0.05). In the pAd-CMV-HSP70transfected groups and pAd-CMV-Null transfected groups, the IL-1βconcentration were higher significantly in the different duration of cold stress (p＜0.05).4Influence of adenovirus-mediated HSP70on release of Th1cytokines (IL-2, IFN-γ) in serumof cold stress ratsIn the blank groups and pAd-CMV-Null transfected groups, the IL-2concentrationsignificantly increased in acute cold stress6h and12h (p＜0.05). In the pAd-CMV-HSP70transfected groups, the level of IL-2enhanced significantly compared with control group under thenormal temperature and the duration of chronic cold stress (p＜0.05). In the blank groups andpAd-CMV-Null transfected groups, the IFN-γ concentration was enhanced significantly comparedwith control group in the acute cold stress24h and chronic cold stress9d,12d (p＜0.05). In thepAd-CMV-HSP70transfected groups, the IFN-γ concentration had no significant differencecompared with control group in the duration of cold stress (p＞0.05).5Influence of adenovirus-mediated HSP70on release of Th2cytokines (IL-4, IL-10) in serumof cold stress ratsAmong the blank groups, the pAd-CMV-Null transfected groups and the pAd-CMV-HSP70transfected groups, the IL-4concentration had no significant difference in the duration of coldstress (p＞0.05). In the blank groups, the IL-10concentration had no significant difference in theduration of cold stress in the each experimental group (p＞0.05). In the blank groups,pAd-CMV-Null transfected groups and pAd-CMV-HSP70transfected groups, the IL-10concentration enhanced significantly in the different duration of cold stress (p＜0.05).6Influence of adenovirus-mediated HSP70on release of chemokine IP-10in serum of coldstress ratsComparing with control group, the IP-10concentration was significantly higher in theduration of chronic cold stress and acute cold stress24h in the blank groups (p＜0.05). In thepAd-CMV-Null transfected groups, the IP-10concentration had no significant differencecompared with control group in the chronic cold stress6d (p＞0.05), and the trend of IP-10weresimilar with the blank groups in the other duration of cold stress. In the pAd-CMV-HSP70transfected groups, the IP-10concentration did not find significant difference compared withcontrol group in the duration of cold stress (p＞0.05).7Influence of adenovirus-mediated HSP70on release of ACTH and corticosterone in serum ofcold stress rats Comparing the control group, the ACTH concentration was significantly higher in the acutecold stress6h,12h in the blank groups (p＜0.05), and the ACTH concentration was significantlylower in the duration of chronic cold stress in the blank groups (p＜0.05). In the pAd-CMV-Nulltransfected groups and pAd-CMV-HSP70transfected groups, the ACTH concentration increasedsignificantly compared with control group in the duration of acute cold stress. The trend of ACTHwas similar between pAd-CMV-Null transfected groups and pAd-CMV-HSP70transfected groupsin the duration of chronic cold stress. In the blank groups, pAd-CMV-Null transfected groups andpAd-CMV-HSP70transfected groups, the corticosterone concentration increased significantlycompared with control group in the different duration of cold stress except the chronic cold stress12d in the blank groups. The corticosterone concentration was lower in the pAd-CMV-HSP70transfected groups compared with the blank groups and the pAd-CMV-Null transfected groups inthe acute cold stress6h,12h (p＜0.05), and there were not significant difference between thepAd-CMV-Null transfected groups and the blank groups (p＞0.05).8Influence of adenovirus-mediated HSP70on expression of HSP70, ACTHR, GR in the bloodlymphocytes of ratsIn the acute cold stress6h and24h, the expression levels of HSP70protein in thepAd-CMV-HSP70transfected groups were reduced compared with the blank groups and thepAd-CMV-Null transfected groups (p＜0.05). In the pAd-CMV-HSP70transfected groups, theexpression levels of ACTHR protein were higher compared with the blank groups in differentduration of cold stress (p＜0.05). In the duration of acute cold stress, the expression levels of GRprotein were higher in the pAd-CMV-HSP70transfected groups compared with the blank groupsand pAd-CMV-Null transfected groups (p＜0.05).In conclusions, the recombinant adenovirus mediated HSP70over-expressed on liver andspleen could improve the protection of acute cold stress in the rats. It mainly via the follow ways:the recombinant adenovirus mediated HSP70could induce down-regulation of pro-inflammatorycytokines TNF-α and IL-6to suppress the occurrence of inflammatory response caused by coldstress in the rats. And stimulate the secretion of IL-2in the serum of cold stress rats to activate thecellular immunity. Moreover, the recombinant adenovirus mediated HSP70could cause a decreaseof ACTH and corticosterone in cold stress rats to reduce the levels of the stress response.