Experimental And Clinical Study Of Jaundai Granule In Intervention Of Damp - Heat Syndrome Intrahepatic Cholestasis

Purpose:Through the "external wet and internal wet + drug" to establish model of combination of diseases and syndromes, and to observe the impact of Dan Qing granules on the general condition of rats in the model of damp heat syndrome of liver cholestasis. Effects of changes in pharmacodynamic indexes(ALT、AST、ALP、TBIL、DBIL、TBA、Bile flow and flow rate, Bile component analysis, LP-X, Liver tissue pathological changes)and the expression of drug action mechanism.index(Na~+-H~+ exchange body, NTCP、BSEP), To investigate the effect of Dan Qing granules on the rats which suffered damp heat syndrome of hepatic bile and its mechanism.Material and method:Experimental part:1. Experimental materials:Healthy and clean Wistar 60 rats of male were selected. The treatment group with Dan Qing granules, control group with Yinzhihuang granules.2. Grouping method:The 60 Wistar rats were randomly assigned to six groups: blank control group, model group 1, model group 2, Yinzhihuang granules group, Dan Qing granules of high dose group and low dose group, with 10 rats in each.3. Modeling methods: external WET + internal dampness + drugs.In the long summer heat season 50 rats were supposed to place in damp heat molding box, feeding of glucose and lard mixed liquid for 21 days, made the injection of ANIT which contributes to acute intrahepatic cholestasis.4. Drugs intervention methods:Yin Zhi Huang granules group and Dan Qing granules of low dose group were given to stomach of 1.62g/kg irrigation, Dan Qing granules in high dose group was given to gavage of dosage 6.48g/kg, blank control group, model group and the model group 2 were given 0.9% saline by 10 ml / kg, all experienced 7 consecutive days, once a day.5. Observation index:5.1 The general situation of rats5.2 Pharmacodynamics indexObserve ALT, AST, ALP, TBIL, DBIL, TBA, bile flow and bile flow rate, component analysis, LP-X and pathological changes in liver tissues.5.3 The mechanism of drug targetsObserve Na~+-H~+ NTCP, permutoid, BSEP in liver tissue and expression level of m RNA of rats.Clinical parts:1. Packet60 Patients which Would have been included in accord with the diagnosis were randomly divided into control group and treatment group, each concluded 30 cases.2. Therapeutic method:The control group was given intravenous drip of glutathione of 1.2 grams, 1 grams of s-ademetionine intravenous and oral administration of Ursodeoxycholic Acid Tablets, 250 mg/, 3 /d. The treatment group was given intravenous injection,and based on the control group,added Dan Qing granules, 6g/ time, 3 times /d.3. Observation items3.1 Patients general items3.2 Efficacy observation item(1) Fill out the TCM symptoms rating scale(2) Detection of serum ALT, ALP, GGT, TBIL, 5 ’-NT, DBIL, TBA and CHOL3.3 The mechanism of observation item(1) Detection of inflammatory cytokines, including TNF- alpha, IL-1 and IL-6(2) Detection of indicators of oxidative stress, including SOD and MDA.3.3 Safety observation itemResults:The experimental part:1. Effect on the general condition of ratsThe rats in the control group were in good spirits, water and food consumption.Body weight increased, urine is pale yellow. Rats were put in heat molding box,on the severnth day they experienced dullhair, slightly yellow ears and tail skin, deep yellow urine, loss of appetite, loose stool, lack of energy, less dynamic, irritability. rats were given intragastric administration with ANIT,after half a day the water and food consumption were significantly decreased,and experienced listlessness. In the course of the experiment we found that after 3 days theatment of the granules in high and low dose group and Yinzhihuang granules group,their appetite, mental state of rats began to improve, Every status of rats improved with the duration of treatment.2. The influence on the rat biochemistryModel group 1, model group 2 compared with other 4 groups, the liver enzyme index were significantly increased(P<0.05), and the model group was higher in group1 than group 2, the difference was statistically significant(P<0.05). Compared with the other 4 groups except model group, they showed no significant difference(P>0.05). That indicated that high and low dose of Dan Qing granules can reduce the hepatic enzyme, which would be close to the blank control group, the curative effect is consistent with Yinzhihuang group.Compared with blank control group, high and low dose of Dan Qing granules groups, TBIL, DBIL, TBA index of model group 1 and model group 2 and Yinzhihuang group were significantly increased(P<0.05), but the increase extent of Yinzhihuang group was lower than model group 1 and model group 2, the difference was statistically