Protective Effect And Mechanism Of Total Saponins Of Panax Japonicus On Acetaminophen-induced Liver Injury

Panax japonicus is the dried rhizome of Panax japonicus C.A.Mey,which has a variety of pharmacological effects such as antioxidant,anti-inflammatory,improving glucose and lipid metabolism.It is known that total saponins are the main active components of Panax japonicus.However,there is currently less systematic and in-depth study on the chemical properties of Panax japonicus and the activity of preventing liver damage.In this thesis,the total saponins of Panax japonicus?PJST?were extracted firstly,and the total saponin content was determined by vanillin method to be 64.3%.Combined with HPLC analysis,it mainly contained ginsenoside Re,ginsenoside Ro,Chikusetsusaponin Pjs-2,Chikusetsusaponin?and Chikusetsusaponin?a.The relative percentages were 8.73%,7.72%,44.10%,22.71%and 7.50%,respectively.The antioxidant activities of PJST and total polysaccharide of Panax japonicus?PSPJ?were evaluated using superoxide anion radical?O₂·^-?and hydroxyl radical?·OH?scavenging systems in vitro.Vitamin C?Vc?was used as a positive control.The results showed that the scavenging activity of PJST on superoxide anion radical?O₂·^-?and hydroxyl radical?·OH?radical was significantly better than that of PSPJ in the concentration range,which showed a dose-dependent relationship.As the concentration of the sample increased,the clearance rate of PJST to free radicals increased.In vitro,a model of HepG2 cell injury induced by acetaminophen?APAP?was established.The effects of PJST on the viability of HepG2 cells were evaluated by MTT assay and LDH kit.The morphology of the cells was evaluated by micrographs.The reactive oxygen species?ROS?were detected by DCFH-DA fluorescent probe.The effect of PJST on the morphology of apoptosis was observed by Hoechst 33258 fluorescence staining.Western Blot?WB?was used to detect the expression of protein kinase B?Akt?,glycogen synthase kinase 3 beta?GSK3??,Nuclear factor erythroid-2-related factor 2?Nrf2?,heme oxygenase-1?HO-1?,quinone oxidoreductase 1?NQO1?and glutamate-cysteine ligase catalytic subunit?GCLC?.The results showed that PJST?30,100?g/mL?pretreatment group promoted the phosphorylation of Akt and GSK3?,up-regulated the expression of Nrf2,HO-1,NQO1 and GCLC,and then inhibited the overproduction of ROS and LDH induced by APAP in HepG2 cells to ameliorate APAP-induced cell damage in HepG2.This suggests that PJST may activate the Akt/GSK3?-mediated Nrf2 pathway to attenuate APAP-induced oxidative stress and apoptosis in HepG2 cells.To further evaluate the protective effect of PJST on APAP-induced liver injury,a model of liver injury in mice induced by APAP was established.Mice were randomLy divided into normal group,APAP group and PJST?50,100 mg/kg?group.The mice were intragastrically administered with PJST?50,100 mg/kg?once a day,and the other groups were given an equal volume of normal saline for 7 days.After 2 hours of gavage on the7th day,the mice in the other groups were intraperitoneally injected with 400 mg/kg APAP except the normal group,and the mice were sacrificed after 12 h.The liver index was calculated and the levels of alanine transfer?ALT?,aspartate transferase?AST?and lactate dehydrogenase?LDH?in serum were detected.The kits were used to detected Glutathione?GSH?,malondialdehyde?MDA?,total superoxide dismutase?T-SOD?and total antioxidant capacity?T-AOC?levels in liver tissue.The levels of inflammatory factors such as interleukin-6?IL-6?,interleukin-1??IL-??and tumor necrosis factor-??TNF-??in tissues were measured using an ELISA kit.H&E staining was used to observe liver pathological changes.TUNEL and Hoechst 33258 fluorescence staining were used to observe the effect of PJST on hepatocyte apoptosis.The expressions of CYP2E1,Akt,GSK3?,Nrf2,NQO1,HO-1,GCLC,Bax,Bcl-2,NF-?B,and Nuclear-NF-?B were detected by immunohistochemistry?IHC?,immunofluorescence?IF?and WB.The results showed that the PJST?50,100 mg/kg?pretreatment group significantly reduced serum levels of ALT,AST,LDH,and MDA in liver tissue,increased the levels of GSH,T-SOD and T-AOC in liver tissue,and inhibited the expression of CYP2E1 compared with the APAP group.TUNEL and Hoechst 33258 fluorescence results showed that PJST can significantly reduce the number of apoptotic cells,which may be because PJST can reduce Bax expression and increase Bcl-2 expression.PJST activated phosphorylation of Akt and GSK3?,which shifted Nrf2 into the nucleus,then regulated the expression of downstream proteins of Nrf2 such as NQO1,HO-1 and GCLC.In addition,PJST significantly inhibited the expression of NF-?B,inhibited the transfer of NF-?B into the nucleus,thereby inhibited the activity of downstream inflammatory factors such as IL-6,IL-?and TNF-?.Histopathological results showed that PJST significantly improved necrosis and apoptosis of liver tissue,reduced necrotic areas,and reduced cellular inflammatory infiltration and bleeding.It indicated that PJST has a protective effect on APAP-induced liver injury in mice,possibly by regulating the metabolic pathway of APAP,enhancing antioxidant enzyme activity,increasing anti-inflammatory activity and inhibiting hepatocyte apoptosis.In summary,the results of this study demonstrate that the PJST have certain preventive and protective effects on APAP-induced liver injury in vitro and in vivo.The main mechanism may be achieved by regulating APAP metabolic pathway,enhancing antioxidant enzyme activity,increasing anti-inflammatory activity and inhibiting hepatocyte apoptosis,which provides a theoretical basis for the clinical application of Panax japonicus.