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ObjectiveTo explore the possible mechanisms of Tongfengning （痛风宁， TFN） in treating hyperuricemia （HUA） of spleen deficiency with exuberance of dampness syndrome. MethodsTen of 60 mice were randomly selected， and were fed with regular diet as the control group， while the remaining 50 mice were fed with high-fat and high-sugar diet combined with excessive exercise and potassium oxonate-allopurinol suspension to establish an HUA animal model of syndrome of spleen deficiency with exuberance of dampness. After the successful modeling， in order to better observe the effects of TFN on the intestinal microbiota of the model mice， a mixed antibiotic suspension was administered by gavage to induce further dysbiosis of the intestinal microbiota in the model mice. Fifty sucessfully modeled mice were randomly divided into model group， TFN group， allopurinol group， probiotics group， and an allopurinol + probiotics group， 10 in each group. The TFN group was administered TFN liquid at a dosage of 19.11 g/（kg·d） by gavage. The allopurinol group was administered allopurinol suspension at a dosage of 78 mg/（kg·d） by gavage. The probiotics group was administered live combined Bifidobacterium and Lactobacillus tablets suspension at a dosage of 3 g/（kg·d） by gavage. The allopurinol + probiotics group was administered allopurinol at a dosage of 78 mg/（kg·d） and live combined Bifidobacterium and Lactobacillus tablets suspension at a dosage of 3 g/（kg·d） by gavage. The control group and model group were administered normal saline at a dosage of 19.11 ml/（kg·d） by gavage. The interventions were continued for 21 days. In order to maintain a stable high blood uric acid state， all groups but the control group continued modeling while receiving drug intervention. The changes in spleen deficiency syndrome scores， blood uric acid levels， microbial community structure， acetic acid and butyric acid content in intestinal lavage fluid， adenosine deaminase （ADA） and xanthine oxidase （XOD） content in small intestine tissue， as well as ATP-binding cassette transporter G2 （ABCG2）， glucose transporter 9 （GLUT9） protein and mRNA expression in the small intestine tissue were compared among the groups of mice. ResultsCompared with the control group， the model group showed increased spleen deficiency syndrome scores， blood uric acid levels， relative abundance of phylum Firmicutes， Firmicutes/Bacteroidetes ratio， abundance of Bacteroides genus， Klebsiella genus， and Enterococcus genus， acetic acid content in intestinal lavage fluid， ADA and XOD content in small intestine tissue， as well as GLUT9 protein and mRNA expression （P<0.05）. The number of operational taxonomic units （OTUs） of intestinal microbiota， relative abundance of Bacteroidetes phylum， abundance of Lactobacillus genus and uncultured Bacteroides genus， butyric acid content in intestinal lavage fluid， and ABCG2 protein and mRNA expression in small intestine tissue were significantly decreased （P＜0.05）. Compared with the model group， in the group treated with TFN， probiotics， and allopurinol + probiotics， the spleen deficiency syndrome score， blood uric acid level， relative abundance of Firmicutes， acetic acid content in intestinal lavage fluid， ADA and XOD content in small intestine tissue， GLUT9 protein and mRNA expression significantly decreased. The number of gut microbiota OTUs， relative abundance of proteobacteria， butyric acid content in intestinal lavage fluid， ABCG2 protein and mRNA expression in small intestine tissue significantly increased （P＜0.05）. In the probiotics group， the ratio of Firmicutes to Bacteroidetes decreased. In the TFN group， the abundance of Lactobacillus and uncultured Bacteroidetes significantly increased， while the abundance of Parabacteroides， Klebsiella， and Enterococcus significantly decreased （P＜0.05）. Compared with the TFN group， allopurinol group and the probiotics group showed elevated blood uric acid levels， abundance of Bacteroidetes， ADA and XOD levels in intestinal tissue， and GLUT9 mRNA expression. The relative abundance of Firmicutes， abundance of lactobacilli， and ABCG2 mRNA expression significantly decreased. The probiotics group showed elevated GLUT9 protein expression in intestinal tissue. The probiotics group and the allopurinol plus probiotics group showed significantly higher scores for spleen deficiency syndrome in mice， and lower levels of butyric acid in mouse intestinal lavage fluid. The allopurinol group showed decreased numbers of OTUs in mouse intestinal flora， decreased abundance of proteobacteria， and butyric acid levels in intestinal lavage fluid. The allopurinol group also showed decreased ABCG2 protein expression in intestinal tissue， increased acetic acid levels in intestinal lavage fluid， increased abundance of Klebsiella， and significantly elevated GLUT9 protein expression （P＜0.05）. ConclusionsThe treatment of HUA with TFN may be associated with the regulation of intestinal probiotics （such as lactobacilli） and pathogenic bacteria （such as Klebsiella）， as well as the production of bacterial metabolites such as acetic acid and butyric acid. It may also involve reducing the expression of ADA and XOD in the intestines， decreasing intestinal uric acid production， upregulating the expression of intestinal epithelial urate transporter ABCG2， downregulating GLUT9 expression， and promoting intestinal uric acid excretion. These factors are related to the syndrome of spleen deficiency with exuberance of dampness.
