| A widely used dietary spice and herbal medicine, Zanthoxylum L. contained many active ingredients and was used to treat various diseases. It was favorite type edible spices and was one of the eight traditional condiments in our country. Zanthoxylum L and their extraction had a weak toxicity, could block the cell cycle, such as gastric cancer, lung cancer. The physiological efficacy contained analgesic effects, relieved pain, antioxidant. The antioxidant effect could reduce lipid content in the human body. Zanthoxylum had attracted great attention due to its extensive biological and physiological properties that were nature, low toxicity and high efficiency. Sanshool was a kind of unique biological active ingredients that had anti-inflammatory analgesic, anesthesia, insecticidal, anti-wrinkle, and regulate gastrointestinal system properties, which could be used in medicine and cosmetic industry. At present, the research on the medicinal function and effects of sanshool in liver cells were deep not enough, especially the mechanism was unclear. Hence, the paper aimed to study physiological functions and explore the mechanism of sanshool in liver cell, being based on HepG2cells as a carrier. The content of the research was divided into three parts, the main research results are as follows:1. The antioxidant effect of sanshool was studied. Different-dose sanshool in vitro was determined four antioxidant index including, the reducing power, FRAP total antioxidant capacity, the scavenging ability on hydroxyl free radical and DPPH free radical. Then the change of SOD activity and MDA content was determined after different-dose sanshool treated HepG2cells for24h. The results showed that sanshool had a certain reducing power and oxidation resistance. The effect was present concentration dose relationship(P<0.05). But the scavenging ability on DPPH and hydroxyl free radical was bit weaker. The mesured method for reducing power and FRAP total antioxidant capacity of sanshool had the highest correlation (p<0.01), the correlation was weakest between Fe2+reduction force and the scavenging ability on-OH of sanshool. And, with the increasing of sanshool concentration, SOD activity was a trend of first being down then being up, was significantly different (p<0.05). When up to50μg/mL concentrations of sanshool, MDA content begun to be decline. In general, sanshool was a kind of potential antioxidant compounds, but the ability in specific free radical was not strong.2. The antitumor effect of sanshool was studied, sanshool induced HepG2cells apoptosis. Cell proliferation was analyzed by MTT to investigate HepG2treated sanshool, the morphology was observed by scanning electronic microscopy (SEM), apoptotic ratio of HepG2cells were measured by flow cytometry. Nucleus staining with DAPI was performed, and mitochondrial membrane potential (△ψm) with rhodamine-123was observed by laser confocal microscopy. P53and Caspase-3gene expression were assayed by reverse transcription-quantitive PCR (RT-qPCR), P53and Caspase-3protein expression by western blot. The results showed that the apoptotic bodies were observed by scanning electron microscopy. The nucleus, together with△ψm, was altered in a dose-dependent manner. The cell cycle was further analyzed by flow cytometry, showed that sub-apoptosis occurred. Reverse transcription polymerase chain reaction further showed that the RNA and protein expressions of P53and Caspase-3were up-regulated. Sanshool-induced apoptosis in HepG2cells was associated with disrupted mitochondrial functions. This result suggested that apoptosis was triggered via a mitochondrion-dependent pathway. Furthermore, the anti-proliferation activity of sanshool was stimulated by apoptosis. Thus, the results the morphology of cell apoptosis triggered that was caused by molecules pathways of apoptosis.3. Cholesterol-lowering physiological effect of sanshool was studied. The liver was the main organ endogenous cholesterol synthesis. Choosing high cholesterol HepG2cells as model, explored the cholesterol-lowering effect of different doses sanshool, and cholesterol metabolism related gene expression mechanism, as well as observerd the variation of [Ca2+]i concentration.With specific antagonisst (TRPV1antagonist and TRPA1antagonist) as TRPV1and TRPA1receptors intervened sanshool (excited) affect the role of the body cholesterol metabolism link. The results showed that high cholesterol HepG2cells was treated sanshool for24h, reduce the total cholesterol in HepG2cells and culture media in a dose-dependent manner. The total bile acid content was increased in culture medium, when concentration of sanshool was20μg/mL the effect was better. The lower cholesterol in the liver cell possible mechanisms are:low doses of sanshool down-regulated the gene and protein expression of HMG-CoA R, reducing the endogenous cholesterol synthesis in the liver; high doses of sanshool not only down-regulated the gene and protein expression of HMG-CoA R, also could up-regulated the gene and protein expression of CYP7A1, promoted the transformation of cholesterol into bile acid. Specificity of TRPV1antagonists and TRPA1antagonists could block the sanshool influence on cholesterol metabolism related gene expression, sanshool might be through activating TRPV1and TRPA1receptors was at the same time to adjust cholesterol metabolism related genes. |
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