Preventive Effect And Mechanism Of Camel Whey Protein On Liver Injury Induced By Heat Stress

Heat stress(HS)is one of the most serious stress factors that endanger human and animal health in hot seasons and regions of the world.Liver injury is the most common and fatal complication in heat stress(HS)cases,and its mechanism involves oxidative stress and inflammatory responses.Developing safe,effective,and inexpensive nutritional additives to prevent HS has become a current research hotspot.A few studies have noted that camel whey protein(CWP)has stronger antioxidant and anti-inflammatory activities than bovine and other whey proteins,and thus it has anti-HS potential.However,whether and how dietary CWP can reduce HS-induced liver injury remains unclear.Therefore,the present study evaluated the ability of rats supplemented with CWP to resist HS-induced liver injury.The research contents are as follows:1.Model of liver injury induced by HS in Sprague-Dawley(SD)rats.HS status of the rats was assessed by using tail artery systolic blood pressure(SBP)and body core temperature(TC),and the degree of liver injury was assessed by histopathological changes in the liver and serum levels of alanine transaminase(ALT)and aspartate transaminase(AST).Interleukin1β(IL-1β)levels in rat serum and liver were measured by enzyme-linked immunosorbent assay(ELISA).The results showed that HS treatment raised rat SBP to a peak of 128.37 ± 2.04 mm Hg after 7 d HS treatment and declined after8 d HS treatment,and Tc increased to 43 ℃.Under this HS treatment,rat liver tissue sections were stained with hematoxylin and eosin(HE),showing a large number of hepatocytes with edema and necrosis,meanwhile,rat serum ALT and AST activities as well as serum and liver IL-1β levels were elevated,indicating that the rat liver had undergone an inflammatory response and injury.Ultimately,the HS procedure established for this study was: rats were placed in an artificial climate chamber(set temperature of 40 ± 0.2 ℃ and relative humidity of 60%-65%)for 2 h per day for 8days.2.The preventive effect of CWP on HS-induced liver injury was evaluated.Six week old SD rats were gavaged daily with 100,200,and 400 mg/kg of CWP.After 2weeks,HS treatment was performed according to the procedure described above at 1 h after each CWP gavage.At the end of the last HS treatment and after 24 h recovery in the standard environment,blood and liver samples were collected.HE staining was applied to evaluate the effects of CWP on histopathological changes in the liver of HS treated rats.Rat serum ALT activity was measured using a commercial kit.IL-1β in rat serum and liver was measured by ELISA assay.Hepatocyte apoptosis was analyzed with a TUNEL staining kit.Rats underwent the entire HS procedure,and Tc was recorded with a thermometer every 3 h.The results showed that CWP alleviated HS-induced histopathological changes and apoptosis in rats in a dose-dependent manner,inhibited the increase of serum ALT activity and IL-1β levels,and promoted the recovery of Tc after HS treatment.These results suggest that CWP has preventive effects against HS-induced liver injury in rats.3.The preventive mechanism of CWP on hepatic inflammatory injury induced by HS in rats was investigated.NLR pyrin domain containing 3(NLRP3)inflammasome plays a critical role in mediating heat stress-induced liver injury.Because of this,the present study evaluated the ability of CWP to inhibit hepatic NLRP3 inflammasome activation in a HS rat model.The mechanism of action of CWP was confirmed by intraperitoneal injection of high mobility group box 1(HMGB1)antagonist or NLRP3 inhibitor before HS treatment.Histological sections were prepared,and the histopathological changes of rat liver suffered HS were observed after HE staining.IL-1βcontent,ALT activity,and Caspase-1 activity were measured using kits.HMGB1,receptor for advanced glycation end products(RAGE),NLRP3 inflammasome,Caspase-3,and Bcl-2 protein expression in rat liver was determined by immunohistochemistry or western blotting assay.We demonstrated that HS activated the NLRP3 inflammasome in rat liver,which was evidenced by increased Caspase-1 activity and IL-1β level.Inhibition of NLRP3 inflammasome activity reversed the HS induced aberrant expression and nuclear translocation of Bcl-2 and Caspase-3 proteins in rat liver,thus alleviating hepatocyte apoptosis,necrosis,and liver injury.The enhanced NLRP3 inflammasome activity by HS was dependent on the cytoplasmic translocation of HMGB1.Glycyrrhizic acid,a specific antagonist of HMGB1,which is also a commonly used anti-inflammatory agent,inhibited HS-induced NLRP3 inflammasome activation in rat liver.And notably,CWP reversed the abnormal expression of HMGB1,RAGE,NLRP3 inflammasome,IL-1β,ALT,Bcl-2 and Caspase-3 in the liver of rats subjected to HS and inhibited Caspase-1 activity,thereby alleviating hepatocyte apoptosis and liver histological changes.The combined application of CWP with glycyrrhizic acid completely blocked rat hepatocyte apoptosis and injury caused by HS.These results demonstrated that CWP alleviated HS-induced liver injury in rats by suppressing the HMGB1/RAGE/NLRP3 inflammasome signaling pathway.4.The mechanism of CWP inhibiting HMGB1 release was evaluated.First,the use of the reactive oxygen species(ROS)inhibitor NAC confirmed that oxidative stress is the key inducing HMGB1 release.Second,the antioxidant capacity of CWP was evaluated.The results showed that CWP suppressed HS-induced hepatic oxidative stress damage by decreasing ROS and malondialdehyde(MDA)levels,while enhancing GSH/GSSG and NADPH/NADP+ ratios and superoxide dismutase(SOD),glutamylcysteine ligase(GCL),heme oxygenase(HO-1),catalase(CAT),and glutathione peroxidase(GSH-Px)expression.And notably,CWP could alleviate the liver injury by inhibiting the cytoplasmic translocation of HMGB1 via activating the Nrf2/HO-1 signaling pathway,as Nrf2 inhibitor or HO-1 inhibitor could inhibit the activation of CWP on above pathway.Meanwhile,CWP also enhanced the phosphorylation level of serine/threonine kinase(Akt),while LY294002(a specific PI3 K inhibitor)blocks the activation of Nrf2 pathway.Thus,CWP could prevent HS-induced liver injury by activating the PI3K/Akt/Nrf2/HO-1pathway.CWP combined with NAC could better eliminate the adverse effects of HS on liver.In summary,CWP has a good preventive effect on HS-induced liver injury in rats,and its possible mechanism is associated with the activation of PI3K/Akt/Nrf2/HO-1pathway to enhance hepatic antioxidant capacity,and the inhibition of HMGB1/RAGE axis mediated NLRP3 inflammasome activation.