The Application Of Inflammatory Animal Models In Evaluation Omedication On Colitis And Investigation The Toxicity Of Polygonum Multiflorum Thunb

Objective: Digestive system diseases are always associated with immune disorders, often characterized by acute paroxysm and hard to treat with. The inflammatory response is involved in the occurrence and development of the digestive system diseases. Thus, it is vital to establish animal models of inflammation caused by immune disorders for drug efficacy and toxicity evaluation of the digestive system diseases. Animal models of inflammation include conventional animal models for drug efficacy evaluation and idiosyncratic toxic animal models for drug toxicity assessment. Inflammatory animal models are used to supplement the disadvantage of normal animals in incapability of simulating idiosyncratic toxicity. Inflammatory animal models can not only be used to evaluate drug efficacy, but also to assess drug toxicity. Therefore, we focus on the immune-associated digestive system diseases, inflammatory bowel diseases（IBDs） and drug-induced liver injury（DILI）, to establish inflammatory animal models with similar characteristics shown in humans and to screen out therapeutic drugs for IBD and Potential Mechanisms and biomarkers for drug induced idiosyncratic liver injury. Firstly, IBD, including Crohn’s disease（CD） and ulcerative colitis（UC）, are chronic remitting and relapsing inflammatory disorders. Accompanied by abdominal pain, diarrhea, bloody stools, patients with IBD course up to 20 years suffer from both physical and psychological tortures which seriously affect the quality of their lives. The precise etiology of IBD are still partially uncertain. Until now, there is no reasonable clinic solution for curing the diseases and especially preventing the recurrence. Therefore, it is necessary to innovate new therapies and drugs for IBD. Serious or long-term physiological and psychological stimulation cause the body’s nerve, endocrine and immune system disorders to induce inflammation. Immuno-modulatory drugs, nutrition support therapies, and neuroendocrine regulation measures can effectively prevent the immune dysfunction induced by stress reaction. In addition, depression and anxiety may have certain effect on the occurrence and recurrence of IBD. Mental factors could be both the cause of IBD and the secondaryperformance of IBD recurrence. Therefore, the first part of this paper is intended to establish a rat model for acute IBD. Based on the regulation among nerve, endocrine and immune systems, vitamin E was chosen as a therapy for DSS-induced rats IBD because of its antioxidant stress function. Meanwhile, a novel selective corticotropin releasing factor receptor1（CRFR1） antagonist P16, namely a potential antidepressant medication, was screened out to treat DSS-induced rats IBD. Secondly, Drug-induced liver injury（DILI） has become the most common cause of withdrawal from the market as a result of safety concern. Characterized by acute paroxysm and liver failure, the hepatotoxicity of Polygonum multiflorum Thunb（ PMT） is commonly reported both in China and in other countries of the world in recent years. Even from the perspective of the TCM protection, the first notice of amendments to oral formulations containing PMT is given by the SFDA in October 2013, and has developed to a clear prompt attention to its risk of liver injury in July, 2014. Former research indicates that PMT-DILI as the idiosyncratic liver injury type may be related to immune-toxicity. The unclear pathogenesis of hepatotoxicity, and the complex chemical composition undoubtedly make it difficult to unveil the mechanisms. One thing need to notice for the conventional animal models for PMT-DILI is that they usually cost a long period up to at least one month. What’s more, the administration dosage is over 200 times more than that of the clinical usage. Therefore, the aim of this work is to provide an animal model for PMT with similar characteristics shown in humans, to investigate its potential mechanism, and to verify the effectiveness of serum micro RNA-122 as a candidate biomarker for Drug-induced Liver Injury（DILI）.Materials and Methods: 1. Establishment of inflammatory animal models for IBD and pharmacodynamics evaluation of the therapies for IBD.（1） Establishment of dextran sulfate sodium（DSS）-induced rats acute IBD model and the effect of vitamin E on this model. Experimental rats were divided into 6 groups as follows: control group（Ctrl）, DSS group, prednisolone group（P）, vitamin E with a low dosage group（EL）, vitamin E with a median dosage group（EM） and vitamin E with a high dosage group（EH）. IBDs model were induced by 5% DSS dissolved in the drinking water of male Wistar rats for seven consecutive days. On the 8th day, half rats in the ctrl and DSS groups were anesthetized to be tested. Indexes, such as body weight（BW）, organ to body weight ratio, clinical observation（CO） and histopathological examination, are used to determine the success of the model establishment. After the model issuccessfully established, prednisone, as the positive control drug, and three-dosage of vitamin E are used to treat the inflammation animal model IBD for seven days. On the 15 th day, all the left rats were anesthetized to be tested. On the basis of the indexes, such as the body weight, organ to body weight ratio, clinical observation and histopathological examination are used to determine the efficacy of the treatment. After the model is established successfully and the end of the treatment, rats blood were collected to test hematologic indexes. Inflammatory cytokines IL- 1 beta, IL-18, IL- 6, IL- 12, TNF alpha, IFN- gamma in rats’ colon were measured by ELISA to determine the potential mechanism of vitamin E on IBD.