significant(P<0.05), the increase extent in model group 2 was lower than model group 1, the difference was statistically significant(P<0.05). Compared with the blank control group, Dan Qing granules with high dose group and low dose group, there showed no significant difference(P>0.05).That indicated that Dan Qing granules of high and low dose could significantly reduce the TBIL, DBIL, TBA index of rats, which would close to the blank control group, curative effect is better than that of Yinzhihuang group.3. Effect of bile flow and bile composition in ratsCompared with the other 4 groups, the bile flow rate were significantly decreased in model 1group and model group 2,(P<0.05), and compared each index there showed no significant difference(P>0.05), compared with the other 4 groups except model group, they showed no significant difference(P>0.05). The results indicate that rats can not improve bile flow and flow rate by self repair, which need early drug intervention. Dan Qing granules can improve the bile flow and rate of intrahepatic cholestasis in rats, which would be close to the control group, and the effect was consistent with Yinzhihuang group.Compared with the other 4 groups, LEC and CHOL in model group 1, model group 2 were decreased significantly(P<0.05), the concentration of TB and TBA increased significantly(P<0.05), and there were no significant differences between the two model groups referring to each index(P>0.05). Compared with the other 4 groups except model group, they showed no significant difference(P>0.05). The results indicated that rats can not improve the components of bile by self repair, which need early drug intervention. Dan Qing granules can improve the bile composition of intrahepatic cholestasis in rats, and make it close to the blank control group, the curative effect was consistent with Yinzhihuang group.4. Effect of LP-X on rats:Compared with the control group, the LP-X in serum in the remaining 5 groups increased, but the rising extent of Yinzhihuang granules group and Dan Qing granules in high and low dose group were lower than model group 1 and model group 2, the difference was statistically significant(P<0.05), and the rising extent of model group 2 was lower than model group 1, the difference was statistically significant(P<0.05). There showed no significant difference within the Yinzhihuang granules group, Dan Qing granules in high and low dose group.(P>0.05). That indicated that Dan Qing granules can reduce the LP-X content of damp heat syndrome of rats who suffered intrahepatic cholestasis, but not close to the blank control group, the curative effect is consistent with Yinzhihuang group.5. Effect of Na~+-H~+ on the expression of m RNA in rats.Compared with the blank control group, the expression of m RNA was decreased in the other 5 groups. but the decreasing extent of group of Yinzhihuang and Dan Qing granules in high and low dose group was lower than the model group 1, model group 2, the difference has statistical significance(P < 0.05),and the decreasing extent of model group 2 was lower than the model group 1,the difference was statistically significant(P<0.05).The expression level of dose group Na~+-H~+ exchanger in Dan Qing granules in high and low dose groups was significantly higher than that of Yinzhihuang group(P<0.05).That indicated that Dan Qing granules can improve Na + / H + antiporter m RNA expression of rats which suffered intrahepatic cholestasis, but failed to close to the blank control group, curative effect is better than that of Yin Zhi Huang group.6. Effect of m RNA on NTCP expression in ratsCompared with the blank control group, there showed a decrease of expression of NTCP m RNA in model group 1, model group 2, group of Yinzhihuang, Dan Qing granules in low dose group except Dan Qing granules in high dose group.the difference was significant(P < 0.05).But Dan Qing granules in low dose group showed lower deceasing extent than the model group 1, model group 2 and Yinzhihuang group, the difference is statistically significant(P < 0.05),and the decreasing extent of model group 2 was lower than the Yinzhihuang group, difference was not statistically significant(P>0.05), the difference between model group 2 and Yinzhihuang group was not statistically significant(P>0.05). The expression of Dan Qing granuless in low dose group was significantly lower than the Dan Qing granules in high dose group, significant difference(P<0.05).That indicated that they showed little effect on the expression of NTCP m RNA in Yinzhihuang granules,while Dan Qing granuless can not only improve the expression of NTCP m RNA content, and high dose group can make the expression close to the blank control group, curative effect is obviously superior to low dose group and the group