Secondary Development Research Of Baicalin And Demonstration Study On Safety Evaluation Of Traditional Chinese Medicines Contained Aristolochic Acid

Study One:secondary development research of baicalinBackground and aim:Liver diseases, serious threat to human health, are the main causes of death due to illnesses, and the mortality rate is only less than cardiac and cerebrovascular diseases and tumors. China has a high incidence of liver disease. There are more than 5 million new cases of end-stage liver disease due to liver cirrhosis each year, and the annual mortality rate is 20%-50%. It is estimated that the cost of treating hepatitis B and its related diseases is up to 100 billion RMB every year, resulting in a heavy economic burden both to the state and to individuals. Therefore, it is very important that actively developing low cost, good efficacy of innovative technologies and medicines to prevention and cure end-stage liver disease. Orthotopic liver transplantation is currently the only effective method for end-stage liver disease. However, because of surgical complex, high cost, lower success rate, just a few liver donors, and needing to take immunosuppressive drugs for life, greatly limited the clinical application. Bone marrow mesenchymal stem cells (MSCs) possess the ability of self-renewal and multi-differentiation, without immune rejection, and treatment costs of MSCs transplantation are far lower than orthotopic liver transplantation. Therefore, transplantation of MSCs may be a promising therapeutic strategy in the treatment of end-stage liver diseases in the future. But just MSCs transplantation for treatment of liver diseases is not the best therapeutic strategy. Therefore, the aims of this study were developing a compound from traditional Chinese medicines which may intervene hepatic differentiation of MSCs both in vivo and in vitro, and finding an optimal therapeutic strategy for liver diseases.Methods:1. We identified the liver protective effect of compounds from traditional Chinese medicines as a secondary development of candidate compounds through literature research. Then, we found that baicalin could promote MSCs differentiating into hepatic cells from candidate compounds, using drug screening model of MSCs and computer-aided.2. In vitro differentiation:MSCs were divided into three groups, including fibroblast growth factor-4 (FGF-4) group, hepatocyte growth factor (HGF) group and baicalin group. We detected the following indicators to illustrate that baicalin could promote MSCs differentiating into hepatic cells:the protein expression of alpha-fetoprotein (AFP), Albumin (ALB) and cytokeratinl8 (CK18); the gene expression of HGF, c-met, AFP, ALB, CK18, transthyretin (TTR), hepatocyte nuclear factors (HNF)-3p and cytochrome P450 (CYP)-2B1; (CYP450)-dependent activity and inducibility; cellular uptake of low density lipoprotein (LDL); urea synthesis; glycogen storage.3. The curative effect of baicalin for hepatic fibrosis and its mechanism:Hepatic fibrosis of rats was induced by subcutaneous injection of carbon tetrachloride (CCl4) twice a week for 8 weeks. Starting from the 5th week, baicalin was intragastric administrated everyday, and silymarin was used as a positive control drug. Then, we detected some indicators to illustrate the curative effect of baicalin for hepatic fibrosis, including sero-enzyme, histopathology, hydroxyproline, glutathione, inflammatory factor, and so on. In addition, detecting the related genes and proteins expression of HGF/TGFβ1 and PPARy/TGFβ1 signal pathway were used to certify the mechanism of baicalin for hepatic fibrosis.4. To optimize the therapeutic regimens for hepatic fibrosis:We designed five different therapeutic regimens using different combinations of baicalin and MSCs:baicalin treatment group, MSC treatment group, pre-differentiated MSC transplantation group, baicalin plus MSC treatment group, and baicalin plus pre-differentiated MSC treatment group. Then, we compared the curative effect of these five therapeutic regimens through detected some indicators, including sero-enzyme, histopathology, hydroxyproline, glutathione, inflammatory factor, to find the optimization therapeutic regimen.Results:1. There are proved that salidroside