The Mechanistic Approach Of Saponins From Panax Japonicus For Treatment Of Alcohol-induced Hepatic Injury

Alcoholism is a social problem, and is also a medical issue. Acute alcoholism, chronic alcoholism, alcoholic fatty liver, chronic hepatitis and cirrhosis caused by prolong and heavy alcohol intake seriously harms people's health. After heavy drinking, blood alcohol concentration was significantly increased, and emergence of various drunken symptoms. Ethanol is mainly metabolized in liver by ethanol oxidation system, while producing a large number of free radicals O2-·, OH·and ethanol production of free radicals C2H5O-, C2H5OH-. When free radicals beyond the body scavenging capacity, it will cause the body tissue injury. So generally considered that one of the mechanisms of the alcohol-induced liver damage is through activating the O2 and produce free radicals, then cause hepatic cell membrane lipid peroxidation and injury.Panax japonicus (Panax japonicus. C.A.Mey), as one of traditional Chinese medicines and recorded in "Chinese Pharmacopoeia", is usually considered to have many effects such as anti-inflammatory, analgesic, sedative, anti-aging, anti-fatigue, anti-virus, anti-tumor, and protection of central nervous system, cardiovascular system, endocrine system, immune system and so on, the main active components of Panax japonicus are the saponins. Pharmacological studies found that total saponins of Panax japonicus can significantly increased the activities of SOD, CAT, GSH-PX, while reducing the MDA content in serum and liver in various animal models (such as:sports, aging, cerebral ischemia reperfusion injury, hyperlipidemia, etc.), thus inhibiting lipid peroxidation. Therefore, the total saponins used in this study were extracted from artificial cultivated Panax japonicus by circular sonication, the composition and its content of different saponins were analyzed with high performance liquid chromatography- evaporative light scattering detector- mass spectroscope (HPLC-ELSD-MS). Antioxidant activities as a starting point, we investigated the protective effect of saponins from Panax japonicus on ethanol-induced mice liver and human hepatic cell L-O2 injury, and its potential mechanisms of hepatoprotection involved, the protective effect of different purity saponin from Panax japonicus on ethanol-induced hepatic cell L-O2 injury and its possible mechanism are also studied in this paper. The main results are as follows:1. Total saponins were extracted from Panax Japonics, tentative identification of the saponins was achieved by comparing their retention times with those of the authentic standards, and further identification of the structures of these saponins by LC-ELSD-MS. The results were that the saponins used in this paper were identified to be ginsenosides Rg1, Re, Rf, F3, Rg2 and Rd, and the contents of which were 12.23%,16.24%,16.95%, 7.51%,8.53% and 11.47%, respectively.2. Saponins from Panax Japonics (SPJ) were given to mice by gavage 1 hour prior the alcohol treatment, the alcohol concentrations in serum and urine were determined by GC; the levels of malondialdehyde (MDA), alanine transaminase (ALT), aspatate transaminase (AST), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) etc were measured by spectrophotometry; histopathological examination were observed through Lecia-DM2500 microscope and JEOL-1230 transmission electron microscopy; Results:SPJ may not inhibit ethanol absorption in the gastrointestinal tract. The AST, ALT activities and MDA content were decreased distinctly in serum in SPJ (50 mg/kg) treated mice, and the antioxidant as GSH, GSH-PX, CAT and SOD were evidently increased in serum and liver. Pretreated with SPJ, all the levels could restore to normal (50mg/kg b.w) in serum. In liver determination, the administration of SPJ significantly increased the activities of GSH-PX and SOD, and the final values were restored to normal at the dose of 50mg/kg b.w. (P>0.05). The levels of CAT and GSH were also significantly elevated compared to alcohol treatment mice, but still lower evidently than control (P<0.01). In histopathological studies, SPJ (50 mg/kg b.w.) pretreatment significantly prevented alcohol-induced abnormalities in dose-dependent manner, in which the hepatic lobule structure, liver cord and sinusoidal were remedied and showed normal histology. The cell structure were gradually restored nearly normal, karyon chromatin distributed uniform, nuclear membrane became smooth, mitochondrial structure returned to normal, the cristae and outer membrane became integrity, the persistent damage disappeared and most of mitochondria restored back to normal, whiles a few portion of mitochondria still