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Objective:Study on the mechanism of Tongfengning in promoting uric acid excretion from the perspective of urate transporter and mRNA in renal of hyperuricemia （HUA） model rats. Method:The 80 sprague-dawley rats were randomly divided into two groups， the blank group with 20 rats and the model group with 60 rats. Rats in model group were established as hyperuricemia （HUA） models by intraperitoneal injection of oxonic acid potassium salt （OAPS） and intragastric administration ethambutol hydrochloride （EMB） once a day for 21 days. After successful modeling， rats in the model group were divided into the model group， Tongfengning group and benzbromarone group， with 20 rats per group. Tongfengning solution （15.3 g·kg-1·d-1） was administered to the Tongfengning group by gavage feeding. Rats in benzbromarone group were administered 5.2 mg·kg-1·d-1 benzbromarone suspension， whereas those in the blank group and the model group were administered the equivalent amount of normal saline for 21 days. On days 14th and 21st following intervention， urine， blood， and kidney were collected from rats， serum uric acid （SUA） and urinary uric acid （UUA） levels， blood urea nitrogenand（BUN） and creatinine（CRE） levels and the expression of urate transporter proteins and their mRNAs of all rats were detected by enzyme-colorimetric method， urease method， sarcosine oxidase method， Western blot and Real-time quantitative PCR（Real-time PCR）， respectively. Result:On days 14th and 21th following intervention， compared with blank group， SUA， CRE and BUN levels， and urate transporter 1（URAT1），glucose transporter 9（GLUT9） expression increased（P<0.05，P<0.01）， whereas UUA level， and adenosine triphosphate-binding cassette transporter protein G2（ABCG2）， organic anion 1（OAT1）， organic anion 3（OAT3） expression decreased in the model group（P<0.05，P<0.01）. Compared with model group， SUA， CRE and BUN levels， and URAT1， GLUT9 expression decreased in Tongfengning group and the benzbromarone group（P<0.05）， whereas UUA level， and ABCG2， OAT1， OAT3 expression increased（P<0.05）. Creatinine and BUN levels decreased in the Tongfengning group（P<0.05，P<0.01）， with the trend much better than the benzbromarone group（P<0.05）. On day 21st， except for the BUN level did not change much compared with day 14th， all the rest indicators got improved obviously. Conclusion:Intraperitoneal injection of OAPS and intragastric administration of EMB can cause HUA models with renal dysfunction. Tongfengning reduced URAT1， GLUT9 mRNA and protein expression， and upregulated ABCG2， OAT1， OAT3 mRNA and protein expression in the rat kidney， which may be one of the mechanisms of promoting uric acid excretion. Tongfengning has a certain protective effect on renal function.
