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OBJECTIVE: To investigate the hepatotoxicity induced by subchronic crotonaldehyde exposure in male rats and analyze its possible mechanism. METHODS: The specific pathogen-free male Wistar rats were randomly divided into control group, low-, medium-and high-dose crotonaldehyde groups, with 10 rats in each group. The crotonaldehyde solution at doses of 0.0, 2.5, 4.5, and 8.5 mg/kg body weight were given by intragastric administration, once a day, 5 days per week, and continuous for 90 days. The body weight of the rats were weighed during the exposure period. After the exposure, the liver organ coefficients and histopathological changes of the rats were observed. The activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) and the level of total bilirubin(TBIL) in the serum of rats were determined by colorimetry. The levels of malondialdehyde(MDA) and reduced glutathione(GSH), and the activities of glutathione peroxide(GSH-Px) and superoxide dismutase(SOD) were determined by colorimetry. The levels of interleukin(IL)-1β, IL-6, tumor necrosis factor alpha(TNF-α) and interferon-γ(IFN-γ) was detected by enzyme-linked immunosorbent assay. RESULTS: At the end of exposure, the increment of body mass of rats in the low-, medium-and high-dose crotonaldehyde groups was lower than that in the control group(P<0.05). The organ coefficients of rats in the middle-and high-dose groups were lower than that in the control group(P<0.05). The liver tissues of the three doses crotonaldehyde groups showed varying degrees of inflammatory cell infiltration. The activities of ALT, AST and the level of TBIL in the serum of rats increased with the increase of the crotonaldehyde exposure dose(P<0.01). With the increase of the crotonaldehyde dose, the level of MDA in rat liver tissue increased(P<0.01), and the level of GSH and the activities of GSH-Px and SOD decreased(P<0.01). The levels of IL-1β, IL-6, TNF-α and IFN-γ in rat liver tissues increased(P<0.05). CONCLUSION: Crotonaldehyde exposure can cause liver tissue damage in rats. Its mechanism of action may be related to the changes of oxidative balance and upregulation of the expression of inflammatory factors in liver tissue. These changes have the dose-effect relationship.
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Objective@#To observe the lung injury of male rats induced by sub-chronic exposure to crotonaldehyde, and to explore the possible mechanism of injury.@*Methods@#Forty SPF male Wistar rats were randomly divided into control group and 3 groups in each group, and each group received 0.0, 2.5, 4.5, 8.5 mg/kg body weight crotonaldehyde solution for continuous intragastric administration. 120 d, once a day. After the end of the exposure, the body weight of the rats was measured, and the lung tissues were quickly separated after cervical dislocation. The organ coefficients were calculated and histopathological examination was performed to determine malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione. Peroxidase (GSH-Px) content; ELISA was used to measure interleukin (IL) -6, IL-1β, and tumor necrosis factor (TNF) -α in lung tissues.@*Results@#Compared with the control group, the weight gain of the rats in the 4.5 and 8.5 mg/kg exposure groups was small, and the lung weight and organ coefficient of the exposed group decreased, the difference was statistically significant (P<0.05). In the exposed group, the lung tissue structure was disordered, the alveolar wall was thickened, and inflammatory cell infiltration was observed. Compared with the control group, the MDA activity in the serum of the rats in the 4.5 mg/kg and 8.5 mg/kg groups increased, and the SOD and GSH-Px activities decreased, the difference was statistically significant (P<0.05). TNF-α levels in the lung tissues of rats exposed to 4.5 mg/kg and 8.5 mg/kg, and levels of (IL) -6 and IL-1β in the lungs of rats in the 2.5, 4.5, and 8.5 mg/kg groups. Significantly increased, the difference was statistically significant (P<0.05) .@*Conclusion@#Crotonaldehyde may induce inflammatory and oxidative stress damage in rats by up-regulating the expression of inflammatory factors in lung tissue and changing the oxidative balance.
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OBJECTIVE: To observe the conditions of sub-chronic crotonaldehyde exposure-induced pulmonary inflammation,oxidative stress and apoptosis in male rats,and to explore the related mechanisms. METHODS: The specific pathogen free Wistar male rats were randomly divided into control group,low-,medium-and high-dose crotonaldehyde groups,with 10 rats in each group. Rats were treated with crotonaldehyde at the concentrations of 0. 0,2. 5,4. 5 and 8. 5 mg/kg body weight by intra-gastric administration,once per day for 120 consecutive days. After the end of treatment,rats were sacrificed,the lungs were weighed and histopathological examination was performed. The levels of malondialdehyde( MDA),superoxide dismutase( SOD) and glutathione peroxidase( GSH-Px) in the serum of rats were determined by colorimetry. The relative expression of reactive oxygen species in lung tissues was detected by fluorescence probe. The apoptosis rate was detected by Tunel staining. The relative expression of B-cell lymphoma( Bcl)-2,Bcl-2 associated X protein( Bax) and cysteine aspartic acid protease-3( Caspase-3) proteins in lung tissue was detected by western blotting.RESULTS: The body weight of the rats in the high-dose group began to decrease after 30 days of exposure( P < 0. 05),and the body weight in the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05),when compared with that of the control group at the same time. The body weight of the high-dose group was lower than that of the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05). After exposure,the lung tissue of the three doses groups showed different degrees of inflammatory change in a dose-effect relationship. The level of serum MDA in rats increased with the treatment of crotonaldehyde( P < 0. 01). The activities of serum SOD and GSH-Px decreased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of ROS and apoptosis rate in rat lung tissue increased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of Bcl-2 protein and the ratio of Bcl-2/Bax in the lung tissue of rats decreased with the treatment of crotonaldehyde( P < 0. 01). The relative protein expression of Bax and Caspase-3 increased with the treatment of crotonaldehyde( P < 0. 01). CONCLUSION: Crotonaldehyde sub-chronic exposure can cause apoptosis in lung tissue by altering the oxidative balance,leading to inflammatory pathological changes in the lung.
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Objective:To express and purify the fusion protein of GST-FILIP-1L and prepare its polyclonal antibody. Methods:The constructed recombinant expression vectors pGEX-4T3-FILIP-1L were transformed into Escherichia Coli BL21. FILIP-1L fusion protein was induced by IPTG and purified by Glutathion Sepharse 4B . The rabbit was immunized by the purified fusion protein,and pro-duced serum with anti-FILIP-1L antibody. The titer of polyclonal antibody was detected by ELISA, the anti-FILIP-1L polyclonal antibody was purified by Active and its combining specificity with FILIP-1L protein was further identified by Western blot. Results:The GST-FILIP-1L fusion protein was highly expressed in E. coli, and its specific polyclonal antibody was obtained after the immunization. The polyclonal antibody purified by Active Ester Agrose was able to combine specially with FILIP-1L protein and transformed FILIP-1L protein in 293 cells and FILIP-1L protein of liver cancer cells, respectively. Conclusion: The GST-FILIP-1L fusion protein was expressed successfully,the anti-GST-FILIP-1L polyclonal antibodies with high titer and specificity are successfully prepared,these antibodies provide an useful experimental tool to research the biological function of the FILIP-1L protein.