Inflammation Signal Analysis Of TCBQ-Induced Nurotoxicity In PC12 Cells

Hexachlorobenzene(HCB) and Pentachlorophenol(PCP) are typical persistent organic pollutants, which have the properties of teratogenic, carcinogenic, mutagenic and pose serious threat to human health. Although the chemical properties of HCB and PCP are relatively stable in the environment, they still can be degraded to other metabolites. HCB undergoes cytochrome P450 metabolic pathway for the formation of active Tetrachlorohydroquinone(TCHQ) and Tetrachlorobenzoquinone(TCBQ). PCP was degraded by Sphingobium chlorophenolicum to TCBQ, then further reduced to TCHQ. There are a large number of ROS in these metabolic process, which disturbing the biological redox balance, resulting in lots of toxic effects, such as genetic toxicity, liver toxicity, immune toxicity, neurotoxicity, etc.With the increasing of human population aging, the prevalence of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and other diseases are also increasing. These diseases are caused by many factors, mainly including environmental, genetic and aging related endogenous factors, but they can be summarized basically as that the production of oxidative stress leads to the formation of neuroinflammation. The neurological effects of HCB and PCP have been investigated widely in recent years. However, the information of TCBQ-induced inflammatory pathology mechanism is not currently available. Studying the mechanism of inflammation induced by TCBQ in PC12 cells can provide new ideas for the prevention and treatment of central nervous system diseases. So we select the PC12 cells as the subjects, assuming the inflammation induced by TCBQ is related to the production of ROS, and do the following two-part experiments.PartⅠStudy of the mechanisms of neuroinflammation induced by TCBQ through IKK/IκB/NF-κB signalingThis experiment is aimed at the comparison of cytotoxicity, ROS formation and inflammatory mediators/cytokines expressions induced by HCB, PCP, TCHQ and TCBQ in differentiated PC12 cells, then explores the mechanisms of NF-κB signaling pathway activated by TCBQ. The comparison of cytotoxicity of HCB, PCP, TCHQ and TCBQ in different concentrations and time was performed by CCK-8 assays. Result is that all four compounds caused concentration- and time-dependent increase of cytotoxicity in differentiated PC12 cells, which is closely related to ROS formation and TCBQ, TCHQ display stronger cytotoxicity then HCB, PCP. By DCFH-DA probe detecting ROS level and Western blot probing inflammatory cytokines expressions, the results showed that TCBQ and TCHQ were stronger than HCB and PCP in the role of ROS formation and inflammatory cytokines expressions. To investigate the inflammatory cytokines protein and mRNA expressions in concentrations induced by TCBQ through IKK/IκB/ NF-κB in PC12 cell, western blotting assay and RT-qPCR analysis were accomplished. TCBQ-induced NF-κB activation was investigated by pretreatment p65 Inhibitor, PDTC. The results above show that TCBQ induces the protein expressions of inflammatory cytokines related to NF-κB signaling and pro-inflammatory cytokines m RNA expressions, however, PDTC significantly inhibits TCBQ-induced NF-κB nuclear accumulation and DNA-binding activity of NF-κB. The antioxidants NAC, vitamin E and curcumin can remove ROS induced by TCBQ in differentiated PC12 cells, and also partly suppress TCBQ-induced protein expressions related to IKK/IκB/NF-κB signaling. Thus, these results suggested that TCBQ-induced IKK/IκB/NF-κB signaling activation is ROS-dependent.PartⅡ Study the antagonistic mechanisms of melatonin on TCBQ-activated HMGB1/TLR4/MyD88 signalingThe pro-inflammatory and pro-oxidative properties of TCBQ have been confirmed in the first part experiment; many literatures have introduced the importance of HMGB1/TLR4/MyD88 in inflammation disease; Melatonin(N-acetyl-5-metyoxytr-yptamine), a secretory product of the pineal gland, is a powerful endogenous antioxidant, has anti-inflammatory, anti-oxidant, anti-apoptotic effects. Based on three points above, this experiment aims to link TCBQ, melatonin and HMGB1/TLR4 /MyD88 signaling to explore their mutual interaction mechanism.Firstly, to screen the optimal concentration of melatonin ameliorated TCBQinduced cytotoxicity in PC12 cells, CCK-8 assays is performed. We pretreated PC12 cells with melatonin(200 μM) for 1 h, followed by treatment with 25 μM TCBQ for 6 h in the subsequent experiment. We used Western blot, Co-IP and RT-qPCR assays to research the effects of TCBQ on TLR4, MD2, CD14, MyD88, and examine the role of melatonin in the process. The results showed that TCBQ can induce TLR4, MD2, CD14, MyD88 protein expression and TLR4, MyD88 mRNA levels, promote the combination of TLR4 with MD2, CD14, MyD88. However, this pattern was significantly reversed by melatonin treatment. Then effects of melatonin on TCBQ-induced MAPKs activation and pro-inflammatory cytokines expression were investigated. We found that melatonin treatment can attenuate MAPKs inflammatory activity induced by TCBQ in PC12 cells. Next, we use TLR4 si RNA, MyD88 si RNA transfected PC12 cells to observe inflammatory cytokines protein expression, and TLR4-KO mice to observe the HE staining, immunohistochemistry and immunofluorescence figures of mouse brain. The results show that TLR4 and MyD88 deficiency partly protect TCBQ-induced inflammatory injury, emphasize the importance of TLR4 signal pathway on the TCBQ induced neuroinflammation.In addition to LPS, HMGB1 can also be used as a ligand of TLR4, and there are lots of reports about ROS involving the HMGB1 activation. So we explore the reasons why TCBQ can activate TLR4 signal pathway. By immunofluorescence, Western blotting and Co-IP assays, it is proved that TCBQ induces hyperacetylation and phosphorylation state of HMGB1 in the nucleus, and translocation of HMGB1 from nucleus to cytoplasm, which in turn releases to culture medium. We examine the effect of TCBQ on HMGB1 and its receptor by Western blotting, immunofluorescence staining, RT-qPCR and Co-IP assays. The results showed that TCBQ can induce the expressions of HMGB1 and its receptors, also enhance the binding of HMGB1 with its receptors. Interestingly, the interaction between HMGB1 and TLR4 has been most up-regulated upon TCBQ-exposure. Using sucrose density gradient centrifugation, lysates are divided into 12 segments, then we confirm TCBQ, HMGB1 and LPS can induce TLR4/MD2 translocating to lipid raft fractions. Melatonin as an antioxidant displays the same result with NAC and lipid rafts inhibitors including MβCD and nystatin, which show that TCBQ induces TLR4 recruitment to lipid raft domain independent on ROS. To observe the related protein expressions, siRNA HMGB1 is transfected into PC12 cells, which indicates that melatonin could inhibit the regulation of HMGB1 on TCBQ-induced TLR4 signaling in PC12 cells. By TLR4-KO mice assay, we find that TLR4 deficiency partially protect against TCBQ-induced brain injury, and also is responsible for the inhibition of HMGB1 translocation, release.The two-part experiments above indicate that TCBQ exerts neurological pro-inflammatory activity by ROS-mediated IKK/IκB/NF-κB signaling and promoting HMGB1 release, which induces TLR4 clustering within the lipid raft.