Protective Effects And Mechanisms Of Sheng Mai San Against Heat Stress Induced Liver Injury In Rats

As global warming worsens,extreme high temperature is becoming an increasing threat not only for human health worldwide but also for livestock production.Liver damage is one of the most common complications in humans and animals after heat stress（HS）.Sheng Mai San（SMS）,a traditional Chinese medicine prescription,exert a therapeutic effect on HS.However,how SMS prevents liver injury after heat exposure remains unknown.Thus,in this study,SMS as object,the model of HS-induced liver injury was built in vivo and in vitro,and the water extract of SMS was utilized to treat it.We systematically investigated the pharmacological effect and molecular mechanisms of SMS on HS-induced liver injury from an energy metabolism and mitochondria function homeostasis perspective.Overall,the principal aim of the study was to explore the role and regulatory mechanism of SMS in the response to liver damage after heat exposure and provide theoretical basis for future applications.The main methods and results were as follows:1.The main active component determination and antioxidant capacity evaluation in vitro of SMS.The total polysaccharides,total flavonoids,and total saponins content were determined based on the method of Phenol-sulfuric acid,Na NO2-Al Cl₃ and Vanilla aldehyde-sulfuric acid in SMS.The results indicated that the content ratio of total polysaccharides,total flavonoids,and total saponins were respectively57.41%,0.42%and 10.98%in SMS decoction powder.The main chemical components of SMS were characterized by HPLC for quality control.The methodological examination was evaluated through linear relationship,precision,stability,reproducibility and add sample collection experiments,etc.The results showed that the detection method was accurate and reliable.The concentration of six major compounds（ginsenoside Rb1,ginsenoside Rh1,ginsenoside Rg3,ophiopogonin D,ruscogenin and schisandrin A）in SMS decoction powder,were 76.67±1.44μg/g,461.10±8.46μg/g,298.35±1.48μg/g,52.75±9.36μg/g,134.79±2.42μg/g and 248.32±1.21,respectively.The antioxidant capacity in vitro of SMS was evaluating using different methods（DPPH,ABTS,OH radicals and reducing power）.The result showed that the IC₅₀ of the scavenging activities of DPPH,ABTS and OH radicals,were respectively3.280 mg/m L,7.503 mg/m L and 4.706 mg/m L.For the reducing power of SMS decoction powder,the slope was 0.0007179 using iron potassium ferricyanide reduction method,and the total antioxidant capacities were 0.0596 mmol Fe SO₄ per gram SMS decoction powder using FRAP assay.2.HS induces liver dysfunction and oxidative stress in different recovery stages in vivo and in vitro.For in vivo experiment,to induce HS,the animals were placed in a prewarmed incubator that was exposed to HS（38±1℃,75±5%relative humidity,2 h/day）for 7 consecutive days.For in vitro experiment,the BRL-3A cells were subjected to HS in an incubator,in which the desired temperature 43℃was maintained,for 2h.After heat exposure,the treated rats and cells were then allowed to recover for 0 h,3 h,6 h,9 h,and 12 h in conventional environment or 37℃.The result indicated the serum ALT and AST concentrations and the De Ritis ratio（AST/ALT ratio）as measure for liver damage severity were markedly increased after heat exposure and progressively increased in a time-dependent manner（P<0.05）.More obvious extensive hepatocyte edemas,ballooning degeneration,inflammatory infiltration and diffuse hemorrhage occurred at 6～12 h after heat exposure.Elevated levels of 8-OHDG and MDA were observed at 6～12 h after heat exposure,but the CAT level was significantly decreased at 6 h（P<0.05）.The cell viabilities of BRL-3A were significantly decreased after heat exposure in a time-dependent manner,and closely48.54%at 9 h（P<0.05）.The change of ROS activities was similar with the cell viabilities,was obviously increased（P<0.05）.3.The therapeutic effects of SMS on