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Acid Sensing Ion Channels Blocker Amiloride Protected Articular Cartilage in Adjuvant Arthritis Rat and Its Mer-mechanisms

Author: ZhangTengYue
Tutor: ChenFeiHu
School: Anhui Medical University,
Course: Pharmacology
Keywords: Acid-sensitive ion channels Amiloride Adjuvant arthritis Chondrocytes Apoptosis
CLC: R96
Type: Master's thesis
Year: 2010
Downloads: 49
Quote: 0
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Abstract


Objective: To clarify the acid-sensitive ion channel blocker amiloride (Amiloride) on adjuvant arthritis (adjuvant arthritis, AA) rat articular cartilage protective effect; Amiloride from the angle of chondrocyte apoptosis research on AA rat articular cartilage protective effect may be the link; by detecting Amiloride AA rat articular cartilage extracellular free calcium concentration changes in expression of calcium-dependent apoptosis-related enzymes, analysis Amiloride inhibition of AA rat articular chondrocyte apoptosis The possible mechanism of action. Methods: The rats were randomly divided into normal group, the AA model group, Amiloride (2.5,5.0,10.0 mg · kg -1 · d -1 ) group and aspirin ( 50 mg / kg) control group. Complete Freund's adjuvant (CFA) induced AA after 10 days, the rats secondary inflammation of the Amiloride, aspirin ig consecutive days; normal group and model group were given an equal volume of sterile water for injection . To detect the degree of joint swelling in rats with secondary side change, optical microscopy joint pathological changes of immunohistochemical techniques Determination Amiloride AA rats protein synthesis of articular cartilage collagen type II (COII), Alcian staining Determination of Amiloride on AA rat articular cartilage proteoglycan (PG) synthesis, transmission electron microscopy, the Tunel method Amiloride AA rat articular chondrocyte apoptosis was observed. Collagenase - trypsin digestion were isolated and cultured articular chondrocytes, extracellular acidification induced by AA rat articular chondrocyte apoptosis in vitro model of apoptosis by TUNEL staining and PI single staining observed the AA rats joint cartilage cells, calcium imaging the the detection [Ca 2 ] the i level the immunocytochemistry detection the AA rats joint chondrocytes the calpain, the calcineurin, iNOS expression. Results: 1. Protective effect of the acid-sensitive ion channel blocker amiloride (Amiloride) AA rat articular cartilage Amiloride each dose group on the the AA rats inflammation secondary side joint foot swelling no significant impact; pathology observed The smooth surface of the articular cartilage chondrocytes level yet clear increase in the number of mature cartilage cells; immunohistochemistry and Alcian staining showed increased expression levels of the articular cartilage matrix composition, the COII protein and PG. Acid-sensitive ion channel blocker amiloride (Amiloride) AA rat articular chondrocyte apoptosis in vivo experimental TEM observations the edge set Amiloride cartilage within the nucleus of each dose group heterochromatin mild intracellular crude mild expansion of the endoplasmic reticulum, swelling of mitochondria mild, with the model group cartilage nuclear membrane shrinkage, nuclear chromatin margination, nuclear membrane rupture, apoptotic bodies were no obvious apoptosis feature; Tunel method In situ detection of the number of apoptotic cells significantly reduced (P lt; 0.01). Vitro PI staining Amiloride administration group and PcTx1 group rate of apoptosis with the model group was significantly lower; the Tunel method situ detection the Amiloride dose group and PcTx1 of apoptotic positive cells decreased. Acid-sensitive ion channel blocker amiloride (Amiloride) AA rat articular chondrocytes free calcium (ionized free calcium the the of Ca ) levels and calcium-dependent apoptosis-related enzymes expression calcium imaging results show the the administered dose group for Amiloride and PcTx1 of cytoplasm Ca 2 F340/F380 ratio relative to the model group, the difference was statistically significant; Immunocytochemistry results show that calcium Amiloride group ion-dependent apoptosis-related enzymes the calpain the calcineurin iNOS expression was significantly lower (P lt; 0.01). Conclusions: 1. Adjuvant arthritis, ASICs blocker amiloride, no significant inhibition of its inflammatory response, has a protective effect on cartilage cells and extracellular matrix. Acid-sensitive ion channel blocker amiloride (Amiloride) AA rat articular cartilage has a protective effect. 2. The articular chondrocytes's acid-sensitive ion channel blocker amiloride (Amiloride) the AA rat articular chondrocytes by extracellular acidification vitro culture apoptosis inhibition. Amiloride by blocking ASICs to inhibit apoptosis AA rat articular chondrocytes, which play a protective effect of the AA rats joint chondrocytes. Acid-sensitive ion channel blocker amiloride (Amiloride) on [the of Ca 2 ] i levels and calcium-dependent apoptosis-related enzymes calpain, the calcineurin inhibition iNOS expression. Amiloride AA rat articular chondrocytes apoptosis inhibition by blocking of ASICs inhibit Ca 2 overload, thereby inhibiting the calcium-dependent apoptosis-related enzymes the calpain, the calcineurin, iNOS The expression to achieve.

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