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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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BACKGROUND:At present, the separation and culture technique of chondrocytes has been mature, but the chondrocytes grow slowly which are prone to degenerate using the present technique. It is not conducive to the fol ow-up test. OBJECTIVE:To investigate and improve the separation and culture method of articular chondrocytes of New Zealand rats at 4 weeks of age. METHODS:New Zealand rats aged 4 weeks were selected to take cartilage tissues from the bilateral knees that were resected under aseptic condition. Chondrocytes were isolated by type Ⅱ col agenase enzyme digestion and mechanical isolation method. The cells were cultured and passaged, and then identified by morphologic observation, toluidine blue staining and type Ⅱ col agen enzyme immunohistochemical methods. Growth curve was pictured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS AND CONCLUSION:Inverted microscope observation showed that the primary cultured chondrocytes adhered at 6 hours after cultivation. The monolayer formation occurred at 72 hours after cultivation, and the cells were ready to be passaged at 96 hours after cultivation. In the fourth generation, some cells represented a spindle-like appearance. In the fifth generation, most cells turned into irregular shape appearance, and cellproliferation capacity diminished. Toluidine blue staining showed that the nuclei of cultured chondrocytes were blue and cytoplasm was pale blue. Immunofluorescent staining showed that cultured chondrocytes had a positive expression of col agen type Ⅱ and the color was tawny. Proliferative rate of chondrocytes in the first to third generations had no differences (P<0.05), while differences were found compared with the fourth generation in 4-7 days (P<0.05) and the fifth generation in 1-7 days (P<0.05). The results indicate that type Ⅱ col agenase enzyme digestion and mechanical isolation method is successful for isolating, cultivating New Zealand rat articular chondrocytes in vitro, and the first to third generations can be the best choice for the experiments of knee osteoarthritis.
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Objective:To observe the effect of different concentration of Qianggubao decoction extract on proliferation of osteoblastic cells cultured by high glucose in vitro. Methods: osteoblastic cells was isolated from the skull of 1-2 day newly born SD rats by means of Trypsin-collagenase digestion and identified by image analysis,V-G collagen staining,ALP staining, calcification nod staining etc. Different concentrations of Qianggubao decoction extract were added to the osteoblastic cells cultured by high glucose in vitro(final concentration:300mg/dl)and incubuted.The effects of Qianggubao decoction extract on the proliferation of osteoblasts was monitored by MTT analysis. Results:Qianggubao extract of 100, 50, 10?g/ml all promoted for osteoblastic cell proliferation, the 100?g/ml and 50?g/ml had the better effects (P