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Objective: Study on the mechanism of Tongfengning in reducing serum uric acid from the perspective of renal urate transporter. Method: The human renal tubular epithelial cells(HK-2)was randomly divided into normal group, model group, Tongfengning low, medium and high dose group (7.65,15.3,30.6 g·kg-1) and benzbromarone group (50 μmo1·L-1),different culture media were given for intervention.HK-2 and cell supernatant were collected after 24 h of intervention. The expressions of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporter 1(OAT1), organic anion transporter 3(OAT3), and ATP-binding cassette superfamily G member 2 (ABCG2) protein and mRNA were detected in HK-2 of all groups by Western blot and Real-time PCR. Result: Compared with normal group, the expression of URAT1, GLUT9 protein and mRNA was significantly increased(PPPPPConclusion: Tongfengning can regulate the reabsorption and secretion of uric acid in renal tubules, promote the excretion of uric acid in kidney and reduce the level of serum uric acid by down-regulating the expression of URAT1, GLUT9 protein and mRNA in HK-2 and up-regulating the expression of ABCG2 protein and mRNA. It is suggested that the regulation of renal uric acid transporter protein may be one of the specific mechanisms of Tongfengning to reduce serum uric acid by promoting dampness and turbid removal. OAT1, OAT3 protein and mRNA were not expressed in HK-2 cultured in vitro.
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Objective:To discuss the influence of Tongfengning Capsule(TFN)in the rats with acute gouty arthritis,and to clarify the curative effect and mechanism of TFN in the acute gouty arthritis rats.Methods:The rat model of acute gouty arthritis was induced by injection of microcrystal sodium urate solution into articular cavity of the ankle joint.A total of 48 mice were divided into blank control group,model group,low(100 mg·kg-1),middle(200 mg·kg-1)and high(550 mg·kg-1)doses of TFN groups,colchicine(0.63 mg·kg-1)positive drug control group;8 rats in each group.The degrees of joint swelling of the rats were monitored at different time after modeling.The number of white blood cells(WBC),lymphocytes and neutrophils in the whole blood of the rats in various groups were detected,and the nitric oxide(NO)levels in the serum and homogenate as well as serum uricacid(UA)were detected.The levels of IL-1βand TNF-αin the soft tissue and synovial fluid around the ankle joint of the rats in various groups were measured.Results:Compared with model group,the swelling degrees of ankle joint of the rats in positive drug control group and high dose of TFN group were significantly decreased in 0-8 h(P<0.05 or P<0.01),especially in 4 h(P<0.01);but there were no significant differences in low and medium doses of TFN groups(P> 0.05).Compared with model group,the levels of WBC, lymphocytes and neutrophils in the blood of the rats in positive drug control group and TFN groups were decreased, especially in high dose of TFN group(P<0.05).The UA levels in serum of the rats in positive drug control group and high dose of TFN group were significantly lower than that in model group(P<0.05 or P<0.01),and the levels of serum NO was increased(P<0.05);but there were no significant differences in low and medium doses of TFN groups(P<0.05).The levels of NO,IL-1βand TNF-αin soft tissue and synovial fluid of the rats in positive drug control group and high dose of TFN group were significantly lower than those in model group(P<0.05 or P< 0.01);but there were no significant differences in low and medium doses of TFN groups(P<0.05). Conclusion:TFN has a remarkable effect in the treatment of gouty arthritis,and this effect may be associated with inhibiting the inflammatory reactions.
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Objective: To discuss the influence of Tongfengning Capsule (TFN) in the rats with acute gouty arthritis, and to clarify the curative effect and mechanism of TFN in the acute gouty arthritis rats. Methods: The rat model of acute gouty arthritis was induced by injection of microcrystal sodium urate solution into articular cavity of the ankle joint. A total of 48 mice were divided into blank control group, model group, low (100 mg · kg-1), middle (200 mg · kg-1) and high 550 mg · kg-1) doses of TFN groups, colchicine (0.63 mg · kg-1) positive drug control group; 8 rats in each group. The degrees of joint swelling of the rats were monitored at different time after modeling. The number of white blood cells (WBC), lymphocytes and neutrophils in the whole blood of the rats in various groups were detected, and the nitric oxide (NO) levels in the serum and homogenate as well as serum uricacid (UA) were detected. The levels of IL-1β and TNF-a in the soft tissue and synovial fluid around the ankle joint of the rats in various groups were measured. Results: Compared with model group, the swelling degrees of ankle joint of the rats in positive drug control group and high dose of TFN group were significantly decreased in 0 - 8 h (P 0.05). Compared with model group, the levels of WBC, lymphocytes and neutrophils in the blood of the rats in positive drug control group and TFN groups were decreased, especially in high dose of TFN group (P<0.05). The UA levels in serum of the rats in positive drug control group and high dose of TFN group were significantly lower than that in model group (P<0.05 or P<0.01), and the levels of serum NO was increased (P<0.05); but there were no significant differences in low and medium doses of TFN groups (P<0.05). The levels of NO, IL-1β and TNF-a in soft tissue and synovial fluid of the rats in positive drug control group and high dose of TFN group were significantly lower than those in model group (P<0.05 or P< 0.01); but there were no significant differences in low and medium doses of TFN groups (P<0.05). Conclusion: TFN has a remarkable effect in the treatment of gouty arthritis, and this effect may be associated with inhibiting the inflammatory reactions.
