ABSTRACT
Objective:To investigate the protective mechanism of TAK242, a specific inhibitor of Toll-like receptor 4 (TLR4), on the liver of septic rats.Methods:Eighteen male Sprague-Dawley (SD) rats were randomly divided into three groups ( n = 6 in each group). The septic model was established by intraperitoneal injection of lipopolysaccharide (LPS) 15 mg/kg. The rats in the TAK242 intervention group received intraperitoneal injection of TAK242 (5 mg/kg) before modeling, while the rats in the septic model group and the control group were injected with the same amount of solvent [10% dimethyl sulfoxide (DMSO) + 90% corn oil]. Six hours later, the blood of abdominal aorta was collected and the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by enzyme linked immunosorbent assay (ELISA). The rats were sacrificed to obtain liver, the expression levels of TLR4, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cysteinyl aspartate-specific proteinase-3 (caspase-3), nuclear factor-κB p65 (NF-κB p65) and phosphorylated NF-κB p65 (p-NF-κB p65) were detected by Western blotting. Immunohistochemical staining was used to observe NF-κB p65 protein expression in liver, and hepatocyte injury was assessed by hematoxylin-eosin (HE) staining. Results:Serum ALT and AST levels in the septic model group were significantly higher than those in the control group [ALT (μg/L): 26.639±7.814 vs. 2.847±2.150, AST (μg/L): 28.442±8.417 vs. 5.779±3.019, both P < 0.01]. The ALT and AST levels in the TAK242 intervention group were significantly lower than those in septic model group [ALT (μg/L): 7.269±3.398 vs. 26.639±7.814, AST (μg/L): 3.580±3.115 vs. 28.442±8.417, both P < 0.01]. Light microscopy showed that the hepatocytes in the septic model group were disordered, with obvious cell edema and increased inflammatory cells infiltration; the hepatocytes in the TAK242 intervention group were more neatly arranged, with significantly reduced hepatocyte edema and reduced inflammatory cells infiltration. Western blotting results showed that caspase-3 protein expression in hepatic tissue of septic model group was significantly higher than that in the control group (caspase-3/GAPDH: 0.794±0.164 vs. 0.482±0.055, P < 0.05), and caspase-3 protein expression in the TAK242 intervention group significantly decreased than that in the septic model group (caspase-3/GAPDH: 0.482±0.056 vs. 0.794±0.164, P < 0.05), which indicated that TAK242 could attenuate hepatocytes apoptosis of septic rats. The expression of IL-6, TNF-α and TLR4 protein and the ratio of p-NF-κB p65/NF-κB p65 in hepatic tissue of septic model group were significantly higher than those in control group (IL-6/GAPDH: 1.442±0.204 vs. 1.019±0.024, TNF-α/GAPDH: 1.089±0.098 vs. 0.806±0.005, TLR4/GAPDH: 1.292±0.085 vs. 0.941±0.087, p-NF-κB p65/NF-κB p65 ratio: 1.936±0.081 vs. 1.579±0.183, all P < 0.05), IL-6, TNF-α and TLR4 protein expression and p-NF-κB p65/NF-κB p65 ratio in the TAK242 intervention group were significantly lower than those in septic model group (IL-6/GAPDH: 1.035±0.042 vs. 1.442±0.204, TNF-α/GAPDH: 0.572±0.096 vs. 1.089±0.098, TLR4/GAPDH: 0.984±0.078 vs. 1.292±0.085, p-NF-κB p65/NF-κB p65 ratio: 1.484±0.255 vs. 1.936±0.081, all P < 0.05), it is suggested that LPS-induced sepsis could activate the inflammatory response mediated by TLR4/NF-κB pathway in liver, and the activation of TLR4/NF-κB pathway was inhibited by TAK242 through the TLR4 pathway, therefore, the inflammation of liver in septic rats was reduced. Immunohistochemical staining showed that the positive expression of NF-κB p65 in liver was significantly increased in the septic model group compared with the control group; the positive expression of NF-κB p65 was significantly reduced in the TAK242 intervention group compared with the septic model group, and there was almost no positive expression in the nucleus. Conclusion:TAK242 could reduce liver function injury and protect the liver by inhibition TLR4/NF-κB pathway in septic rats.
