| Backgrounds and Objective:Gout is a clinical syndrome which is attributed to precipitation and deposition of monosodium urate (MSU) crystals on the tissue or organ caused by purine dysbolism and /or excretion reduction and continuous elevation of uric acid. Some researches have showed that inflammation and immune both involved in the pathogenesis of gout. Nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) is an important member of innate immunity which can identify the formation and activation of NLRP3 inflammasome caused by MSU, and then induce the inflammatory reaction caused by maturation and release of IL-1β and so on. We already have known that alternative splicing Pre-mRNA is an important mechanism for controlling gene expression and producing protein diversity, the variations of alternative splicing are relative to cell physiology, developmental regulation and diseases occurrence, the specific alternative splicing in particular environment tend to be the molecular signals of certain diseases and also can be the cause of certain diseases.However, the role of NLRP3 inflammasome genes transcript variants in the pathogenesis of patients with primary gout in different TCM syndromes is unclear yet, so this study regards this as the pointcut to explore mechanism of inflammasome genes transcript variants in the pathogenesis of patients with primary gout (PG) in different TCM syndromes.Methods(1):The expression of NLRP3 inflammasome genes transcript variants mRNA in peripheral blood monocytes (PBMCs) of 96 patients with PG and 30 healthy persons as healthy controls (HC) was measured by semi quantitative polymerase chain reaction (RT-PCR)and/or real-time quantitative polymerase chain reaction (qRT-PCR), among them 44 patients were in acute phase (APPG group) and 52 patients were in non-acute phase(NAPPG group). The expression of NLRP3, PYCARD, CASP1 and NF-κB(p50) protein in patients with PG and HC were measured by western blot, the expression of plasma IL-1β, IL-2 and TNF-α protein in patients with PG and HC were measured by enzyme-linked immuno sorbent assay(ELISA). The expression of plasma CRP was measured by lmmunoturbidimetry. The data were compared between multiple groups with One-way ANOVA, two groups with least significant difference (LSD), the variables of different groups were analyzed with t-test or rank transformation analysis.Methods(2):Peripheral venous blood was collected from 12 patients in APPG,13 patients in NAPPG and 8 healthy persons in HC, then were cultured and stimulated respectively by PBS (0.2μg/mL), MSU, LPS and MSU+LPS for 6,12 and 24h. The plasma was separated and PBMCs was extracted after stimulation. The expression of NLRP3 inflammasome genes transcript variants mRNA in each group were measured by RT-PCR and/or qRT-PCR before and after stimulation. The expression of NLRP3, PYCARD, CASP1 and NF-KB(p50)protein in PBMCs of each group were measured by western blot, the expression of plasma IL-1β protein in each group was measured by ELISA. The expression of plasma CRP was measured by lmmunoturbidimetry. The statistical method was the same as before.Methods(3):The levels of White blood cell count (WBC), neutrophil absolute value (GR), lymphocyte absolute value (LY), monocyte absolute value (MO), alanine transaminase(ALT), aspertate transaminase(AST), albumin (ALB), globulin (GLOB), urea (Ur), blood uric acid (UA), creatinine (Cr), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), low density lipoprotein cholesterol (VLDL-C), apolipoprotein B100 (apoB100), apolipoprotein Al (apoAl) were tested in 44 patients in APPG group,52 patients in NAPPG group and 30 persons in HC group. The difference of those indicators were compared and the correlation of those indicators with the expression of NLRP3 inflammasome genes transcript variants mRNA were also analyzed, the data were compared between multiple groups with One-way ANOVA, two groups with least significant difference (LSD), the variables of different groups were analyzed with t-test or Rank transformation analysis, the correlation between variables were analyzed by spearman correlation analysis.Method (4):Peripheral venous blood was collected from 3 patients in APPG,3 patients in NAPPG and 4 healthy persons in HC, then cultured and stimulated respectively by berberine with different concentration which was 5μg/mL and 10μg/mL. The plasma was separated and PBMCs were extracted after the blood cultured for 6 and 12h respectively, then the expression level of NLRP3 inflammasome gene transcript variant mRNA was detected by qRT-PCR and the expression level of plasma CASP1 protein was detected by ELISA before and after stimulation in every group. The statistical method as the same as before.Results(1):The expression of NLRP3 inflamasome genes and part of their transcript variants mRNA in APPG, NAPPG, IPSBS, ODHS, PDIDS and QBDS groups all had statistical differences compared with HC group (P<0.05,P<0.01); the expression of NLRP3, PYCARD, CASP1 and NF-KB(p50) protein in patients with PG in different phases and TCM syndromes had statistical differences compared with HC group (P<0.05, P<0.01); the expression of plasma IL-1β, IL-2, TNF-a and CRP protein in patients with PG in different phases and TCM syndromes also had statistical differences compared with HC group (P<0.05, P<0.01), and also had statistical differences between each group (P<0.05,P<0.01).Results(2):After stimulated by MSU, LPS and MSU+LPS, the expression of NLRP3 inflammasome genes and part of their transcript variants mRNA in patients with PG in different phases and TCM syndromes had statistical differences compared with HC group and before stimulated,which we called control group (CG) (P<0.05, P<0.01); the expression of NLRP3 inflammasome and NF-KB(p50) protein in patients with PG in different phases and TCM syndromes after stimulation had statistical differences compared with HC and CG group (P<0.05, P<0.01); the expression of plasma IL-1β protein in patients with PG in different phases and TCM syndromes after stimulation also had statistical differences compared with HC and CG group (P<0.05, P<0.01), and also had statistical differences between each group (P<0.05,P<0.01).Results(3):The testing results of clinical lab indicators showed the levels of part of blood routine test indicators, liver and kidney function indicators and blood lipids in APPG and NAPPG group had statistical differences compared with HC group (P<0.05, P<0.01); the correlation analysis results showed that the expression of part of NLRP3 inflammasome genes and their transcript variant mRNA in APPG group had corrrelation with part of clinical lab indicators (P<0.05), and also had corrrelation with IL-1β mRNA in PBMCs and plasma IL-1(3 protein (P<0.05), but the expression of NLRP3 inflammasome genes and their transcript variants mRNA in NAPPG and HC group had no corrrelation with IL-1β mRNA and plasma IL-1β protein (P>0.05).Results(4):After stimulated by barbering (5μg/mL) and barbering (10μg/mL), the expression of NLRP3, PYCARD, CASP1, NLRP3-4 and PYCARD-1 gene transcript variants mRNA in patients with PG in different phases and TCM syndromes had statistical differences compared with before stimulated,which we called control group (CG) (P<0.05, P<0.01); the expression of CASP1 protein in patients with PG in different phases and TCM syndromes after stimulation had statistical differences compared with CG group (P<0.05, P<0.01).Conclusion:NLRP3 inflammasome genes and their transcript variant mRNA may play an important role in the regulation of inflammatory reaction of patients with PG in different phases and TCM syndromes; MSU has a certain regulation effect on the expression of NLRP3 inflammasome genes and their transcript variants mRNA and this may be the important tache of the occurrence and development of PG induced by MSU; The change of TCM syndromes of PG may be relative to the regulation of the expression of NLRP3 inflammasome genes and their transcript variants mRNA caused by MSU; Berberine play an important role in regulating the NLRP3 inflammasome and part of their gene transcript variant of patients with gout. |
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