HS-induced liver injury in vivo and in vitro.The high（SMS-H,5.04 g/kg）,medium（SMS-M,2.52 g/kg）and low（SMS-L,1.26 g/kg）doses were administrated orally intervened HS-induced rat model,and NAC（150 mg/kg）was used as a positive control.The result showed that SMS could significantly decreased the serum ALT,AST,and De Ritis ratio（AST/ALT ratio）and repaired liver injury in HS-induced rat model（P<0.05）.The imbalance of antioxidant defense system was improved after SMS intervened,the levels of SOD,CAT and GSH were significantly increased,while the MDA content reduced（P<0.05）.The administration of SMS to HS rats significantly decreased the level of MPO in serum and liver tissue,and downregulated the m RNA expression of IL-1β,IL-6,TNF-αand IL-10（P<0.05）.Meanwhile,the overexpression of HSP m RNA（including HSF-1,HSP27,HSP90 and HSP70）was reversed to maintain HSP homeostasis（P<0.05）.In vitro,SMS could upregulate the BRL-3A cell viabilities after heat exposure,and inhibit the ROS accumulation（P<0.05）.4.The energy metabolism change of SMS on HS-induced rat liver.The 40 key metabolites involved in the TCA cycle,glycolytic pathway,oxidative phosphorylation and corresponding cofactors were detected simultaneously by UPLC-MS/MS.Meanwhile,the expression of rate-limiting enzymes was quantitatively detected to investigate the therapeutic effect of SMS on HS-induced energy disorder in the liver using RT-q PCR.The result showed that HS leads to severe energy crisis,which has a close relationship with glycolysis and TCA cycle.The content of fructose 6-phosphate,pyruvate and glucose-6-phosphate,were significantly changed in HS rat liver,whereas SMS could improve the abnormal situation,meanwhile the isocitrate,succinate,and fructose 6-phosphate levels were significantly increased in SMS-treated HS-induced rats（P<0.05）.After SMS intervened,the expression of Pklr,Pgk1,Pfk1,Gck m RNA of the rate-limiting enzymes were significantly upregulated in glycolysis pathway（P<0.05）.Meanwhile the key enzymes m RNA expressions were also increased in TCA cycle,including Cs,Idh3a,Ogdh,Fh,Sdhb and Mdh2,but the Ldha m RNA expression level was decreased（P<0.05）.The PAS staining result showed that the glycogen loss was inhibited in HS-induced rat liver after SMS treatment to improve energy metabolism disorder.5.The therapeutic mechanisms of SMS on HS-induced rat liver based on AMPK/Drp1 pathway.The protective mechanisms of SMS were evaluated from a perspective of mitochondria function and energy metabolism through using the TEM,western-blot and fluorescent probe etc.techniques in vivo and in vitro based on targeted energy metabolism.The results showed that SMS could improve the mitochondria distortion in shape along with mitochondrial swelling and inhibit cytochrome c（Cytc）released into cytoplasm in HS-induced rat liver,meanwhile,in HS-induced BRL-3A cells,which upregulate the mitochondrial membrane potential（?Ψm）and inhibit the opening of mitochondrial permeability transition pore（MPTP）（P<0.05）.SMS could phosphorylate AMPKα^Thr172 and further activate Drp1^Ser616in HS-induced rat livers,which coordinates mitochondrial fission and mitophagy in the response to mitochondrial insults to protect liver.Conclusion:The standard of the main active component determination and antioxidant capacity evaluation in vitro of SMS were constructed.SMS attenuates HS-induced liver damage through regulating liver function,restoring histological injury,altering abnormal energy metabolism,and sustaining mitochondria structure and function homeostasis.Notably,we have preliminarily proved that the protective mechanisms of SMS are attributed to an AMPK-mediated Drp1-dependent mitophagy and mitochondria rebuilding process,which in turn alleviate HS-mediated rat liver damage and energy expenditure.Targeting AMPK,mitochondrial fission,and mitophagy may serve as effective approaches to inhibit the HS-induced liver injury,and our study lends further support to the use of SMS in treating HS condition.