Effect Of Heat-Shock Protein B7 On Oxidative Stress In Adipose Tissue Of Ketotic Dairy Cows

Perinatal dairy cows often display a state of negative energy balance induced by a low intake of dry matter and an increase in demands for energy.Excessive fatty acids absorbed by liver from plasma are metabolized into ketone bodies in hepatocytes,thereby inducing ketosis.Dairy cows with ketosis display systemic oxidative stress.However,the redox state of adipose tissue in ketotic cows remains unclear.Heat shock proteins（HSPs）are a kind of proteins that can resist stress when the body is stimulated by chemical,physical and biological factors.In recent years,heat shock protein B7（HSPB7）has been found to play an important role in anti-oxidation and protection of body damage.However,whether HSPB7 plays a role in adipose tissue of ketotic dairy cow remains unclear.In addition,the nuclear factor E2-related factor 2（NFE2L2）signaling pathway is a key mechanism of protection of the cell against oxidative stress.Whether HSPB7 is associated with NFE2L2 signaling pathway remains unclear.Therefore,in this study,adipose tissue of clinical ketosis dairy cows and healthy dairy cows were collected in vivo to detect redox state and expression of HSPB7 and NFE2L2.The effects of oxidative stress on HSPB7,NFE2L2 and its downstream target genes,heme oxygenase-1（HMOX1）and NADH quinone oxidoreductase-1（NQO1）,were studied by isolating and culturing calf adipocytes in vitro.Adenovirus overexpressing HSPB7 and small interfering RNA silencing HSPB7 were transfected to study the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes.An in vivo study consisting of healthy and clinically ketotic cows was performed to detect redox state and expression of HSPB7 and NFE2L2.The results showed that the malondialdehyde（MDA）content was greater but the activity of glutathione peroxidase（GSH-Px）and superoxide dismutase（SOD）lower in adipose tissue of clinically ketotic cows,suggesting oxidative stress exists in adipose tissue of ketosis dairy cows.At the same time,the expression of HSPB7 and NFE2L2 was measured,the results showed that the mRNA and protein level of HSPB7 and NFE2L2 in adipose tissue of dairy cows with ketosis was higher than that of healthy dairy cows.These results indicated that HSPB7 played a role in adipose tissue of dairy cows with ketosis and the NFE2L2 signaling pathway was activated.Calf adipocytes were cultured in vitro and treated with different concentrations of H₂O₂（0,25,50,100,200 and 400μM）for 2 hours.The results showed that H₂O₂treatment induced the overproduction of reactive oxygen species（ROS）and MDA,and inhibited the activity of antioxidant enzymes GSH-Px and SOD in adipocytes.Furthermore,25,50,100μM H2O2 increased the abundance of HSPB7,NFE2L2,HMOX1 and NQO1,but 200 or 400μM H₂O₂ reduced the abundance of HSPB7,NFE2L2,HMOX1 and NQO1.Calf adipocytes were cultured in vitro and transfected with adenovirus（Ad-HSPB7）overexpressing HSPB7 for 48 hours,and treated with 200μM H2O2 for2 hours.The results showed that overexpression of HSPB7 attenuated the effect of H₂O₂ on the expression of NFE2L2,HMOX1 and NQO1.Furthermore,HSPB7overexpression increased SOD and GSH-Px activity but decreased MDA and ROS production,which improved the H₂O₂-induced oxidative stress.The adipocytes were transfected the small interfering RNA（si-HSPB7）for 48 hours,and 200μM H2O2was added for 2 hours.The results showed that HSPB7 knockdown exacerbated the downregulation of NFE2L2,HMOX1 and NQO1 in H₂O₂-treated adipocytes.Furthermore,HSPB7 knockdown exacerbated the H₂O₂-induced decreased SOD and GSH-Px activity,and MDA and ROS production,suggesting that silencing HSPB7could aggravate oxidative stress in calf adipocytes.Our results indicated that the oxidative stress in adipose tissue of clinically ketotic cows was significantly increased.Abundance of HSPB7 and NFE2L2 was greater in adipose tissue of clinically ketotic cows.Activation of HSPB7 activates NFE2L2 pathway,thereby alleviating oxidative stress in calf adipocytes.