| Liver fibrosis is one of the important pathological processes that chronic liver disease shifting to liver cirrhosis, which is mainly resulted from the excessive synthesis of extracellular matrixcomponents (ECM), and the key lies in the over activation and proliferation of hepatic stellate cells. What is lately confirmed is that oxidative stress (OS) takes part in most kinds of liver injuries. ROS includes oxygen free radicals and superoxide dismutase (SOD), such as hydroxyl free radicals (.OH), singlet oxygen free radicals (02), Super oxygen anion free radical (02-) and so on. Angiotensin II (Ang Ⅱ), the main biological active substances of the renin-angiotensin system (RAS), plays an important role in the activation, contraction and proliferation of HSC.Electron spin resonance (ESR) is the most accurate and effective method to study free radicals. The spin complement technology may also be good at completing the determination of free radicals and clearing the drug antioxidant protection mechanism and the mechanisms of cell membrane phospholipid membrane peroxidation and membrane protein conformational changes resulted from the attack of free radicals’ attack on cell membrane.It has also reported that the turtle shell decoction oral liquid showed anti liver fibrosis effects. In the present study, the researchers explored the types of Ang Ⅱ induced free radicals from human hepatic stellate cells (HSC-LX2), the effects of turtle shell decoction oral liquid at different time point on free radicals generated from HSC-LX2, and its effects on ROS-ACE-AngⅡ-AT1R/ACE2-Ang(1-7)-Mas related pathways. All of those would provide a new mentality for liver fibrosis early clinical intervention.To begin with, simulating the oxidative stress conditions and inducing the proliferation of HSC-LX2by Ang II. In addition, to detecte the cell proliferation situation by MTT, and capture the free radicals in the nutrient solution and observe the characteristics of free radicals on different stages of HSC-LX2proliferation via ESR. Finally to figure out the HSC-LX2proliferation and oxidative stress related signaling pathway by protein chip technology, and then, the expression of ACE,ACE2, Mas, ATI R,Ang II and Ang (1-7) by immunohistochemical technology, and apply the method of RT-PCR for the expression of ACE2, Mas which belong to RAAS.The effects of Ang II on HSC-LX2cell vitality while inducing oxidative stress: the cells grew well after recovery. And compared with the normal group, cell’s vitality levels significantly increased (P<0.05) when oxidative stress was induced by Ang II in HSC-LX2cells, besides concentration dependence have been detected.The results of the types of free radical signals which were generated in HSC-LX2after oxidative stress was induced by Ang II:Capturing different types of free radicals generated in HSC-LX2by a variety of free radical scavenger using ESR. Compared with the normal group, two type free radicals which are singlet oxygen free radicals and hydroxyl radicals, have been detected in oxidative stress groups.The effects of different time delivery of drug on generating of free radicals HSC-LX2cells are that both before and after cell oxidative stress delivery of drug showed antioxidant effects on HSC-LX2, and inhibited the expression of free radicals in HSC-LX2cells stimulated by Ang II, while the effects of after cells oxidative stress delivery showed a more powerful effect.The effects of BOL on the HSC-LX2cells’expression position of ACE, ACE2, AT1R, Mas, Ang II and Ang (1-7) during the oxidative stress stimulated by Ang II are that compared with the normal group, the expression position of ACE, ACE2, AT1R, Mas, AngⅡ and Ang (1-7) are explicit, and the expression of mRNA of ACE2and Mas are dramatically different.The effects of different time delivery of BOL on generating of free radicals:that both treat BOL before and after oxidative stress are showing dramatical antioxidant effects on HSC-LX2, and inhibiting the expression of free radicals in HSC-LX2cells stimulated by Ang II, while the effects of after cell oxidative stress delivery showed a more powerful effect.Conclusion:1, HSC-LX2, induced oxidative stress by Ang II can produce two free radicals, that are hydroxyl radicals and singlet oxygen radicals;2, The application of traditional Chinese medicine Biejiajian oral liquid compound(BOL) in different times can effectively inhibite the produce of hydroxyl radicals during the oxidative stress induced by Ang II, but the effect on singlet oxygen free radicals is weaker;3, Applying Protein Chip can figure out583targets during the BOL against Ang Ⅱ-induced oxidative stress of HSC-LX2. And among them, MAPK pathway which is main pathway, TGFβ-Smad pathway, and NF-κB pathway make more contribute to the liver fibrosis. |
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