（2） Effect of CRFR1 antagonist P16 on DSS-induced rats IBD and its potential mechanisms. Rats were randomly divided into 9 groups, control group（Ctrl）, DSS group（DSS）, Prednisolone group（P）, precaution P16 L group（pre-P16L）, precaution P16 M group（pre-P16M）, precaution P16 H group（pre-P16H）, P16 L group（P16L）, P16 M group（P16M）, P16 H group（P16H）. Rats except in the ctrl group were administrated with 5% DSS（w/v） in drinking water for seven days to induce colitis. During this induction period, rats in pre-P16 L, pre-P16 M and pre-P16 H groups were administrated with p16 for prevention via intraperitoneal injection. After successful establishment of DSS-induced IBD, rats in P and P16 L, P16 M and P16 H groups were respectively treated with prednisolone by gavage and p16 via intraperitoneal injection for 7 consecutive days while the ctrl,DSS, pre-P16 L, pre-P16 M and pre-P16 H groups received tap water without DSS nor P16. Body weight, clinical observation score, histopathological observation, colon to length ratio and inflammation-related cytokines expression by ELISA were used as indicators for evaluates the effect of P16 on treating DSS-induced colitis. Three different doses of P16 were administrated for both prevention and treatment therapies, respectively.2. Establishment of a new animal model for PMT-induced liver injury in rats and speculation of its potential mechanisms.（1） In preliminary screening model, we use 3.5% DSS and 4 mg/kg LPS as inducers to activate hepatotoxicity of PMT. SD rats were administrated with 20-folds clinical dosage PMT for 7 consecutive days.（2） The modified Karber’s method was applied to determine the median lethal dose（LD50）, maximum tolerated dose（MTD） and the maximum letha dose（MLD） of alcohol extract from PMT on SD rats via oral administration. Rats were randomly divided into 6 groups that the concentration of PMT were 30g/kg, 39g/kg, 51g/kg, 69g/kg and90g/kg（r=1.32） respectively. There were half male and half female. Rats were given 4mg/kg LPS via intravenous injection. 2 hours before the oral administration of PMT at the dosages of 10ml/kg, 13ml/kg, 17ml/kg, 23ml/kg and 30ml/kg once daily respectively. Acute toxicity of symptom spectrum was observed for 14 consecutive days. The cumulative death number and weight of rats were recorded. Rats blood were collected to test ALT and ALP at the end of the experiment. Meanwhile, liver to body weight ratio of rats were determined.（3） Male SD rats were divided into 8 groups as follows: control group（ctrl）, LPS group, acetaminophen group（APAP）, LPS + acetaminophen group（LPS+APAP）, Polygonum-low dosage group（PMT-L）, Polygonum-high dosage group（PMT-H）, LPS + Polygonum-low dosage group（LPS+PMT-L） and LPS + Polygonum-high dosage group（LPS+PMT-H）. A dosage of 4mg/kg LPS was injected to the caudal vein of rats in the LPS, the LPS + APAP, the LPS + PMT-L and the LPS + PMT-H groups. 2 hours later, rats were administrated by intragastric route with APAP（325 mg/kg）, PMT（3g/kg, equivalent to 15 times the dose of clinical medication） and PMT（6 g/kg, equivalent to 30 times the dose of clinical medication） respectively for 7 days according to each group once a day. Indexes, such as body weight（BW）, organ to body weight ratio, clinical observation（CO） and histopathological examination, were used to determine the success of the model establishment.（4） On the Hour 2, Hour 14, Day 5 and Day 8, after getting the organ to body weight ratio for each rats, liver samples were treated for histopathological observation, detected m TLR-4 expression by RT-PCR and screened out proteins with significant differential expression by i TRAQ technique.（5） Serum samples in each group were collected for ALT/ALP test and Taq Man detection of micro RNA-122 expression.Results: 1. Establishment of inflammatory animal models for IBD and pharmacodynamics evaluation of the therapies for IBD.（1） Compared with the rats in the control group, rats treated with 5% DSS performed obvious diarrhea and bloody stool, CO increased significantly（p < 0.05）. Meanwhile, colonic ulceration and swelling appeared by histopathological examination. Inflammatory cytokines IL-1β,IL-18,IL-6,IL-12,TNF-α,IFN-γ in colon increased significantly. The DSS-induced rats IBD model is established successfully. In colitis rats, administration of threedosage vitamin E and prednisolone decreased the CO score respectively, and remarkably alleviated the severity of inflammation of the colon compared with untreated DSS group in histopathological examination. Furthermore, administration of three-dosage vitamin E and prednisolone both suppressed the level of TNFα, IL-18 and IL-12 in colon significantly, while they showed different effects on other histological inflammation markers such as IL-6 and IFN-γ.