Study On The Mechanism Of Glycyrrhizic Acid Alleviating Semen Strychni-induced Neurotoxicity Based On The Regulation Of PRDX1 On HMGB1 Release

Backgrounds:Semen Strychni has been found to have various pharmacological effects in Chinese medicine theory and modern clinical practice,such as analgesic and anti-inflammatory,anti-tumor,etc.However,its clinical application is limited by its severe neurotoxicity.Our group found that glycyrrhizic acid has great potential to alleviate Semen Strychni induced neurotoxicity,and the nucleoplasmic translocation of high mobility group protein B1(HMGB1)is involved in the mechanism of Semen Strychni induced neurotoxicity,and might be an important part of the detoxification effect of glycyrrhizic acid.Therefore,the regulation of HMGB1 nucleoplasmic translocation and release may be the key to Semen Strychni induced neurotoxicity and the alleviation by licorice.Objectives:This research focus on the regulation of PRDX1 on HMGB1 nucleoplasmic translocation and release.We investigated the detoxification mechanism of glycyrrhizic acid based on PRDX1/HMGB1-TLR4/NF-κB pathway to alleviate Semen Strychni induced neurotoxicity Methods:This study can be divided into two parts: cell and animal experiments.Animal experiments were conducted to investigate whether there is a protective effect of glycyrrhizic acid on the neurotoxicity caused by Semen Strychni extract and its effect on PRDX1/HMGB1-TLR4/NF-κB pathway.(1)Construction of Semen Strychni poisoned SD rat model:6-week-old SD rats were selected and randomly divided into blank group(Control),glycyrrhizic acid group(GA),Semen Strychni group(SS),and detoxification group(SS+GA).After 30 min of intraperitoneal injection of glycyrrhizic acid,the rats were injected with Semen Strychni extract intraperitoneally on the other side of the abdomen,and the control group was injected with 0.5% CMC-Na,and the brain tissue and serum were collected after 5 days of continuous administration.Neurological deficit score(NDs)and Racine ratings were performed 30 min after drug administration.(2)HE staining and Nissl staining were used to observe neuropathological changes in the hippocampus of rats,and Iba-1 and GFAP immunohistochemical staining were used to assess microglia activation and neurological damage.WB was used to detect the levels of inflammatory mediators such as TNF-α,IL-1β,HMGB1 and PRDX1 in serum for neuroinflammatory evaluation of rats.(3)Immunoblotting was used to detect the levels of TLR4,Iba-1,GFAP,IL-1β and i NOS in hippocampal whole proteins,and the distribution of PRDX1,HMGB1 and NF-κB in the cytoplasm and nucleus.Cell experiments used BV2 cells and PC12 cells,main cotents were as follows:(1)The protective effect of glycyrrhizic acid on brucine induced BV2/PC12 cell injury: the appropriate concentration and intervention time of glycyrrhizic acid and brucine were screened by CCK-8 method;(2)The levels of IL-1β and HMGB1 in cell culture medium supernatant were measured,and immunoblotting was used to the change of proteins of PRDX1/HMGB1-TLR4/NF-κB pathway in whole protein or cytoplasm or nucleus.(3)Investigation on the effects of glycyrrhizic acid and brucine on the nucleoplasmic translocation and release of HMGB1 based on PRDX1 regulation.BV2 cells were transfected with negative control or PRDX1 si RNAs respectively,then pretreated with glycyrrhizic acid for 6h,followed by glycyrrhizic acid/brucine treated for 24 h.PRDX1 level in whole protein and the levels of PRDX1 and HMGB1 in the cytoplasm and nucleus were detected.IL-1β/HMGB1 levels in the culture medium supernatant were detected.Results:The results of animal experiments showed that:(1)The weight gain of rats in the SS group and the SS+GA group was significantly lower than that of the Control group after 5 days of continuous administration;(2)The neurological deficit score of rats in the SS group was significantly lower compared to the Control group,and the neurological deficit score of the SS+GA group was significantly improved;(3)The hippocampus of rats in the SS group showed significant pathological changes,microglia and astrocytes were activated,Iba-1,GFAP levels were significantly increased,and the same indexes in the SS+GA group were significantly decreased.(4)HMGB1,PRDX1,IL-1β,TNF-α levels in serum were increased significantly in SS group,and i NOS,IL-1β levels in hippocampus were increased significantly;and serum HMGB1,PRDX1,IL-1β,TNF-α levels and hippocampal i NOS,IL-1β levels decreased significantly in the rats of the SS+GA group.(5)In the SS group,hippocampal PRDX1 level in nuleus was significantly increased,nucleoplasmic translocation of HMGB1 was also significantly increased;In the SS+GA group,hippocampal PRDX1 level in nuleus and nucleoplasmic translocation of HMGB1 was significantly decreased.(6)TLR4 receptor levels and NF-κB levels in nucleus in the rats of the SS group were increased,while they were decreased in the SS+GA group.The results of cell experiments showed that(1)the damaging effects of brucine on BV2 cells and PC12 cells were dose-and time-related,and the cell viabilities(CVs)gradually decreased with increasing dose and time,the IC50 of brucine on both cells for 24 h was about 250 μM.500μM glycyrrhizic acid significantly increased the cell viability which was decreased by brucine.(2)The levels of HMGB1 and IL-1β in the culture media were significantly increased with brucine treatment,and glycyrrhizic acid could dose-dependently reduce the levels of HMGB1 and IL-1β in the culture supernatant after brucine treatment in a certain concentration range.(3)The PRDX1 and levels in nucleus of BV2 and PC12 cells in the BR group showed a significant increase,and there was a significant rise in HMGB1 expression,whose nucleoplasmic transportation and release into the culture supernatant were also upregulated.Glycyrrhizic acid dose-dependently decreased the level of PRDX1 in the nucleus and reduced the nucleoplasmic translocation of HMGB1.(4)TLR4 levels were significantly upregulated and nuclear NF-κB levels were significantly increased in the BR group;glycyrrhizic acid dose-dependently decreased the level of NF-κB in the nucleus,and reduced the level of TLR4.(5)After silencing PRDX1,the promotion effect of brucine on HMGB1 nucleoplasmic transfer was partially inhibited,and the release of HMGB1 and IL-1β into the culture media was significantly decreased;the inhibitory effect of glycyrrhizic acid on HMGB1 nucleoplasmic translocation was significantly reduced,and the levels of HMGB1 and IL-1β in the culture supernatant of the glycyrrhizic acid/brucine group after silencing PRDX1 were not significantly different compared with the unsilenced group.Conclusions:Semen Strychni/brucine promotes the oxidative secretion of HMGB1 out of the cell by upregulating the PRDX1 content in the nucleus;glycyrrhizic acid downregulates PRDX1 to reduce the release of HMGB1 and inhibits the TLR4/NF-κB pathway to exert its protective effect on Semen Strychni/brucine-induced neurological damage.