Preliminary Study On The Specific Liver Injury Of Polygonum Multiflorum Based On Immune Stress

Polygoni Multiflori Radix (PM), a herb derived from dry tuberous root of polygonaceae plant Polygonum multiflorum Thunb., is a common traditional Chinese medicine (TCM) used in clinic. A traditionally view hold that heshouwu is not a poisonous TCM. However, in recent years, the increasing reports about the adverse reaction of PM and its pharmaceutics causing liver injury are at an epidemic proportion both in China and foreign countries. And warning about which PM and its pharmaceutics can cause liver damage and relevant policy have been unveiled by the drug administrations in England, Canada, Australia and China, which has aroused a huge public attention worldwide.Based on the idiosyncrasy and immune characterization in fundamental diseases found in the previous analysis of clinical PM liver injury cases and associated with influence of immune factor in medicinal idiosyncratic liver injury, the idea that the idiosyncratic liver injury by PM is immune stress-mediated is proposed. Preliminarily, the animal model representing idiosyncratic liver injury by PM, relevant substance and mechanism were investigated. The main results are as follows:1. Objectivity of immune stress-mediated idiosyncratic liver injury by Polygonum multiflorum based on objective epidemiological survey of drug.With the retrospective statistics of medicinal liver injury cases in 302 hospital, it could be observed that the total morbidity of liver injury by PM was relatively low. There was no relationship between liver injury by PM and dosage and time, regardless of irrational drug use and toxicity increase by drug combination, indicating that the liver injury might be idiosyncratic. Currently, the evaluation and systematic study pattern of idiosyncratic liver injury by non-toxic Chinese herb are still in shortage. And it was found that immune cytokines including IL-1, IL-6 and TNF-a were up-regulated in previous clinical samples, which indicated that immune stress might be an important inducement. Further analysis demonstrated that incidence of liver injury by PM was higher in the patients with some basic immune diseases like leukotrichia, skin itch, leucoderma, osteoarthrosis, psoriasis and systemic lupus erythematosus. A part of immune cytokines increased slightly and might be higher because of immune enhancement of PM, which increased the risk of liver injury. This phenomenon indicates that patients above may be susceptible population of liver injury by P. multiflorum.In this study, we established a rat model to evaluate the PM-induced liver injury (PMILI) firstly, then used it to screen hepatotoxic substances by applying target constituents removing and adding strategy, Furthermore, we explored mechanisms and related biomarkers by means of biochip as well as metabolomics. What we did not only provides the scientific evidence to elucidate the compound and its mechanism of PMILI, but share a methodology for the studies on other herbs supposed same characteristics as an potent reference. The main research contents and results were as follows:2. The objective evaluation idiosyncratic liver injury by Polygonum multiflorum based on immune stress-mediated animal modelsReferencing an internationally recognized assessment on idiosyncratic drug-induced liver injury and improvement, we had established a rat model which were applicable to the PMILI, and investigated the dose-toxity and time-toxity relationship. The results showed that there was no significant injury when SD rats were intragastrically administered with mega-dose (equivalent of 75.6 g crude drug per kg body weight) of 50% alcohol extracts of PM alone, agreed with the ones in the literature, suggesting that PM had no obvious toxity on normal animals; no significant alterations of plasma ALT and AST activities were observed in the groups of solo-administration of LPS (1.4 mg/kg,2.8 mg/kg, iv), compared to normal control group (P>0.05), causing slight infiltration of inflammatory cells in portal area but no evident hepatocytes injury. While intragastrically administered once with alcohol extracts of PM (75.6-1.08 g crude drug per kg body weight) based on the LPS groups (2.8 mg/kg, iv), significant liver injury and the obviously pathological changes were all observed and it also showed significant dose-toxity and time-toxity relationships appearing that the lowest toxic dose was 2 times the amount normally used in clinic, approximately in agreement with positive liver injury drug monocrotaline. The present results suggest that animal models established well characterized idiosyncratic PMILI, which might be a critical tool for the evalution of PMILI, the research of toxic constituents as well as scientific mechanisms, with the advantages of simple in operation and stable in result.3. The research of relevant cytokines in immunological stress-mediated idiosyncratic liver injury of Polygonum multiflorumThe research was designed to investigate cytokines in plasma and liver tissue related to PMILI screening by protein chip technology. Cotreatment with PM (1.08 g crude drug per kg body weight)/LPS led to high expression of 13 cytokines including proinflammatory cytokines (IL-6, IL-1 and TNF-a, ect.), chemokines (MIP-2, IP-10 and MCP-1, ect.), cell growth factors and the like in liver tissue and plasma, compared to LPS