Protective Effect Of Huanglianjiedu Decoction On Microcystin-LR Induced Nerve Injury

BACKGROUND: Microcystin-LR(MC-LR)in eutrophic water has recently been found to trigger severe toxicity of nervous system,but there is no effective protection.Our previous study in neuroendocrine PC-12 cell line found that MC-LR induced cytoskeletal architecture disorder and abnormal phosphorylation of the cytoskeletal-associated proteins,and the mechanism involved the oxidative stress and activation of protein phosphatase 2A(PP2A)-mitogen-activated protein kinases(MAPKs)signaling.Huanglianjiedu Decoction(HLJD)from the Tang Dynasty,Wang Tao's Wai Tai Mi Yao Fang,composed of four crude drugs: Rhizoma Coptidis,Radix Scutellariae,Cortex Phellodendri and Fructus Gardeniae,is a classical Chinese traditional formulas,which could clear away heat-evil and toxic material,attending the triple energizer excessive heat syndrome.Recent studies demonstrated the neuroprotective effect of HLJD is based on the antioxidant effects of active ingredients in HLJD.So,we supposed that HLJD is a potential medicine for MC-LR-induced neurotoxicity.METHODS: We used PC-12 cell line to study the dose and time course of MC-LR's neurotoxicity,cell apoptosis,protein phosphatase 2A(PP2A)enzyme activity and MAPKs expression were determined by MTT assay,enzyme activity kits and Western Blot,respectively.Animal model of nerve injury in C57BL/6J mice were induced by continuous intraperitoneal injection of MC-LR.These nerve injury model mice were treated with 3 mg/(kg·d)donepezil,2 g/(kg·d),4 g/(kg·d),and 8 g/(kg·d)HLJD,respectively.Open field experiment and Morris water maze experiment were used to test the memory and cognitive ability of these mice.Cerebral cortexes were harvested to do immunohistochemistry staining and Western Blot analysis.RESULTS: In PC-12 cells,apoptosis was induced by 30 ?M and higher dose of MC-LR,which was rescued by JNK inhibitor.MC-LR inhibited PP2 A activity in dose-dependent manner.3 ?M MC-LR significantly inhibited PP2 A activity at 12 h and 24 h.10 ?M MC-LR significantly inhibited PP2 A activity at 6h,12 h and 24 h.Time-effect and dose-effect experiment of MAPKs showed that 10 ?M MC-LR significantly induced phosphorylation of JNK,ERK1/2 and p38.HLJD can attenuate the inhibition of PP2 A activity at 6h and the activity of 5mg/m L group is similar with control group.1.25mg/m L,5mg/m L and 5mg/m L HLJD can attenuate the abnormal phosphorylation of JNK,ERK1/2 and p38.HLJD can also reverse the reaction of Nuclear Factor Erythroid-2 Related Factor 2/Antioxidant Response Element(Nrf2/ARE)endogenous defense system activated by 10 ?M MC-LR.In vivo study,the results revealed that MC-LR caused cognitive impairment in C57 mice,abnormal phosphorylation of neuronal cytoskeleton and increased phosphorylation of JNK and ERK1/2 in cerebral cortex were observed,whereas HLJD ameliorated the cognitive impairment,the performances of low dose and medium dose were better than that of high dose group,and was comparable to the positive control.Both donepezil and HLJD alleviated MC-LR-induced neuronal skeleton abnormalities,and reduced phosphorylation of JNK and ERK1/2 in cerebral cortex.The neuroprotection in high dose HLJD group was similar with that of donepezil group,but there was no significant difference in the inhibition of MAPKs phosphorylation between donepezil and different dose HLJD groups.CONCLUSIONS: These findings suggested that HJLD had the protective effect on MC-LR induced nerve injury via MAPKs signaling pathway.HLJD will be a promising protector for neurotoxicity of MC-LR.