| Excessive free radicals accumulated in the body produce oxidative stress associated with many diseases.It has been found that the liver diseases caused by free radicals-induced oxidative stress include fatty liver,viral hepatitis,and hepatic fibrosis.The liver is one of the major organs of free radical attack,so its research on oxidative stress injury is representative.Phytochemicals are the main source of natural antioxidants.Clerodendrum cyrtophyllum Turcz(Clerodendrum,Verbenaceae)is a traditional Chinese medicinal plant.It is often used in southern areas to treat many diseases such as inflammation and wounds.Previous research of this group had found that the extract of C.cyrtophyllum Turcz leaves has a strong antioxidant activity in the chemical detection method,so it is worthy to evaluate the antioxidant activity on the cell level.In this study,C.cyrtophyllum Turcz leaves was extracted by ultrasound and extracted with petroleum ether,dichloromethane,ethyl acetate,n-butanol and water into five different polar components.A model of human hepatocellular carcinoma(HepG2)oxidative stress induced by tert-butyl hydroperoxide(TBHP)was used to study the cytoprotective effect and mechanism of the five component extracts and verbascoside(Vb)which is the major constituents of C.cyrtophyllum Turcz leaves.Colorimetric assays were used to determine cell viability,oxidative factors(LDH,MDA,ROS),antioxidants(GSH),and antioxidant enzymes(SOD,CAT).Apoptosis was analyzed using fluorescence microscopy and flow cytometry.The apoptosis pathway caspase-3 was analyzed by Western blot.The main findings are as follows:The study of five different polar components of C.cyrtophyllum Turcz leaves found that ethyl acetate,n-butanol,and water components showed better antioxidant activity in the cell oxidative stress model.The cell viability was measured by MTT assay.It was found that the three components can increase the survival rate of oxidative damage induced by TBHP in HepG2 cells in a dose-dependent manner.At the concentration of 30 ?g/mL,the activity of ethyl acetate showed the largest increase in cell activity,followed by n-butanol and water,and there was no significant effect of dichloromethane and petroleum ether.It's showed that the three components with cytoprotective activity can maintain the normal morphology of HepG2 cells to varying degrees by a microscope.Further studies showed that pretreatment of HepG2 with ethyl acetate,n-butanol,and water extracts can reduce LDH leakage,intracellular ROS content,MDA content,increase intracellular GSH content,and increase intracellular SOD activity and CAT activity.In-depth study found that ethyl acetate,n-butanol and water groups can inhibit the activation of caspase-3 to varying degrees,so that the level of cleaved caspase-3 protein returned to normal,that is,down-regulate the oxidative damage of HepG2 cells caused by TBHP,and then we It was hypothesized that the three component extracts could mediate the caspase-3 dependent mitochondrial pathway and alleviate the apoptosis induced by TBHP by inhibiting the expression of caspase-3.A study of verbascoside was found that its antioxidant activity as a whole was comparable to the positive control butylhydroxytoluene(BHT)and superior to vitamin E(VE).The cell viability was measured by MTT assay.It was found that verbascoside can increase the survival rate of HepG2 cells after oxidative injury in a dose-dependent manner,and the effect is similar to that of BHT,and is superior to VE.Further studies showed that pretreatment of HepG2 cells with verbascoside can reduce the leakage of LDH,which is reduced by 18.4%to 42.4%(BHT:43.7%,VE:32.1%),and the intracellular ROS content is reduced by 8.1%to 34.8%(BHT:32.6%,VE:27.0%),decreased MDA content by 9.4%-39.8%(BHT:36.6%,VE:37.2%),and increased intracellular GSH content by 80.6%-306.8%(BHT:257.3%,VE:197.1%),increased SOD activity by 31.9%-71.5%(BHT:68.2%,VE:59.0%),and increased CAT activity by 12.7%-55.9%(BHT:55.1%,VE:50.0%).In addition,by fluorescence microscopy and flow cytometry,it was found that verbascoside can relieve apoptosis,and with increasing concentration,the remission effect is more obvious.The in-depth study found that verbascoside can inhibit the activation of caspase-3 and restore the level of cleaved caspase-3 protein,ie,down-regulate the oxidative damage in HepG2 cells caused by TBHP.Furthermore,we hypothesized that verbascoside can mediate caspase-3-dependent mitochondrial pathway and relieve apoptosis induced by TBHP by inhibiting caspase-3 protein expression.In summary,with the model of TBHP-induced HepG2 cells oxidative stress,the ethyl acetate,n-butanol and water components of C.cyrtophyllum Turcz leaves extract exhibited better antioxidant activity,and the ethyl acetate component was the most prominent.The butanol and water components followed.Therefore,the ethyl acetate extract can be used as the main research object of future antioxidant activity and it is a good source of natural antioxidant.In addition,verbascoside has good antioxidant activity and is an excellent antioxidant.Its effect is similar to BHT,better than VE,and its antioxidant mechanism is basically the same as the extracts.Therefore,verbascoside can be used as the material basis of the antioxidant activity of C.cyrtophyllum Turcz leaves and provides the basic for its antioxidative efficacy. |
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