could induce MSCs differentiation to hepatocytes by itself, and baicalin could promote MSCs differentiation to hepatocytes in the presence FGF-4.2. Baicalin could significantly enhance the differentiated cells expressed marker genes and protein specific of hepatocytes in a time-dependent manner, and increased functions characteristic of hepatocytes, including urea secretion, glycogen storage, uptake of low-density lipoprotein, and inducible cytochrome P450 (CYP)-dependent activity. The effect of promoting differentiation is related to the signal pathway of HGF/c-met.3. Baicalin could decrease the activity of AST, ALT and ALP; increase the serum content of ALB and TP; restrain the degree of fibrosis by reducing the content of hydroxyproline; enhance the ability of free radical scavenging by increasing the concentration of GSH; depress inflammatory reaction of organism by down-regulating the contents of TNFa and TGF(31 significantly. The mechanism of anti-hepatic fibrosis of baicalin is that activates PPARy and HGF to inhibit the TGFβ1 signal pathway.4. In the transplantation experiment, we found that there is little effect for hepatic fibrosis using low-dose baicalin; transplantation of MSCs alone restored partial liver function and suppressed liver inflammation, but had little effect on reduction of the fibrotic area; the therapeutic effect was greatly boosted after MSC transplantation in conjunction with baicalin administration; the curative effects of transplantation of pre-differentiated MSCs for CCl4-induced liver fibrosis were better than untreated MSCs; the combination of pre-differentiated MSC transplantation with baicalin administration achieved the best therapeutic effects in our study.Conclusion:This study established the drug screening model with MSCs and computer-assisted successfully, confirmed that baicalin has the ability of promoting MSCs differentiate into functional hepatocytes, and generated the curative effects for liver fibrosis through influencing HGF/TGFβ1 and PPARy/TGFβ1 signal pathway. This study achieved secondary development research of baicalin successfully, and found an optimization therapeutic strategy for hepatic fibrosis by comparative pharmacological study.Study Two:Safety evaluation demonstration study on the traditional Chinese medicines contained aristolochic acidBackground and aim:Guanxin Suhe Wan is an effective Chinese patent medicine for acute coronary heart disease. However, long-term use can lead to kidney damage, due to Radix Aristolochiae Debilis. Aristolochia debilis is the dry root of Fructus Aristolochiae Debilis, having the effects of promoting qi circulation to relieve pain, detumescencing by detoxification, dissolving dampness, and so on. The plant-medicines of Aristolochia have been shown to be a nephrotoxicity and a potent carcinogen, due to Aristolochic acid (AA). Therefore, State Food and Drug Administration pronounced that abolished the medical standards of Aristolochia debilis, and used Radix Inulae to replace aristolochia debilis in all Chinese patent medicine of the National Prescription Drug Standards. In our study, we evaluated the safety of Radix Inulae and Guanxin Suhe Wan (with Radix Inulae) to provide data for clinical drug safety, and the safety of aristolochia debilis and Guanxin Suhe Wan (with aristolochia debilis) to illustrate the importance of safety evaluation of the traditional Chinese medicines as a negative example. In addition, we evaluated the safety of Radix Inulae and aristolochia debilis, Guanxin Suhe Wan (with Radix Inulae), Guanxin Suhe Wan (with aristolochia debilis) and Guanxin Suhe Wan (without Radix Aucklandiae) by comparative toxicology, to spread the safety evaluation of traditional Chinese medicine as a demonstration.Methods:1. Acute toxicity tests in mice:all animals were divided into six groups: aristolochia debilis group, Radix Inulae group, Guanxin Suhe Wan (with aristolochia debilis) group, Guanxin Suhe Wan (with Radix Inulae) group, Guanxin Suhe Wan (without Radix Aucklandiae) group and normal control group. All animals were administrated everyday according to groups. We observed toxic reaction of animals in later 14 days, and determined maximum tolerated dose or median lethal dose of every drug.2. Long-term