OBJECTIVETo observe the effect of gut protease activity on visceral hypersensitivity in rats with acute restraint stress.

METHODSSprague-Dawley rats were given 30, 100 or 300 mg/kg camostat mesilate (CM), a protease inhibitor, or saline intragastrically 30 min before acute restraint stress induced by wrapping the fore shoulders, upper forelimbs and thoracic trunk for 2 h. Visceral perception of the rats was quantified as the visceral motor response with an electromyography, and the rectal mucosa and feces protease activity and spinal c-fos expression were measured.

RESULTSCM dose-dependently reduced visceral sensitization elicited by rectal distension, but these doses did not completely inhibit stress-induced visceral sensitization. In normal rats, c-fos expression was found mainly in the superal spinal cord dorsal horn, and after the administration the CM, c-fos-positive cells decreased significantly in all dose groups (P<0.05). In 30 mg/kg CM group, fecal and rectal mucosal protease activity significantly decreased as compared with that in the stress group (P<0.05), and as CM dose increased to 100 and 300 mg/kg, the protease activity decreased even further (P<0.01).

CONCLUSIONThe gut protease is involved in acute stress-induced visceral hypersensitivity, and CM can lower the visceral sensitivity and spinal c-fos expression in rats.

Animals ; Gabexate ; analogs & derivatives ; pharmacology ; Protease Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Sprague-Dawley ; Restraint, Physical ; Spinal Cord ; metabolism ; Stress, Physiological

Objective:To investigate the effects of fluoride exposure on kidney injury in rats and the sirtuin 3 (SIRT3)-fork head protein O3a (FOXO3a)-tensin homolog induced putative kinase 1 (PINK1)/E3 ubiquitin ligase (PARKIN) pathway.Methods:Twenty-four 4-week-old SD rats (clean grade, body mass 100 - 150 g) were selected and divided into three groups according to the randomized numeric table: control group, low fluoride group, and high fluoride group, with eight rats in each group (half male and half female). The control group was given free access to tap water (fluoride ion concentration < 0.5 mg/L), while the low fluoride and high fluoride groups were given free access to tap water and sodium fluoride solutions with fluoride ion concentrations of 5.0 and 50.0 mg/L, respectively, for a period of 180 days. The formation of dental fluorosis in rats was observed and recorded, and the femur, urine and blood samples of rats were collected to measure bone fluoride, urinary fluoride, and blood fluoride levels, and to detect kidney function related indicators (serum uric acid, creatinine, and urea nitrogen contents). Morphological changes of renal tissues stained with hematoxylin-eosin (HE) were observed under a light microscope. Real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression levels of renal SIRT3, FOXO3a, PINK1, PARKIN, microtubule associated protein 1 light chain 3 (LC3), autophagy receptor protein (P62), respectively.Results:Seven and one rats in the low and high fluoride groups were found to haveⅠdegree dental fluorosis, while zero and seven rats were found to haveⅡdegree dental fluorosis. Compared with the control group, rats in the low and high fluoride groups had higher levels of bone fluoride (μg/g: 1.18 ± 0.06, 2.16 ± 0.07 vs 0.52 ± 0.05), urinary fluoride (mg/L: 4.43 ± 0.11, 7.46 ± 0.09 vs 2.58 ± 0.14), blood fluoride (μg/ml: 0.77 ± 0.06, 1.68 ± 0.10 vs 0.52 ± 0.08), serum uric acid (μg/ml: 61.01 ± 4.17, 103.92 ± 5.43 vs 28.68 ± 2.91), creatinine (μg/ml: 74.82 ± 9.61, 132.05 ± 5.35 vs 22.38 ± 4.11), and urea nitrogen (μg/ml: 13.36 ± 1.27, 14.55 ± 0.34 vs 0.29 ± 0.07, P < 0.05). Under the light microscope, the kidneys of the control group showed tight and orderly arrangement of renal tubules and glomerular cells, with complete and clear cell contours. The low fluoride group was similar to the control group and no significant abnormalities were observed. The high fluoride group showed abnormal glomerular structure and atrophy, with some areas of renal tubules showing epithelial cell edema and unclear intercellular boundaries. The results of qRT-PCR assay showed that compared with the control group, the low and high fluoride groups had lower mRNA expression levels of SIRT3 (0.82 ± 0.03, 0.58 ± 0.02 vs 1.00 ± 0.08), P62 (0.75 ± 0.07, 0.28 ± 0.09 vs 1.00 ± 0.07, P < 0.05), and higher mRNA expression levels of FOXO3a (1.35 ± 0.04, 3.01 ± 0.23 vs 1.00 ± 0.08), PINK1 (1.58 ± 0.09, 3.28 ± 0.09 vs 1.00 ± 0.07), PARKIN (1.51 ± 0.04, 1.67 ± 0.10 vs 1.00 ± 0.05), LC3 (1.74 ± 0.07, 2.38 ± 0.18 vs 1.00 ± 0.08, P < 0.05). The results of Western blotting showed that compared with the control group, the low and high fluoride groups had lower protein expression levels of SIRT3 (0.91 ± 0.01, 0.55 ± 0.03 vs 1.00 ± 0.01), P62 (0.94 ± 0.27, 0.66 ± 0.38 vs 1.00 ± 0.19, P < 0.05), and higher protein expression levels of FOXO3a (1.14 ± 0.03, 1.22 ± 0.05 vs 1.00 ± 0.02), PINK1 (1.46 ± 0.03, 1.56 ± 0.03 vs 1.00 ± 0.05), PARKIN (1.98 ± 0.02, 2.33 ± 0.11 vs 1.00 ± 0.06), LC3 (4.10 ± 0.58, 4.93 ± 0.33 vs 1.00 ± 0.13, P < 0.05). Conclusion:Exposure to fluoride can cause renal tissue injury in rats, with downregulation of SIRT3 and P62 expression levels, and upregulation of FOXO3a, PINK1, PARKIN, and LC3 expression levels.

