Cytoprotective Effect Of L-carnitine Against Burn-induced Hepatic Lipotoxicity Through Modulation Of ER Stress-mediated Pyoptosis

Background:Large-area severe burns can break the homeostasis of the environment.In addition to skin damage,the damage can also cause a variety of severe visceral dysfunctions.The liver is one of the most severely damaged organs after large-area severe burns.Hepatocyte steatosis is an important pathological manifestation of burn-induced metabolic dysfunction and can lead to hepatocyte dysfunction and injury.However,the mechanisms underlying hepatocyte steatosis associated with burns remain unclear.L-Carnitine(LC),a natural biological active amino acid derivative,has been reported to show lipid-lowering activity.However,whether it can improve liver steatosis after burns is currently unclear.Therefore,this study focused on the effects of lipid overload on liver injury after severe burns and the effect of LC on it through in vitro and in vivo experiments,and further explored its molecular mechanism.Methods:1.Animal experiment: 54 Sprague-dawley(SD)rats were immediately divided into a control group(Control),a simple burn group(Burn),and a burn + carnitine treatment group(Burn+LC).All rats were anesthetized by intraperitoneal injection of 10% chloral hydrate(3m L/kg body weight),and then subcutaneously injected with the analgesic buprenorphine(0.05 mg/kg body weight),and the back and sides of body hair were removed using a shaving machine.Prepare skin about 30% of the total surface area for scalding.The rats in the Control group were placed in a 37°C water bath for 15 seconds to simulate a scald model,and the rest of the groups were placed in a 98°C water bath for15 seconds to make a 30% total surface area Ⅲ° burn rat model.After the injury,each group received fluid resuscitation immediately(intraperitoneal injection,sodium lactate Ringer’s solution45 m L/kg body weight).The Burn+LC treatment group was intraperitoneally injected with sodium lactate Ringer’s solution + LC(45 m L/kg body weight + 400 mg/kg.d)after scald.Rats in each group were sacrificed at 24 h,48h,72 h after injury,blood was collected from the abdominal aorta and liver tissue from the same part was taken out.Biochemical methods were used to analyze liver injury indicators,and RT-q PCR and Western blot were used to detect changes in the expression of related markers m RNA and protein in rat liver.2.Cell experiment: In vitro,HepG2 cells were treated with palmitic acid(PA)to simulate cell lipid deposition model.The experiment is divided into FBS control group,PA group,PA+LC(0.8m M/L)co-culture group(PA+LC),PA+LC(0.8m M/L)+GSK2656157(10umol/L)co-culture group(PA+LC+GSK2656157).Cell viability was detected with CCK-8,RT-q PCR and Western blotting were used to analyze the m RNA and protein expression of related markers.CCK-8 method was used to detect the changes of cell viability,RT-q PCR and western blot were used to detect the changes in the expression levels of inflammasome signals such as GRP78,CHOP and NLRP3,Caspase-1 in HepG2.The level of IL-1β was detected by enzyme-linked immunosorbent assay(ELISA).Results:1.In vivo,the results also indicated that severe burn injury can cause liver injury,ER stress and activation of pyroptosis in Sprague–Dawley rats.The scald/carnitine group showed significant improvement of the pathological changes in the liver,especially inhibition of the m RNA and protein expression of ER stress and pyroptosis markers.2.The data demonstrated that LC treatment markedly promoted the proliferation of HepG2 cells and reversed the decrease in HepG2 cell survival.3.PA induced ER stress and pyroptosis in HepG2 cells,as evidenced by marked increases in the m RNA and protein expression of the ER stress markers GRP78 and CHOP,as well as the inflammasome markers NLRP3,caspase-1,GSDMD,and IL-1β.However,LC markedly alleviated PA-induced ER stress and pyroptosis in HepG2 cells.4.More importantly,Tunicamycin upregulated the m RNA and protein expression of ER stress and pyroptosis markers similarly to PA,further confirming the crosstalk between ER stress and pyroptosis.5.Taken together,the cytoprotective effect of LC against ER stress and pyroptosis was abolished in HepG2 cells harboring PERK inhibitors.Conclusion:1.Burns can activate the liver endoplasmic reticulum stress and NLRP3 inflammasome signaling pathway,and further promote the expression of inflammation-initiating factors such as IL-1belta,leading to liver damage.2.PA can induce the activation of the NLRP3 inflammasome signal mediated by the endoplasmic reticulum stress of HepG2 cells,and then release the IL-1belta inflammatory factor.3.LC can inhibit the activation of inflammasome NLRP3 and the occurrence of liver endoplasmic reticulum stress,effectively reducing liver damage.