Inhibition Of Compound Tufuling Granules On Xanthine Oxidase In Hyperuricemia

Hyperuricemia is a clinical disease that blood uric acid level supersaturated due to disorders of purine metabolism, characterized by accumulation of uric acid caused by increase synthesis and reduce excretion of uric acid. Supersaturation of uric acid may force precipitation of uric acid crystals. Gout will be developed as the uric acid crystals depositing in the joints, soft tissues and kidneys, etc. It cannot be ignored that morbidity of hyperuricemia is close to the growled of economic and the trend of age is become younger and younger in recent years. It is important that uric acid synthesis pathway affect uric acid levels in the human body. Xanthine oxidase is a major key rate-limiting enzyme of synthetic of uric acid in purine metabolism. The target of current uric acid suppression drugs on the market for many hyperuricemia is xanthine oxidase. Compound Tufuling Granule is a TCM formula which come from Xie-Zhuo-Chu-Bi-Fang, used for hyperuricemia and gout. We has studied the mechanism of multi-faceted of the Granule before, now this paper will study the inhibition of Compound Tufuling Granule on xanthine oxidase.ObjectiveThis topic discusses the inhibition of Compound Tufuling Granule on xanthine oxidase in hyperuricemia through literature review research and experimental studies. The experimental studies section including animal and in vitro experiments. This topic hope to continue to clarify the multi-target mechanism of Compound Tufuling Granule on treatment of hyperuricemia, provide evidence to support the clinical use for the Granule, provided foundation of herbal drug development of Chinese medicine treatment on hyperuricemia.MethodReview:This chapter is divided into two sections, the first section is about Western medicine research progress on hyperuricemia and gout, while the second section about TCM medical research progress on hyperuricemia and gout, and previous studies of Compound Tufuling Granule.Experimental study:(1) animal experimental:48 SPF male SD rats were randomly divided into six groups after feeding adaptation, as the control group, model group, febuxostat group, CTG low-dose group, CTG mid-dose group and CTG high-dose groupExcept the control group, the rest of the groups were given potassium oxonate 100 mg/kg intraperitoneally and hypoxanthine 500 mg/kg orally to induce hyperuricemia. Modeling agent administered once daily, continued until the end of experiment, total of eight days. Four days after the start of the experiment, at 0.5 hours later of given modeling agent, CTG low-dose group, CTG mid-dose group and CTG high-dose group were given 1 g/kg,2 g/kg,4 g/kg administered Compound Tufuling Granule, while febuxostat group received febuxostat 4 mg/kg orally. Model group and the control group were only given the normal saline. The treatments were given once a day, a total of five days of treatment. The entire experiment lasted eight days. After the last administration, blood was collected by cardiac blood, liver was collected as two pieces, one placed in liquid nitrogen, another placed in 4% poly formaldehyde fixation. Blood samples were centrifuged to detect serum uric acid, liver stored in ammonia was made to 10% homogenates and then was detected about xanthine oxidase activity, and theformalin-fixed liver tissue was detected about xanthine oxidase gene expression in situ hybridization detection. (2) Cell experiments:the SPF male rats of 250 g-280 g were given Compound Tufuling Granule twice daily for 5 consecutive days to prepared medicine serum. The template gene of XDH was extracted and amplified from the cells, then was constructs to recombinant plasmid pENTER-XDH, and was confirmed by double enzyme digestion and sequencing amplification. After comfirmation, the plasmid pENTER-XDH was extracted with internal toxins. The BRL 3A hepatocytes were cultured in vitro. When the cells was steady, cell activity test with herbal medicine serum was been made. And then the cells were divided into six groups, as the blank control group, transfection control group, blank rat serum group, medicine serum low-dose group, middle-dose group and high-dose group. The plasmid pENTER-XDH was transfected to the BRL cells except the blank control group.24 hours later, blank rat serum group was added with xanthine and blank rat serum, herbal medicine serum low, medium and high dose groups were added with low, medium and high concentration Chinese medicine serum and xanthine. Another 24 hours after treatment, the cell culture medium was collected to detect uric acid and xanthine oxidase activity, and the cell was lysed, extracting total RNA to be detected XDH-mRNA by quantitative PCR.ResultsExperimental study:(1) Animal experiments:The serum UA level of model group was higher than the control group, the difference was statistically significant (P<0.05), shows that the modeling method is effective. The serum UA levels of febuxostat group was lower than the model group(P<0.05), shows that Western medicine treatment is effective. The CTG low-dose group was not significant differences with the model group (P> 0.05), while the CTG high-dose and mid-dose group were significant higher than model group, the difference was statistically significant (P<0.05), and the high-dose group was more effectively that there was not significant differences between high-dose group and febuxostat group. The results of XO activity were similar to the serum UA level. In situ hybridization, XDH-mRNA expression levels of the model group was much higher than the control group, indicating that after the model rats receiving modeling agent, the liver XDH-mRNA expression is greatly increased (P<0.05). In febuxostat group, XDH-mRNA expression was significantly decreased compared with the model group (P<0.05). In the CTG groups, high-dose group was significantly decreased (P<0.05), no significant difference between high-dose group and febuxostat group (P> 0.05). (2) cell experiments:Plasmid pENTER-XDH was identified by DNA electrophoresis with PCR products and double enzyme digestion products. The insert gene and PCR product were at the same level, which meet the length of the XDH gene, showed that plasmid was constructed successfully. In CCK-8 assay, there were no significant different among the cell culture medium containing 10% fetal bovine serum and rat blank serum and CTG medicine serum (P>0.05). There value was very low, so there was no effect on the OD. There were also no effect on cell activity with rat blank serum and CTG medicine serum (P> 0.05). In qPCR detection, XDH-mRNA expression level in transfected control group was much higher than blank control group, and the difference was statistically significant (P<0.05), showed that transfected plasmid pENTER-XDH success. XDH-mRNA gene expression in rat serum blank control group did not change significantly from transfected group (P> 0.05), showed that normal rat serum had no significant effect on the expression of XDH gene. XDH-mRNA gene expression in the high dose group was significantly decreased with a statistically significant (P<0.05), illustrated medicine serum high-dose group can inhibit XDH gene expression level. Medicine serum mid-dose group was low in expression, but statistics do not show a statistically significant (P> 0.05). Maybe the sample volume was too small. In uric acid and XO activity results, the blank rat serum control group was significantly higher than blank control group and transfection control group, the difference was statistically significant (P<0.05), while the XO activity, the transfection control group and the blank serum rats group were significantly higher than the blank control group. There was no significant difference in uric acid levels between the transfected control group with the blank control group, suggesting that uric acid synthesis is limited when lack of xanthine. XO activity and uric acid levels in CTG mid-dose group and high-dose group were significantly lower than blank rat serum control group, the difference was statistically significant (P<0.05), explained that mid-dose and high-dose of CTG serum can inhibit the production of uric acid and inhibit XO activity. The high-dose group was significantly more effect (P<0.05).ConclusionOne mechanism of treatment of Compound Tufuling Granule in hyperuricemia may be that inhibition on XDH-mRNA levels in the liver and XO activity may reduce production of uric acid. In vitro experiments also confirmed that the medicine serum of the granule could inhibit gene XDH-mRNA overexpression in rat hepatocytes, and also the xanthine oxidase activity, thereby reducing uric acid production.