toxicity test in rat:all animals were divided into six groups:aristolochia debilis group, Radix Inulae group, Guanxin Suhe Wan (with aristolochia debilis) group, Guanxin Suhe Wan (with Radix Inulae) group, Guanxin Suhe Wan (without Radix Aucklandiae) group and normal control group. All animals were administrated everyday according to groups, lasting 180 days, and then drug withdrawal lasting 30 days. We detected some indicators to evaluate the safety of these drugs, including electrocardiogram, a routine blood test, coagulation system, serum biochemical and histopathology of all organs, at 90th day,180th day and 210th day.3. Long-term toxicity test in Beagle:all animals were divided into eight groups: aristolochia debilis high dose group, aristolochia debilis low dose group, Radix Inulae group, Guanxin Suhe Wan (with aristolochia debilis) high dose group, Guanxin Suhe Wan (with aristolochia debilis) low dose group, Guanxin Suhe Wan (with Radix Inulae) group, Guanxin Suhe Wan (without Radix Aucklandiae) group and normal control group. All animals were administrated everyday according to groups, lasting 180 days, and then drug withdrawal lasting 30 days. We detected some indicators to evaluate the safety of these drugs, including electrocardiogram, a routine blood test, coagulation system, serum biochemical and a routine urine test, at 0th day,30th day,60th day,90th day, 120th day,150th day,180th day and 210th day. In addition, we observed pathological changes of all organs by histopathology, at 90th day,180th day and 210th day.Results:1. Acute toxicity test in mice was not found to have acute mortality, and the maximum tolerated dose of drugs were tens or hundreds of times more than the clinical dose.2. Aristolochia debilis and Guanxin Suhe Wan (with aristolochia debilis) had apparent toxicity to rats, including hematological toxicity, nephrotoxicity, hepatotoxicity, reproductive system toxicity, and carcinogenicity. These toxicities mentioned above were gradually stronger along with increased drug cumulant, and were inreversible. Radix Inulae, Guanxin Suhe Wan (with Radix Inulae) and Guanxin Suhe Wan (without Radix Aucklandiae) could influence the some biochemical indicators, hematologic indicators and electrocardiogram indicators after long-term taking. However, most of these indicators were still in the normal range, and the other abnormal indicators would put back after drug withdrawal.3. Animals in aristolochia debilis high dose group, aristolochia debilis low dose group, Guanxin Suhe Wan (with aristolochia debilis) high dose group and Guanxin Suhe Wan (with aristolochia debilis) low dose group were suffered from the toxicity of drugs. Those drugs could affect appetite leading to weight loss, produce hematological toxicity leading to serious malnutrition and anemia, seriously affect the liver function leading to hepatic inflammation and hepatocyte necrosis, produce immune system toxicity leading to thymus involution, produce reproductive system toxicity leading to restrain the development of testis and epididymis, produce lymphatic system toxicity leading to spleen congestion. Radix Inulae, Guanxin Suhe Wan (with Radix Inulae) and Guanxin Suhe Wan (without Radix Aucklandiae) could produce slight anemia and abnormal hepatic function, but these toxic reactions would put back after drug withdrawal.Conclusion:Aristolochia debilis and Guanxin Suhe Wan (with aristolochia debilis) both are toxic drugs, and long-term use can cause systemic toxicity of multi-target and multi-organ, including hematological toxicity, nephrotoxicity, hepatotoxicity, reproductive system toxicity, immune system toxicity, lymphatic system toxicity, and carcinogenicity. These toxicities mentioned above were gradually stronger along with increased drug cumulant, and were inreversible. Radix Inulae, Guanxin Suhe Wan (with Radix Inulae) and Guanxin Suhe Wan (without Radix Aucklandiae) could produce slight anemia and abnormal hepatic function, but these toxic reactions would put back after drug withdrawal. Therefore, these three drugs are relatively safe. Radix Inulae and Guanxin Suhe Wan (with Radix Inulae) can replace Aristolochia debilis and Guanxin Suhe Wan (with aristolochia debilis) to be used in clinical practice.