The Effect Of Acetazolamide On AQP1 And AQP3 Expression In Fibroblasmike Synoviocyte Of Rheumatoid Arthritis

Objective：1. To observe the expression of AQPs in fibroblast-like synoviocyte ofrheumatoid arthritis;2. Comparing to OA, to discuss acetazolamide on the RA FLS AQPs andinflammatory factors expression, and explore the role of AQPs in RApathogenesis.Methods：In this experimental study, 12 RA with knee hydrarthrosis and 10 OAwith knee hydrarthrosis were recruited. FLS were separated from synovia andcultured in vitro. FLS was purified when cells were tansfered to 3～5generations. RA FLS was treated with different concentrations ofacetazolamide（10-4mol/L,10-6mol/L,10-8mol/L）in different time(24h,48h,72h). The MTT method was used to study the growth inhibition ofRA FLS; The expression of AQP1mRNA and AQP3mRNA in RA and OAwas measured by RT-PCR; ELASA was used to detect concentration ofTNF-αand IL-1βin cell-cultured supernatant fluid; To detect the expressionof AQP1 in FLS with the same concentration of acetazolamide(10-4mol/L)indifferent time (24h,48h,72h) by immunofluorescence.Results：1. The MTT method was used to study the growth inhibition of RA FLS.Acetazolamide（10-4mol/L,10-6mol/L,10-8mol/L）had no effect 0f RA FLS on absorbance value(P>0.05).Different concentrations of acetazolamidesignificantly inhibited the proliferation of RA FLS in a time-dependentmanner(P<0.05). That to say, the results of MTT analysis which detected thecytoactive of RA FLS demonstrated that acetazolamide had no effect ondecreasing cellular survival rate. The inhibition was not produced by drugtoxicity.2. The expression of AQP1mRNA and AQP3 mRNA were detected inRA FLS. The level of AQP1mRNA in RA FLS was significantly higher thanOA FLS(P<0.05). Different concentration of acetazolamide（10-4mol/L,10-6mol/L,10-8mol/L）was able to significantly decrease the expression level ofAQP1mRNA in RA FLS (P<0.05). The role was in a time-dependent anddose-dependent manner. There was no significant difference in transcriptionlevels of AQP3mRNA between RA FLS and OA FLS(P>0.05).3. Immunofluorescence showed that, AQP1 protein significantlydistributed plasmalemma. AQP1 protein expression was very apparentwithout treating acetazolamide, whereas the expression of AQP1 protein wassignificantly attenuated by acetazolamide in different time.4. Different concentration of acetazolamide（10-4mol/L,10-6mol/L, 10-8mol/L）could significantly inhibit the expression level of IL-1βand TNF-αincell-cultured supernatant fluid from RA FLS (P<0.05). The role was in atime-dependent and dose-dependent manner.Conclusion：1. AQP1 protein significantly distributed RAFLS plasmalemma. Theabnormal expression of AQP1 resulted in disorder of water metabolism andtransport mechanism, so, that might be one of the mechanism of synovialinflammation and arthroedema. 2. Acetazolamide degraded the expression of AQP1mRNA and AQP1protein in RA FLS while having no effect on the expression of AQP3mRNA,inhibited the proliferation of RA FLS and decreased the secretion of TNF-αand IL-1βin cell-cultured supernatant fluid from RA FLS.