models of rats (P<0.05), which suggested that these 13 cytokines might be relevant to the process of PMILI. When that compared to normal rats,18 of these such as proinflammatory cytokines, chemokines, chemoattractant protein were highly expressed in liver tissue (P<0.05), showing basic immunology micro-environment in liver might be related to the PMILI, and 13 cytokines (TNF-a, MIP-2 and IL-6 ect.) of them were highly expressed in plasma (P<0.05), which could provides a reference for clinical research about PMILI and a further indication that basic immunology micro-environment could be used as entry points to explore PMILI in liver. Besides, proinflammatory cytokines (IL-6, IL-1, TNF-a, IL-12 and IFN-y ect.) play a key role in downstream region of TLR4 signaling pathway, declaring the pathway and PMILI would be relevant.4. The research on components of PMILI based on constituents removing and addingThe hepatotoxic effects were evaluate to carred out parallel comparison of ethanol extracts and its extractive fractions (ethyl acetate, chloroform and other extract) by constituents removing and adding based on the model of PMILI mentioned above. Our results showed, in constituents removing, ethyl acetate extract which was equivalent to the relative content of extract, however, manifested the similar liver injury in extract groups (1.08 g crude drug per kg body weight), compared to the control or model group (P<0.05), while neither chloroform extract nor other extract induce liver injury in rats, compared to the control or model group (P>0.05), suggesting that ethyl acetate extract might be the main ingredients of PMILI; in constituents adding, as we added 0%、25%.50% and 100% ethyl acetate extract to its negative sample (chloroform and other extract) in order, liver injury appeared again when the addition is 100%. Comprehensive analysis implied that ethyl acetate extract was the main constituents of PMILI.The main compositions of ethyl acetate extract using LC-MS were identified as Trans-2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (SG) (more than 99% in content), Cis-SG and dimerization of SG, ect., inferring that stilbene glycosides might be the mian constituents of PMILI. Moreover, the effect of idiosyncratic liver injury in Trans-SG and Cis-SG were compared at animal and cellular level, which indicated that obvious hepatic injury appeared in the rats of Cis-SG groups but the Trans-SG, illustrating Cis-SG could be the correlative component of PMILI. Cis-SG is rarely found in nature, part of that was transformed from Trans-SG in light or ultraviolet radiation. In order to reduce the formation of Cis-SG, light and ultraviolet radiation should be avoided in processing, soaking in wine and storing as liquid of herbs containing stilbene glycosides, which can help reduce the risk of PMILI in clinic.5. The screening of genes and metabolic markers relevant to PMILI227 miRNA genes had been identified expressed differently including 57 up-regulated and 126 down-regulate by gene chip technique that can find out the different expression genes in liver of LPS models treated with Trans-SG and Cis-SG respectively. Then 14 important pathways were slected such as PPAR signaling pathway, TGF-beta signaling pathway, Tryptophan metabolism and so forth by searching KEGG. The results showed that Cis-SG-induced liver injury was particularly relevant to PPAR-y signaling pathway (significant down-regulated, P=1.72E-06) that was consistent with the reference published by Nobuyuki in which the researchers concluded idiosyncratic liver injury was related to PPAR-y signaling pathway. Moreover, LPS-induced inhibition of NF-κB target gene expression could be terminated by PPAR-y signaling pathway down-regulating, prompting that LPS-TLR4-NF-κB signaling pathway might be a important pathway, agreed with cytokines screening results above-mentioned well, which could provides a reference for plausible mechanisms of PMILI.The research was designed to investigate cytokines in plasma and liver tissue related to PMILI screening by protein chip technology. Using the technology of metabonomics. metabolic markers related to Cis-SG-induced idiosyncratic liver injury were sceened. The robust UPLC-HDMS analysis platform provided the determination quantitatively of endogenous metabolite in serum from rats that were Cis-SG/LPS-cotreated, Trans-SG/LPS-cotreated, LPS-induced or normal. Analyzing the difference of those endogenous metabolite,9 characteristic marker including Bovinocidin, Uridine, and D-Ornithine were found to represent the metabolism disorder caused by Cis-SG-induced idiosyncratic liver injury, and 13 metabolic pathways were found by searching KEGG. The discovery of these markers provided valuable reference to realize the earlier warning and distinguish susceptible populations of PMILI.In summary, PM characterized idiosyncratic liver injury, it may induce idiosyncratic liver injury when body is in immunological stress state, which indicated that the body with immunological stress state in clinic may belong to susceptible areas, should be cautious with PM. The study preliminary elucidated the mechanism of PM liver injury, provided scientific data to guide rational drug use, and reduce the risk of clinical medicine, also provided ideas and methodological reference for the research of other traditional chinese medcines which are not considered toxic side effects.