（3） Three different doses of P16 were administrated for both prevention and treatment therapies, respectively. For both therapies, P16 prevented weight loss from established DSS-induced colitis in rats. Meanwhile, colonic ulceration and swelling recovered better in groups treated with P16, and the expression of the inflammatory cytokines in rats colon decreased significantly than those in the DSS group（p < 0.05）. On the hand, histopathological observation of livers in the DSS, three-dosage of pre-P16 and three-dosage of P16 groups showed no obvious hepatotoxicity. 2.Establishment of inflammatory animal models for PMT-DILI and its mechanisms.（1） Compared with the rats in the DSS group, all the rats in the LPS group appear semiliquid stool, abnormal secretion of eyelids. Their liver to body weight ratio increased significantly. Serum ALT in the LPS group raised significantly. From the histopathological observation, a small amount of liver cell degeneration could be seen. Ratio of liver injuries is higher than that of the DSS group. In addition, males were sensitive to LPS-activated PMT hepatotoxicity than that of females. Thus LPS was chosen as the inducer.（2） To determine the PMT dosage of administration, modified Karber method was adopted to test SD rat oral median lethal dose（LD50）, the maximum tolerated dose（MTD） and the maximum letal dosage（MLD） of PMT ethanol-extract after 4mg/kg LPS injection. LD50, MTD and MLD were 30g/kg, 90g/kg, and 53.26g/kg, which were equivalent to 150, 450 and 266.3-fold of clinical dosage respectively. The 95% confidence limit for LD50 was 46.91 60.46g/kg.（3） General clinical observation showed that PMT could induce weight loss of rats after LPS activation. After LPS activation, PMT promote slight liver swelling. Histopathological examination showed that a small amount of hepatocyte degeneration in the LPS group was observed on the 2nd hour. From the 2nd hour to the 8th day, the hepatocyte degeneration recovered gradually in the LPS group. Only a small amount of hepatocyte degeneration was observed in the PMT-L and PMT-H groups; Degrees of liver injury in the LPS+PMT-L and the LPS+PMT-H groups deepened gradually, showing various extent of hepatocyte damage and degeneration. Chronic inflammatory lesions could be seen locally; Livers were damaged partially in the APAP group, and hepatocytes degenerated and grew necrosis.（4） On the 2nd hour, m TLR4 expression in the LPS group showed significant inhibition. The following three time points, compared with the control group, m TLR4 expression appeared no obvious change in the PMT-L and the PMT-H groups. On the 5th day, m TLR4 expression in the LPS+PMT-L and LPS+PMT-H groups increased significantly by contrast to that of the control, LPS, PMT-L and PMT-H groups. While significantly increased in the APAP, LPS+APAP, LPS+ PMT-L and LPS+ PMT-H groups on the 8th day. Together, these data indicated that up-regulation of m TLR4 expression was associated with hepatotoxicity of PMT with activation of LPS. i TRAQ.（5） After activated with LPS, micro RNA-122 expression in rats serum increased significantly in the LPS group（vs Ctrl p<0.05） on the 2nd hour. The following three time points, compared with the control group, micro RNA-122 expression in rats serum appeared no significant change in the LPS group, while significantly increased in the LPS+ PMT-L, LPS+PMT-H, APAP, LPS+APAP groups with time passing by（vs Ctrl p<0.05）. Comparing with the expression of ALT and ALP, micro RNA-122 in rats serum and m TLR4 in rats liver in the LPS+PMT-L and the LPS+PMT-H groups elevated significantly on the 8th day. And the degree of changes was in proportion to the liver damage. The augment of indexes in the LPS+PMT-L and the LPS+PMT-H groups was greater than those in the PMT-L and the PMT-H groups. Although ALT, ALP in rats serum in the LPS+PMT-L and the LPS+PMT-H groups increased significantly at the beginning（vs Ctrl, p<0.05）, the expression of the two varied within normal range of rats serum. This phenomenon indicates that micro RNA-122 in serum can be used as potential biomarker for this model. Perhaps m TLR4 in liver is also a promising candidate. gi|13242301|ref|NP₀77367.1| was screened out as the idiosyncratic protein expressed by rats’ liver in the LPS+PMT-H group. Its gene is estradiol 17-beta-dehydrogenase 2. The protein of human for the gene is HSD17B2.Conclusion: 1. The inflammatory animal model for IBD was established successfully. Oral administration of vitamin E promoted recovery of colitis rats by inhibition of histological proinflammatory cytokines, while its underlying mechanisms might be different from glucocorticoid drugs. Our results suggested that P16 might be a new potential effective drug for IBD therapy with no obvious hepatotoxicity. 2. LPS could activate the hepatotoxicity of PMT. This new animal model for PMT-induced Liver Injury was established successfully. 3. Its mechanism is closely related to m TLR4expression. 4. More sensitive than ALT and ALP, micro RNA-122 was certificated to be an ideal potential serum biomarker for this model. Meanwhile the expression trend of m TLR4 in liver is similar to that of micro RNA-122 in serum. It hints that m TLR4 might also be used as a potential biomarker for this model. While the new model could validate the relation of toxicity mechanism with TLR4 and contribute to further study. 5. It provides a new orient for studying hepatotoxicity mechanism of chemical, herbal and botanical drugs.