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Objective:To discuss the influence of Tongfengning Capsule (TFN) in the levels of uricacid (UC),creatinine (Cr) and urea nitrogen (BUN) in mouse serum and the activities of the xanthine oxidase (XOD),adenosine deaminase (ADA) activity in the liver homogenate of the mice with hyperuricemia,and to observe the improvement effect of TFN on the pathological changes of liver tissue and to clarify its mechanisms.Methods:The models of mouse hyperuricemia were induced by yeast extract with potassium oxonate.Seventy mice were divided into blank control group,model group,low (200 mg · kg 1),medium (400 mg · kg-1) and high (800 mg · kg-1) doses of TFN groups,allopurinol positive drug control group (50 mg · kg-1),Tongfengshu (TFS,600 mg · kg-1) positive drug control group (n=10).The levels of UC,Cr,BUN in serum and the activities of XOD,ADA in homoggenate were detected and the histopathological changes of the kidney tissue of the mice were measured with HE staining.Results:Compared with blank control group,the levels of serum UC,Cr and BUN ofthe mice in model group were significantlyincreased (P＜0.01),and the activities of XOD and ADA in liver tissue were also increased (P＜0.01).Compared with model group,the levels of serum UC,Cr and BUN of the mice in positive drug control groups and different doses of TFN groups were decreased (P＜0.01),and the activities of XOD and ADA in liver tissue were also decreased (P＜0.05),especially in high dose of TFN group.Compared with model group,the pathologic changes such as renal glomerulus atrophy,renal interstitial fibrosis and expansion of renal tubule of the mice in positive drug control groups and high dose of TFN group were improved to a certain extent.Conclusion:TFN has improvement effcet on the hyperuricemia in the mice and its mechanism is related to the inhibition of uricogenesis and the promotion of UC excretion.
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Objective:To discuss the influence of Tongfengning Capsule (TFN) in the levels of uricacid (UC),creatinine (Cr) and urea nitrogen (BUN) in mouse serum and the activities of the xanthine oxidase (XOD),adenosine deaminase (ADA) activity in the liver homogenate of the mice with hyperuricemia,and to observe the improvement effect of TFN on the pathological changes of liver tissue and to clarify its mechanisms.Methods:The models of mouse hyperuricemia were induced by yeast extract with potassium oxonate.Seventy mice were divided into blank control group,model group,low (200 mg · kg 1),medium (400 mg · kg-1) and high (800 mg · kg-1) doses of TFN groups,allopurinol positive drug control group (50 mg · kg-1),Tongfengshu (TFS,600 mg · kg-1) positive drug control group (n=10).The levels of UC,Cr,BUN in serum and the activities of XOD,ADA in homoggenate were detected and the histopathological changes of the kidney tissue of the mice were measured with HE staining.Results:Compared with blank control group,the levels of serum UC,Cr and BUN ofthe mice in model group were significantlyincreased (P＜0.01),and the activities of XOD and ADA in liver tissue were also increased (P＜0.01).Compared with model group,the levels of serum UC,Cr and BUN of the mice in positive drug control groups and different doses of TFN groups were decreased (P＜0.01),and the activities of XOD and ADA in liver tissue were also decreased (P＜0.05),especially in high dose of TFN group.Compared with model group,the pathologic changes such as renal glomerulus atrophy,renal interstitial fibrosis and expansion of renal tubule of the mice in positive drug control groups and high dose of TFN group were improved to a certain extent.Conclusion:TFN has improvement effcet on the hyperuricemia in the mice and its mechanism is related to the inhibition of uricogenesis and the promotion of UC excretion.