Protective Effect And Its Methanism Of Yinning Tablet Against Liver Damage Induced By Hyperthyroidism In Rat

Thyroid hyperfunction abbreviate "hyperthyroidism" is a common endocrine disease, often involving many systems and organs, in which common and often easily overlooked is the liver damage. The incidence of hyperthyroidism hepatic damage up to 37%～76.7%. A considerable number of patients with hyperthyroidism accompanies hepatic damage, causing abnormal liver function, hepatomegaly, jaundice, etc. The liver is the body important metabolic organ, the hepatic damage lead to hepatocellular injury, regeneration and hepatic fibrosis, can develop into cirrhosis and hepatic failure. Therefore, it is very urgent that the study on the mechanism of hepatic damage induced by hypethyroidism and the drug for protection and treatment on hepatic damage, which have been paid attention by many researchers, it has become a hot issue and a difficult subject in domestic and international. Clinical treatment of hyperthyroidism hepatic damage is mainly to control the main clinical symptoms of hyperthyroidism and less drugs are to protect the hepatic function and reduce transacylase and prevent to hepatic damage. Therefore, we tried to study both the hepatoprotective and antithyroid effect of traditional Chinese medicine to fill the gaps. Yinning Tablet, prepared by Nanfang Hospital based on Yiqiyangyin and Ruanjiancanjie theory, is traditional Chinese medicine preparation. Yinning Tablet has obvious advantages in controlling the symptoms and signs of hyperthyroidism, especially in the treating or reducing hyperthyroidism complications, reducing side effects and reducing the recurrence of hyperthyroidism, even has hepatoprotective effect. The previous studies have shown that Yinning Tablet significantly reduced thyroid hormone levels in rat blood, extending anti-hypoxia duration in mice with hyperthyroidism, increasing non-specific immune function in mice, but the mechanism of Yinning Tablet in the treatment of hyperthyroidism hepatic damage is unclear, requiring further study.Therefore, in this thesis we aim our research to exploration of molecular mechanism of Yinning Tablet in treating hyperthyroidism hepatic damage through the animal and cell experiments from the receptor and cellular levels, for the purpose of consolidating the laboratorial evidence on drug development and clinical applicationPart I Experimental study of Yinning Tablet on rat model of hepatic damage induced by hyperthyroidismObjective:To observe the effect of Yinning Tablet on rat model of hepatic damage induced by hyperthyroidism. Serum thyroid hormone level, hepatic function and oxidative stress parameters were detected. To observe the histopathological changes of liver and explore the impact of oxidative stress on hyperthyroidism hepatic damage and provide the laboratorial foundation for further studying its therapeutic mechanism.Methods:Sixty Sprague-Dawley (SD) female rats were used in this study and randomly divided into six groups. Except normal control group, all the other rats were perfused intragastrically with Levothyroxine Sodium Tablet (800μg/(kg-d)) to establish the rat model of hyperthyroidism liver damage for 30 days.After constructing model, rats were treated with Yinning Tablet (0.6 g/(kg·d),1.2 g/(kg·d),2.4 g/(kg·d)) and Jiakangling(1.2 g/(kg·d)) for 30 days, respectively. After executed, blood samples and liver were immediately collected.The serum contents of T3,T4 and TSH were assayed by radioimmunoassay(RIA), the levels of ALT,AST and ALP were measured by automatic biochemical analysator, the concentration of MDA and GSH and activities of SOD,GSH-Px,CAT and Na+-K+-ATP enzyme were detected by spectrophotometry method. All rats were executed to take liver tissue specimens for observation under light microscope and electron microscope alterations.Rssults:(1) Changes of T3,T4 and TSH in serum of each groups:In comparison with those in the control group, the serum T3 and T4 levels in the model group were significantly increased and TSH was markedly decreased (P<0.01).Compared with the model group, Yinning Tablet in low,medium and high doses significantly decreased T3 and T4 levels and increased TSH level (P<0.05).(2) Changes of AST, ALT and ALP in serum of each groups:Compared with the control group, the levels of AST, ALT and ALP in serum in the model group were significantly increased (P<0.01). Compared with the model group, the concentrations of AST and ALT in all Yinning Tablet groups were markedly decreased (.P<0.05 or P<0.01), the level of ALP in Yinning Tablet high dosage group was significantly decreased (P<0.05), but no statistical difference was noted in Yinning low and medium groups(P>0.05).