showed with little distorted cristae. These results indicated that SPJ may exert a protective effect against lipid peroxidation by scavenging reactive oxygen species and elevating the activity of antioxidant enzymes, in consequence prevented the peroxidative deterioration of structural lipids in membranous organelles, especially mitochondria and karyon.3. To investigate the protective effect and its possible mechanism(s) of SPJ on alcohol-induced hepatic cell L-O2 damage, the normal and alcohol-induced injury cell were counted through trypan blue staining, the intracellular MDA, SOD, CAT and GSH-PX levels of L-02 cells were measured by spectrophotometry, the CAT. GSH-PX1, GSH-PX3, SOD1, SOD2 and SOD3 mRNA were detected by Quantitative real-time reverse transcription-PCR. Results:SPJ(100μg/mL) could promote normal cell (L-02) proliferation, but SPJ (400μg/mL) showed a evident toxicity to normal cell (L-02). SPJ (100μg/mL) also showed a significant protective effect on ethanol-induced hepatic cell L-02 injury, and presented evidently inhibitory ability on MDA in L-02 cells. Meanwhile, the SOD, GSH-PX and CAT activities were increased, in which the SOD and GSH-PX activities were restore to normal after treated with SPJ (100μg/ml), but the CAT was still lower than control (P<0.01). RT-PCR results showed that the expression of all the antioxidant enzymes were remarkably decreased with treated with ethanol (200 mmol/1) for 48h. Pretreated with SPJ (100μg/ml), these down-regulated tendencies of all the antioxidant enzymes were reversed, in which GSH-PX3 was evidently increased compared with normal control, SOD1 and SOD3 were restored to normal level, but the CAT and GSH-PX1 mRNA levels were still lower than evidently. These results of RT-PCR studies were in agreement with biochemical analyses in vivo, which indicated that the antioxidant effect of SPJ was associated with up-regulating the expression of SOD, GSH-PX and CAT, especially of GSH-PX 3, SOD1 and SOD3.4. Antioxidant activities of SPJ measured by hydroxyl radicals (·OH) and superoxide anion radical (O2·-) scavenge assays in vitro. Results:SPJ had a higher scavenging hydroxyl radical's effect than Vc, and their scavenging effects increased with increasing concentration. Scavenging effects of SPJ was 3.37-59.47% at amount of 12.5-1600μg/ml, respectively, and that of Vitamin C was about 3.10-31.79%. Superoxide radicals were generated in biphenyl trihydroxybenzene autoxidation system; results showed that the SPJ has little scavenging activities on O2'-. At the amount of 12.5-1600μg/ml. the effect on scavenging superoxide of SPJ was just 1.01-6.87%, while the scavenging activity of Vitamin C for superoxide radical was 0.89-15.98%. These results indicated that the protective effect of SPJ on alcohol-induced damage not only just associated with increasing the activities of GSH-PX, CAT and SOD, but also related with directly scavenging free radical such as·OH,O2-·,or the synergy effect of the two.5. To study the protective effect of different purity saponins from Panax japonicus on ethanol-induced hepatic cell (L-O2) injury and its possible mechanism(s). Survival ratios of hepatic cell (L-02) were determined by MTT, the level of MDA, the activities of SOD and GSH-PX in cell L-02 were measured by spectrophotometer. Result:0.16 mg/mL Rg1,s 1.28 mg/mL Re and 0.64 mg/mL Rf could promote normal cell (L-02) proliferation, the proliferation ratio was 22.68%,34.80%,28.47%(P<0.01). respectively.0.16 mg/mL Rd and 1.28 mg/mL F3 showed a evident toxicity to normal cell (L-02), the inhibition ratio (%) was 49.69%,43.33%(P<0.01).0.16 g/L Rg1,1.28 mg/mL Re and 0.64 mg/mL Rf also showed a significant protective effect on ethanol-induced hepatic cell (L-02) injury, the inhibition ratio (%) was decreased to 23.31%,26.90%,26.58% compare to ethanol-induced control 50.37% (P<0.01), but 0.16 mg/mL Rd and 1.28 mg/mL F3 showed a more evident toxicity to ethanol-induced hepatic cell (L-02), the inhibition ratio was 83.18%,64.79%(P<0.01), respectively. Meanwhile,Rg1. Re and Rf also could decreased the level of MDA (1.17±0.04,1.21±0.03,1.21±0.05 compare to ethanol-induced control 1.35±0.05 mmol/; P<0.01) and increased the activities of SOD (38.32±4.85,35.58±4.43,38.96±3.27 compare to ethanol-induced control 26.45±2.78 kU/L; P<0.01) and GSH-PX (86.30±5.72,78.40±3.54,84.25±4.35 compare to ethanol-induced control 67.04±4.27 kU/L; P<0.01) in intracellular fluid of cell L-02. These results indicated that Rg1, Re and Rf have protective effect against ethanol-induced hepatic cells L-02 injury, the possible mechanism may relate to decline of MDA content, increase of antioxidant enzymes such as SOD and GSH-PX activities.