ABSTRACT
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.
ABSTRACT
Objective:To examinethe absolute numbers of cluster of differentiation (CD4) + T cell subsets in peripheral blood of patients with rheumatism and further to develop a new immunomodulatory therapies which aimed to restore their imbalanced CD4 + T lymphocyte subpopulation. Methods:A total of 6 395 rheumatic patients [4 430 females, 1 965 males, mean age (49±15) years] and 206 healthy controls (HCs) were enrolled in this retrospective cross-sectional study, which included rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), Psoriatic arthritis (PsA), systemic sclerosis (SSc), primary Sj?gren′s syndrome (pSS), Be?het's disease (BD), dermatomyositis/polymyositis (DM/PM), gout and vasculitis. Some patients received treatment combined with immunoregulatory drugs (IMiDs) such as low-dose interleukin (IL)-2, rapamycin, metformin, retinoic acid and coenzyme Q10. The absolute numbers of T helper cell (Th)1, Th2, Th17 and regulatory T cell (Treg) in peripheral blood (PB) of these individuals were measured by Flow Cytometery (FCM). Independent sample t test and paired sample t test were used to compare the levels of CD4 + T cell subsets in PB of patients and HCs, before and after treatment respectively, and P<0.05 was considered statistically significant. Results:Compared with HCs, the mean absolute number of Treg was significantly decreased [(31±15) cell/μl vs (27±17) cell/μl, t=3.407, P<0.01] and the ratio of Th17/Treg was increased in all patients [(0.3±0.2) vs ( 0.4±0.7), t=-9.508, P<0.01]. There was a significant increase in the number of Th17 in patients with AS [(10±8) cell/μl, t=-5.403, P<0.01], PsA[ (11±11) cell/μl, t=-3.829, P<0.01], SSc [(7±6) cell/μl, t=3.114, P<0.01], BD [(11±9) cell/μl), t=-4.774, P<0.01] and gout [(11±9) cell/μl, t= -4.604, P<0.01) , and we observed lower level of Treg in patients with RA[(28±15) cell/μl, t=3.032, P<0.01], SLE [(21±21) cell/μl, t=6.836, P<0.01], AS [(28±15) cell/μl, t=2.216, P<0.05], SSc [(27±16) cell/μl, t=3.698, P<0.05], BD [(27±17) cell/μl, t=2.502, P<0.05], DM/PM [(27±22) cell/μl, t=2.974, P<0.01) and gout [(28±15) cell/μl, t=2.079, P<0.05). After IMiDs combination treatment, the levels of CD4 + T subsets were increased. Interestingly, the expansion of Treg was much more dramatical than those of other effector T cells, resulting in a decrease in ratios of Th17/Treg, especially in SLE [(0.6±1.0) vs (0.5±0.4), t=3.157 , P<0.01]. Conclusion:Impaired balance of pro- and anti-inflammatory immune cells, especially insufficiency of Treg, might be a cornerstone of the pathogenesis of rheumatism. The new immunomodulatory therapies could relatively specifically promote Treg proliferation and restored patients' autoimmune tolerance, which isexpected to provide a new strategy for the treatment of rheumatism.