of Yinzhihuang.7. Effect of BSEP m RNA expression on rats.Compared with the blank control group, there showed a decrease of expression of BSEP m RNA in model group 1, model group 2, group of Yinzhihuang, Dan Qing granules in low dose group except Dan Qing granules in high dose group.the difference was significant(P < 0.05), But Dan Qing granules in low dose group and Yinzhihuang group showed lower deceasing extent than the model group 1 and model group 2, the difference is statistically significant(P < 0.05), and the decreasing extent of model group 2 was lower than the model group 1, difference was statistically significant(P<0.05), and Dan Qing granules in low dose group and the group of Yinzhihuang expression level had no significant difference(P > 0.05), but their expression were lower than those of Dan Qing granules in high dose group, significant difference(P < 0.05).That indicated that Dan Qing granules can not only increase the expression of BSEP m RNA in the rats which suffered damp heat syndrome in the liver cholestasis,and high dose group can make the expression close to the blank control group, curative effect is obviously superior to low dose group and the group of Yinzhihuang.8. Effect on the morphology of rat liverLight microscope showed: the structure of the liver of the blank control group was clear and complete, and the liver cells were arranged in a radial order, and no obvious pathological changes were found. In model group1 and model group 2, the swelling of liver cells can be found, the nucleus became larger, and the fat cells and inflammatory cells were found in both groups. Yinzhihuang granules group and Dan Qing granules in low and high dose group were visible of liver tissue structure, compared with model group,the morphology showed better recovery, liver cell edema reduced,fat cells disappeared and there were a small amount of inflammatory cells in the interlobular, hepatic sinusoids, central vein comparison were regular, skin cells were nomal.Under electron microscope: the blank control group of adjacent liver cells Were arranged in neat rows,nucleus were large and nucleolus could be found, mitochondria were round or long clubbing,and There were a nuclear margination, large nuclei and intranuclear heterochromatin increased in model group 1 and model group 2,mitochondria swelling, cristae rupture is vacuolar degeneration and bile canaliculi slightly expansion and inner tube reduced microvilli, Yinzhihuang granules group and Dan Qing granules group in high and low dose were visible in varying degrees of cytoplasm dissolved,organelles were rich, there showed karyopyknosis, mitochondrial contraction.Clinical part:1. In the aspect of improving the clinical curative effect.Dan Qing granules combined with western medicine has significant advantage in treatment and efficacy than that of pure western medicine treatment.2. In the aspect of TCM syndrome, DanQing granules combined with western medicine treatment was better than western medicine.3. In improving the TCM syndrome integral aspect, Dan Qing granules combined with western medicine treatment was better than western medicine,especially in Improvement of part of Chinese medicine symptoms(Hypochondriac pain, dry mouth pain, abdominal distension nausea, skin itching, irritability, yellow urine and stools),they showed more significant effect.4. In reducing liver enzyme, Dan Qing granules combined with western medicine had the same treatment and efficacy with pure western medicine treatment.5. In the reduction of bilirubin, bile acid and CHOL, Dan Qing Granules Combined with western medicine was better in treatment than pure western medicine.6. In the aspect of inflammatory and oxidative stress reaction, Dan Qing granules combined with western medicine treatment was better than the pure western medicine treatment.Conclusion:1. In the reduction of TBIL, DBIL, TBA, curative effect of Dan Qing granules was obviously superior to Yinzhihuang granules. In the improvement of the general conditions of rats, they showed almost the same treatment effect in the reduction of rats ALT, AST, ALP, increasement of bile flow and flow rate, improvement of bile composition and liver tissue damage, Dan Qing granules could make the treatment of intrahepatic cholestasis in rats through up regulation of Na+- H+ exchange body and NTCP and BSEP m RNA expression.2. Dan Qing granules combined with western medicine was advantageous than pure western medicine in the improvement of TCM therapy and improvement of TCM syndromes,the efficacy of two groups have no significant difference in the western medicine and reduction of ALT、ALP、GGT. Dan Qing granules can be used to treat intrahepatic cholestasis patients through the inflammatory reaction and inhibition of oxidative stress.