(3) Changes of the levels of MDA and GSH and the activities of SOD,GSH-Px,CAT and Na+-K+-ATP enzyme in liver:Compared with the control group, the contents of MDA in liver were significantly increased(P<0.01), but SOD,CAT,GSH-Px and Na+-K+-ATP enzyme activities were markedly decreased (P <0.05), and the content of GSH was lower (P<0.05) in the model group. There was significant difference between the control group and the model group. Compared with the model group, after treatment with Yinning Tablet, MDA value was decreased in a dose-dependent (r=0.449, P<0.01); the activities of SOD in Yinning Tablet medium and high dosage groups were significantly increased than in Yinning Tablet low dodage group (P<0.01), but no statistical difference was noted between Yinning medium and high groups(P>0.05);. the activity of CAT in Yinning Tablet high dosage group was significantly increased than in Yinning Tablet low dodage group (P<0.05), but no statistical difference was noted between Yinning low and medium groups(P>0.05);the activities of GSH-Px and Na+-K+-ATP enzyme and the concentration of GSH were markedly increased(P<0.05), no significant differences were noted between Yinning Tablet groups.(4) Pathological changes of liver under light and electron microscopy: Histological examination showed fatty degeneration, inflammatory cell infiltration, mitochondrion decrement and mitochondrial crista disappearance in the model group. In all treatment groups the pathological changes of liver can be improved:glycogen were increased significantly and the numbers, volumes and, degrees of swelling of mitochondria were reduced.Conclusions:Yinning Tablet plays a protective role on hyperthyroidism hepatic damage induced by Levothyroxine Sodium in rat. It is proposed that the effect is concerned with decreasing the levels of thyroid hormone and reducing the levels of transaminase in serum and improving hepatic pathological damage and inhibiting hepatic oxidative stress in rat.PartⅡEffect of Yinning Tablet on expressions of thyroid hormone receptor, typeⅠiodothyronine deiodinase and cytochrome P450 gene in liver of rat with hyperthyroidism hepatic damageObjective:To examine the effects of Yinning Tablet on expressions of thyroid hormone receptor (TR), typeⅠiodothyronine deiodinase(DioI) and cytochrome P450(CYP450) gene in liver of rat with hyperthyroidism hepatic damage.Methods:Sprague-Dawley (SD) female rats were perfused intragastrically with Levothyroxine Sodium Tablet to establish the rat model of hyperthyroidism hepatic damage.Yinning Tablet of low, medium and high doses and Jiakangling as a positive control treated rats for 30 days. The levels of expression of TRal, TRa2, TRβ1, DioⅠ, CYP1A2, CYP2E1 and CYP3A2 gene in liver were examined by Real-time Quantitative PCR.Results:(1) Changes of the expressions of TRα1mRNA, TRa2mRNA and TRβ1mRNA in rat liver:Compared with control group, the mRNA expressions of TRal mRNA and TRβ1 mRNA were up-regulated and the expression of TRa2mRNA was down-regulated (P<0.01). Yinning Tablet decreased the the expressions of TRα1 and TRβ1 genes (P<0.05), but no statistical difference was noted on the expression of TRα2 mRNA(P>0.05)(2) Changes of the expressions of DioⅠmRNA in rat liver:Compared with the control group, the level of expression of DioⅠgene in the model group was significantly increased (P<0.01).Yinning Tablet significantly inhibited the expression of DioⅠmRNA in a dose-dependent (P<0.01)(3) Changes of the expressions of CYP1A2 mRNA, CYP2E1 mRNA and CYP3A2 mRNA in rat liver:Compared with the control group, the expressions of CYP1A2 mRNA and CYP3A2 mRNA were decreased (P<0.01, P<0.05), but the expression of CYP2E1mRNA was increased in the liver of rat with hyperthyroidism hepatic damage (P<0.01). Yinning Tablet decreased the the expressions of CYP2E1 mRNA (P<0.05), but did not markedly change the expressions of CYP1A2 mRNA and CYP3A2 mRNA (P>0.05)Results:Yinning Tablet may adjust the expressions of TR, DioⅠand CYP450 genes in liver of rat with hyperthyroidism hepatic damage, which may be one of the molecular action mechanisms, that Yinning Tablet prevent hyperthyroidism hepatic damage.PartⅢThe influence of Yinning Tablet on the apoptosis in rat liver with hyperthyroidism hepatic damageObjective:To investigate the effect of Yinning Tablet on expression of Fas,FasL, TNF-α,Caspase-3 and Bcl-2 proteins related to apoptosis in rat liver with hyperthyroidism. To assess the protective action and possible mechanism of Yinning Tablet on liver dysfunction induced by hyperthyroidism.Methods:Sprague-Dawley (SD) female rats were perfused intragastrically with Levothyroxine