The Mechanism Of Mitophagy-mediated Hepatic Injury Induced By Exertional Heat Stroke

Objective: Exertional heat stroke(EHS),because of its high mortality and morbidity,has attached great importance to the people,EHS prevention and control has gradually become the focus of attention.EHS secondary to multiple organ system dysfunction(MODS),the liver function damage,the most serious damage to the central nervous system(CNS)and can affect long-term prognosis and quality of life of patients.Current studies have found that early heat exposure or regular heat acclimatization training can significantly reduce the incidence of EHS.But the exact mechanism is not yet clear.Therefore,this study aims at the mechanism of EHS induced liver injury and Heat Acclimation Training(HAT)that can significantly improve the prognosis of.EHS patients,and preliminary exploration of its potential mechanisms.Methods: To construct an animal model of exertional heat stroke in rats: 65 healthy male S/D rats aged 8 weeks,weighing 200-220 g,and randomly divided into blank group(Con group),Exertional Heat Stroke group(EHS group),and Heat Acclimatization group(HA group).All rats started the experiment after running through the gradient intensity adaptive treadmill.Among them,each rat in the Con group was raised normally after passing through the adaptive treadmill and no longer treadmill.After the HA group went through the adaptive treadmill,the short-term thermal shock training was started at an interval of 48 hours,totaling 3 times,and the formal experiment was started at an interval of 48 hours.Rats in the EHS group and the HA group were randomly selected according to 2h,6h,24 h,48h,72 h after the onset of onset,and blood samples were collected through the abdominal aorta after anesthesia,and the liver and other internal organs were collected for later use.In the experiment,the left liver was retained and then fixed with 10% neutral formalin fixative for pathological experiments;most of the right liver lobe was quickly frozen for molecular biology determination.Use HE staining to observe liver morphology,AB-PAS staining to observe liver glycogen metabolism,TUNEL staining to detect rat liver cell apoptosis,and immunohistochemical(IHC)staining to detect in situ expression of CD34 and Parkin protein in rat liver,western blotting was used to detect the expression of Parkin/ PINK-1,Bcl-2/Bax,HIF-1a,v WF,CD31 and other proteins.Results: 1.Analysis of modeling time and survival rate: All rats in the Con group,EHS group,and HA group passed adaptive treadmill training,and none of the rats died due to adaptive training.The 72-hour survival rate of rats in the Con group was 100%;84.1% in the EHS group(Log-Rank P >0.05),of which 2/44 died within 1 hour after the termination of the experiment due to acute pulmonary edema;93.3% in the HA group and there was no significant difference compared with the Con group and EHS group(Log-Rank P >0.05).2.Pathological results: 2.1 HE: Compared with the Con group,hepatocyte swelling can be observed in the EHS and HA groups at 2h and 6h,and the swelling of each subgroup of the EHS group is more obvious,accompanied by hepatic sinusoidal congestion,and the HA group has hepatocyte swelling Compared with the EHS group,the EHS group and the HA group 2h,6h subgroups compared with the Con group,the cytoplasm of adjacent liver lobules stained lightly,it can be concluded that the above four groups of hepatocellular edema is obvious;There were vacuoles around the nucleus in the 24 h subgroup of the HA group,with less inflammatory cell infiltration,mainly at the junction of adjacent lobules;the 24 h subgroup of the EHS group had fewer perinuclear vacuoles than the HA group,but the inflammatory cell infiltration was obvious.After 48 hours,the vacuolar degeneration of hepatocytes in the EHS group and HA group could be observed almost spread to the whole lobule. 2.2 AB-PAS : Compared with the Con group,the intracellular glycogen was stained purple-red in all subgroups of the EHS group,especially at 2h and 72h;in the HA group,except for the 72 h subgroup,the rest of the subgroups The glycogen content in the liver cells of the rats was lower than that of the EHS group.It can be speculated that after the onset of exertional heat stroke,the glycogen metabolism(gluconeogenesis pathway)in the rat liver may be inhibited.2.3 Immunohistochemical staining: Compared with the Con group,in each subgroup of the EHS group,the number of Parkin expression-positive cells at 6 h and 48 h were significantly reduced(P <0.05).There was no significant difference between the HA group and the Con group(P> 0.05).Compared with the EHS group,the expression level of Parkin in the 24 h subgroup of the HA group was significantly increased(P <0.05),which shows that the ubiquitin-dependent autophagy pathway in EHS is activated 3.Changes in liver cell apoptosis: 3.1 TUNLE: The number of hepatocyte apoptosis in the EHS group and HA group increased significantly within 6 hours(P <0.05),and the number of hepatocyte apoptosis in the EHS group was significantly higher than that in the HA group within 2 hours(P <0.05).This shows that HAT has a protective effect on the liver of EHS rats.3.2 Apoptosis-related proteins: Bcl-2/ Bax and cytochrome C(Cyto-c)protein level detection: the expression of Bax in the 24 h subgroup of the EHS group was significantly lower than that of the Con group(P <0.05),and the expression of the 72 h subgroup in the HA group.The amount was significantly lower than the Con group(P <0.05).There was no statistically significant difference in Bcl-2 expression.The p65 in the 6h,24 h and 48 h subgroups of the HA group was significantly lower than that of the Con group(P<0.05).The Cyto-c content in the 24 h,48h,72 h subgroups of the HA group was lower than that of the Con group,and the difference was statistically significant(P<0.05).The above conclusions suggest that HAT can reduce the number of hepatocyte apoptosis caused by EHS by regulating the process of autophagy.4.Injury and repair of liver vascular endothelium: 4.1 Immunohistochemical staining: The number of CD34 positive cells in the liver of the HA group was significantly more than that of the Con group and EHS group(P <0.05),suggesting that HAT can accelerate the repair of vascular endothelial damage.4.2 Detection of endothelial damage repair marker protein(CD31,v WF)levels: The liver expression of v WF protein in HA group was significantly lower than that in Con group(P <0.05),suggesting that HAT can accelerate vascular repair and reduce the risk of thrombosis;there is no statistics on CD31 expression between groups The scientific difference indicates that CD31 cannot be used as an indicator of vascular endothelial repair in EHS.5.PINK1/Parkin-HIF-1a channel activation status: Detection of PINK1,Parkin,and HIF-1a pathway protein levels: The expression level of HIF-1a in the 6h to 72 h subgroup of the EHS group showed a progressive decrease(P <0.05),and the HA group did not significantly decrease(P >0.05);HA group The level of Parkin in the subgroup from 2h to 72 h was lower than that of the Con group(P<0.05),and there was no significant difference in the PINK1 level between any two groups(P>0.05).Conclusion: Liver dysfunction is very common in the EHS,which is closely related to the patient in the long-term,currently believed to reduce exertional heat stroke incidence by HAT,but the exact mechanism is unknown.This study found that,by activating the HAT PINK1 / Parkin mitophagy pathway regulated process,and is expressed by the target route control of HIF-1a,increase in liver perfusion low tolerance,and can accelerate the rat liver endothelial EHS Damage repair process can also indirectly inhibit the activation of inflammatory response.PINK1/Parkin-HIF-1a pathway can play a hepatoprotective effect by mediating the process of liver mitochondrial autophagy.