Signal Transduction Analysis Of Halogenated Quinone Compounds-Induced Inflammatory Responses, ER Stress And Apoptosis In Heaptic Cells

Halogenated quinone compounds are a class of persistent organic pollutants which widely exist in the environment. They are mainly formed by multi-halogenated aromatic compounds through chemical reaction, enzymatic reaction and biological metabolism. The intermediate products which are formed during the metabolism always can result in the cell toxicity, genetic toxicity, neurotoxicity, immune toxicity and so on, which can damage our health seriously.This thesis is carried out by in vitro and vivo levels to reveal the toxicity mechanism of halogenated quinone compounds, which provides new research findings to consummate the theory of halogenated quinone compounds toxicity mechanism and new ideas for prevention and treatment of diseases caused by halogenated quinone compound.Part I The protective effect of curcumin for Tetrachloro-p-benzoquinone induced liver injury in miceTetrachloro-p-benzoquinone(TCBQ) is one of the main metabolities caused by pentachlorophenol(PCP) which is widely used as wood preservative. And the Tetrachloro-p-benzoquinone(TCBQ) in our body can generate a large amount of ROX through redox active oxygen process that can lead to oxidative damage. Liver is the main organ of metabolism and detoxification in human body. TCBQ may cause liver damage in vivo metabolism.Curcumin, a phenolic natural product isolated from plant Curcuma longa (Linn) rhizomes, has anti-inflammatory, antioxidant, scavenging oxygen free radicals, anticoagulation, hypolipidemic, anti atherosclerosis and other pharmacological effects. It can protect the body from oxidation damage through its antioxidant and scavenging free radical effects. In this experiment, we also examined the protective mechanism of curcumin on TCBQ induced acute liver injury in mice.In the experiment was used TCBQ to establish the model of acute liver injury in mice,.32 male kun-ming mice were randomly divided into four groups with 8 mice in each group:Control group, Curcumin group, TCBQ group, TCBQ+Curcumin group. Control group mice received equal volume of vehicles. Curcumin group mice received curcumin for 5 continuous days. TCBQ group mice received a single dose of TCBQ TCBQ+Curcumin group mice received curcumin for 5 successive days by injection. After 3 h for the final curcumin treatment, Mice were injected with TCBQ. After 24 h the blood and liver samples were collected for detecting the related index of liver injury. We detected the levels of ALT, AST in serum and TBARS, SOD, CAT in liver. These results show that TCBQ can cause serious damage to the liver of mice. Through observing the pathological changes of liver by HE staining, there is a large number of inflammatory cell infiltration in TCBQ group, indicating that the liver exists inflammatory reaction. INOS, COX-2 Immunohistochemical and p65,TNF-α, IL-6, IL-1 β, IκB, P-IκB Western blot results further demonstrates that the main hepatic injury induced by TCBQ is inflammatory reaction. We also used Western blot method to explore the expression of apoptosis related proteins Bax, Bcl-2, Caspase 3, Caspase 8 and Caspase 9. The expression of these proteins in TCBQ group did not significantly change compared to the other groups. Immunohistochemical results of cleaved Caspase 3 did not change obviously either. It illustrates that TCBQ does not induce liver apoptosis.Protective effects of curcumin are attributed to its ability to scavenge free radicals. Hence, we selected the important antioxidant signal pathway Nrf2/Keapl/ARE as the research object. The experimental results show that curcumin activates Nrf2/Keapl/ARE signal pathways and generates a good protective effect on acute liver injury induced by TCBQ.The above studies indicate that the main acute liver injury induced by TCBQ is inflammatory reaction rather than apoptosis in liver cells. Curcumin activates Nrf2/Keapl/ARE signal pathway and regulates the expression of phaseⅡ metabolic enzymes HO-1 and NQO1 in liver tissue to protect the liver.Part II Study on the Mechanisms of Apoptosis Induced by Polychlorinated biphenyls quinone through ER stress pathway in HepG2 cellsPolychlorinated biphenyls quinone (PCBQ) is one of the major metabolites of polychlorinated biphenyls (PCBs). Recently, plenty of research findings illustrat that PCBs metabolites can induce oxidative stress response and cause the oxidative damage in the body. While oxidative stress, endoplasmic reticulum stress response and apoptosis are closely related, which hints us polychlorinated biphenyls quinone may cause endoplasmic reticulum stress and apoptosis as well.In the experiment,we selected the HepG2 cells as the research object to investigate whether the polychlorinated benzoquinone can induce endoplasmic reticulum stress response and apoptosis in HepG2 cells. Firstly, we used fluorescence microscopy and transmission electron microscopy to observe the morphological changes of HepG2 cells and endoplasmic reticulum in without and with 10 μM PCB29-pQ treatment for 48 h The results show that the typical characteristics of apoptosis and endoplasmic reticulum stress appears in the PCB29- pQ treated cells. Then the expression of endoplasmic reticulum stress associated with protein were carried out by the concentration gradient and by time gradient. And after analyzing the mRNA expression level of Bip, GRP94, XBP-1 by RT-PCR, compared with the control group, we find that the expression level of these protein and gene were obviously improved in PCB29-pQ treated cells. Flow cytometry was used to detect changes of the intracellular calcium level induced by PCB29-pQ. Experimental results show that PCB29-pQ can increase intracellular calcium levels. These results indicate that PCB29-pQ can induce ER stress in HepG2 cells. In the morphological study we find that apoptosis happened obviously in PCB29-pQ treated cells. Therefore, we selected three signal pathways based on the literature which involve in the regulation of apoptosis: PERK/eIF2a/ATF4/CHOP, Caspase 12/Caspase 9/Caspase 3, IRE1 α/JNK/c-Jun. A series of experimental results show that PCB29-pQ induced apoptosis is due to activating the PERK/eIF2a/ATF4/CHOP, Caspase 12/Caspase 9/Caspase 3 signal pathways in HepG2 cells. Then we used the calcium chelator BAPTA-AM and ROS scavenger NAC to investigate the reasons of endoplasmic reticulum stress caused by PCB29-pQ. The results show that ER stress induced by PCB29-pQ is mediated by intracellular calcium and ROS levels.According to the whole research, we know that PCB29-pQ can induce ER stress in HepG2 cells, and that can be regulated by intracellular calcium and ROS levels. The apoptosis induced by PCB29-pQ is regulated by PERK/eIF2a/ATF4/CHOP and Caspase 12/Caspase 9/Caspase 3 signal pathways,but IRE1 α/JNK/c-Jun pathway is not activated in this experiment and does not participate in the apoptosis induced by PCB29-pQ.