Sodium Tablet to establish the rat model of hyperthyroidism hepatic damage. Yinning Tablet of low, medium and high doses and Jiakangling as a positive control were treated for 30 days. After treatment, liver samples were immediately collected. The expressions of Fas, FasL and TNF-a were monitored by immunohistochemical method, and the expressions of Caspase-3 and Bcl-2 in liver were detected by Western Blot.Results:(1) Effect of Yinning Tablet on Fas, FasL and TNF-a expressions in rat liver with hepatic damage induced by hyperthyroidism:Positive products in immune response of Fas and FasL were brown,mainly expressed in the cytoplasm.The immunoreactive product of TNF-a was brown, mainly expressed in the cytoplasm, cell membrane and inflammatory cells, that there was scattered small amount of expression in the control group and was significantly increased in the model group, the ranges of expression increased, there were significant statistical differences between the control and the model groups (p<0.01).Compared with the model group, Yinning Tablet medium and high doses groups have decreased the expressions of Fas, FasL and TNF-a (P<0.01)(2) The influence of Yinning Tablet on expressions-of Caspase-3 and Bcl-2 in rat liver with hyperthyroidism hepatic damage:The expression of Caspase-3 was significantly increased and the expression of Bcl-2 was markedly decreased in the model group than in the control group (P<0.01).Yinning Tablet in three groups can significantly down-regulate the Caspase-3 expression and up-regulate the Bcl-2 expression compared with the model group (P<0.01)Conclusions:Fas, FasL, TNF-α, Caspase-3 and Bcl-2 have been showed abnormal expression in rat liver with hyperthyroidism hepatic damage. Yinning Tablets played a protective role in the pathogenesis of the hepatic damage induced by hyperthyroidism in rat. It is proposed that the effect is concerned with inhibition apoptosis.PartⅣYinning Tablet reduce the hepatic damage of BRL hepatocyte induced by T3 in vitroObjective:To observe the effect of Yinning Tablet on proliferation, hepatic function and oxidative stress of BRL hepatocyte induced by T3, and prepare the laboratorial foundation for further study on its therapeutic mechanism.Methods:(1) Preparation of drug serum:Twenty five SD females were randomly divided into 5 groups:normal serum group, Yinning Tablet groups(low, medium and high dose), Jiakangling group. After administration of drug serums were obtained from abdominal vein of all rats.(2) Effect of Yinning Tablet on the hepatic damage of BRL hepatocyte induced by T3:The T3 injury model of BRL hepatocyte was established. Under the action of different drug serum concentrations of Yinning Tablet, the change of the cell viability was detected by MTT method, the levels of ALT and AST in the culture supernatant were determined by automatic biochemical analyzer, the values of intracellular MDA and SOD were detected by absorption spectrometry.Results:(1) Changes in cell survival rate of BRL:With the increasing of concentration of Yinning Tablet, the BRL hepatocytes survival rate increased in a dose-dependent manner. Compared with the drug serum in low concentration of Yinning Tablet, the drug serum in medium concentration markedly increased cell survival rate (P<0.05). The drug serum in high concentration of Yinning Tablet tended to increase cell survival rate, but did not have statistical difference than the drug serum in medium concentration(P>0.05).(2) Changes of ALT and AST in the culture medium of BRL hepatocyte: Yinning Tablet can reduce the release of ALT and AST compared with the model group (P<0.05). There was statistical difference between the low and the medium drug serum concentrations of Yinning Tablet (P<0.05). The drug serum in high concentration of Yinning Tablet tended to decrease AST and ALT, but did not have statistical difference than the drug serum in medium concentration (P>0.05).(3) Changes of MDA and SOD in the cell suspension of BRL hepatocyte Compared with the control group, the contents of MDA were significantly increased(P<0.05), but SOD activities were markedly decreased in the cell suspension of model group (P<0.05). Jiakangling group and Yinning Tablet in medium and high groups can reduce the content of MDA significantly and Yinning Tablet in low,medium and high doses recover the activity of SOD markedly (P<0.05). Conclutions:The drug serum concentrations of Yinning Tablet play a protective role in the pathogenesis of the hepatic damage of BRL induced by T3, increase the BRL hepatocytes survival rate injured by T3, decrease transaminase levels in the cell culture supernatant, reduce oxidative stress of hepatocytes.