【Objective】 To investigate the expressions of NLRP1 and NLRP3 in the colon of ulcerative colitis (UC) patients and analyze the correlation of the expressions with severity of UC, endoscopic manifestations and related laboratory indicators. 【Methods】 We collected biopsical specimens obtained with colonoscopy in 46 patients with UC (22 mild cases and 24 moderate to severe cases) and 20 cases of normal control group. We used the disease activity index to evaluate the Mayo UC inflammatory activity and immunohistochemical method to detect the protein expression levels of intestinal mucosal NLRP1 and NLRP3 in the tissue. RT-PCR was used to detect the expressions of NLRP1 and NLRP3 mRNA in intestinal mucosal tissues. Meanwhile, the colonoscopy, serum uric acid, C-reactive protein, serum sedimentation rate, platelet count, low-density lipoprotein, and cholesterol of UC patients were also counted to further analyze the relationship between NLRP1 and NLRP3. 【Results】 The expressions of NLRP1 and NLRP3 protein and mRNA in colonic mucosal tissues of UC patients were significantly higher than those of normal controls (P<0.05). Compared with that in mild UC, the expression of NLRP1 in colonic mucosal tissues of moderate and severe UC patients was significantly increased (P<0.05). There was no significant difference in the expressions of NLRP1 and NLRP3 in colonic mucosal tissues of UC patients with different lesion ranges. NLRP1 expression was positively correlated with Mayo overall score, Mayo endoscopic score, erythrocyte sedimentation rate, and C-reactive protein (P<0.05), NLRP3 expression was positively correlated with C-reactive protein (P<0.05), but not correlated with Mayo overall score, Mayo endoscopic score, or erythrocyte sedimentation rate. NLRP1 expression was positively correlated with low-density lipoprotein and platelet (P<0.05), but not with uric acid or cholesterol. NLRP3 was positively correlated with low-density lipoprotein, uric acid and cholesterol (P<0.05), but not with platelet. 【Conclusion】 NLRP1 and NLRP3 may be involved in the pathogenesis of UC and related to disease activity. Therefore, they can be used as molecular targets for targeted therapy, and NLRP1 can be used as a predictor of mucosal healing.