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Objective:To investigate the role of Toll-like receptor 4 (TLR4) pathway on myocardial injury and cardiac dysfunction in septic rats.Methods:According to the random number table, 18 male Sprague-Dawley (SD) rats were divided into control group, lipopolysaccharide (LPS) group and TLR4 specific inhibitor TAK242 pretreatment group (TAK242+LPS group) with 6 rats in each group. The rat model of septic cardiac dysfunction was induced by intraperitoneal injection of LPS 15 mg/kg, and the control group was given the same amount of normal saline. The TAK242+LPS group was intraperitoneally given injection of TAK242 [it was injected intraperitoneally at a dose of 3 mg/kg and dissolved in 10% dimethyl sulfoxide (DMSO) and 90% corn oil according to the concentration of 0.2 g/L] 3 hours before LPS stimulation. The control group and LPS group were given the same amount of 10% DMSO and 90% corn oil. The cardiac function of rats in each group was examined by Doppler echocardiography 14 hours after injection of LPS. The blood of abdominal aorta was taken and the level of serum troponin (cTn) was measured by enzyme linked immunosorbent assay (ELISA). Myocardial tissue was harvested for hematoxylin-eosin (HE) staining, and the morphological changes of myocardial tissue were observed under light microscope. The mRNA expressions of interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α) in myocardial tissue were detected by real-time fluorescence quantitative polymerase chain reaction (qPCR). The protein expression of TLR4 in myocardial tissue was observed by immunohistochemical method. Western blotting was used to detect the levels of TLR4, nuclear factor-κB p65 (NF-κB p65) and its phosphorylation (p-NF-κB p65) in myocardial tissue.Results:① The cardiac function and myocardial injury: Doppler echocardiography showed that the levels of left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) in the LPS group were significantly higher than those in the control group, while left ventricular ejection fraction (LVEF) and left ventricular shortened fraction (LVFS) were significantly lower than those in the control group. The degeneration, necrosis and inflammatory cell infiltration of cardiomyocytes were found with light microscope in the LPS group, and the levels of serum cTn were significantly higher than those in the control group, indicating that LPS-induced sepsis could cause cardiac dysfunction and myocardial injury. TAK242 blocking TLR4 pathway had a protective effect on cardiac function and myocardium during sepsis. LVEDV and LVESV in the TAK242+LPS group were significantly lower than those in the LPS group [LVEDV (μL): 71.25±21.16 vs. 118.01±11.89, LVESV (μL): 9.57±5.75 vs. 32.70±9.22, both P < 0.01]. LVEF and LVFS were significantly higher than those in the LPS group [LVEF: 0.868±0.075 vs. 0.722±0.095, LVFS: (59.88±8.46)% vs. (42.37±8.71)%, both P < 0.05]. Myocardial tissue injury was significantly reduced, and the serum cTn level was significantly lower than that in the LPS group (μg/L: 107.85±21.38 vs. 152.25±27.46, P < 0.05). ② Inflammatory parameters: the results of qPCR, Western blotting and immunohistochemistry showed that the mRNA expressions of IL-6 and TNF-α, the expression of TLR4 protein and the p-NF-κB p65/NF-κB p65 ratio in the LPS group were significantly higher than those in the control group, indicating that LPS-induced sepsis could activate the inflammatory response mediated by TLR4/NF-κB pathway in the heart. However, blocking the TLR4 pathway by TAK242 could inhibit the TLR4/NF-κB pathway and reduce the myocardial inflammation in septic rats. The mRNA expressions of IL-6 and TNF-α, the expression of TLR4 protein and the p-NF-κB p65/NF-κB p65 ratio in the TAK242+LPS group were significantly lower than those in the LPS group [IL-6 mRNA (2 -ΔΔCT): 10.44±3.30 vs. 107.50±29.48, TNF-α mRNA (2 -ΔΔCT): 2.38±0.68 vs. 3.77±0.56, TLR4 protein (TLR4/GAPDH): 0.39±0.01 vs. 0.58±0.04, p-NF-κB p65/NF-κB p65 ratio: 1.21±0.11 vs. 2.10±0.18, all P < 0.05]. Conclusion:TAK242 can protect LPS-induced cardiac dysfunction and myocardial injury by blocking the TLR4 mediated inflammatory response.
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Objective To investigate the effect of the peritoneal lymphatic stomata on intra-abdominal infection and the regulatory mechanism of angiotensin Ⅱ receptor specific inhibitor PD123319 on peritoneal lymphatic stomata.Methods The experimental study was adopted.Forty rats were divided into the control group,sham operation group,intra-abdominal infection group and intra-abdominal infection drug intervention group by the random number table,every group had 10 rats.The classic appendix perforation (CLP) intraabdominal infection model was established in the abdominal infection group.After establishing the model of abdominal infection,PD123319 solution was injected intraperitoneally immediately (0.2 g/kg) in the abdominal infection drug intervention group.Abdominal cavity of the rats in the sham operation group was opened,and then was shut after flipping the intestine.The rats in the control group,sham operation group and intra-abdominal infection group were treated with intraperitoneal injection of 1ml stroke-physiological saline solution.After 2 hours,the rats were sacrificed,and peritoneal tissue was taken for the following tests.(1) The aperture size and distribution density of peritoneal lymphatic stomata were observed by scanning electron microscope (SEM).(2) The nitric oxide (NO) concentration in the peritoneal tissues was detected using nitric oxide nitric acid reduction method.(3) The expressions of endothelial nitric oxide synthase (eNOS) and Phospho-eNOS (P-eNOS) were detected by the Western blot.(4) The intracellular Ca2+ concentration were detect by flow cytometry.Measurement data with normal distribution were presented as-x ± s.The comparison among groups was analyzed using the ANOVA and pairwise comparison was analyzed by the LSD test.Results (1) The aperture size and distribution density of the peritoneal lymphatic stomata in the control group,sham operation group,intra-abdominal infection group and intra-abdominal infection drug intervention group were respectively (2.3 ± 0.4) μm,(2.5 ± 0.5)μm,(4.7 ±0.5)pm,(3.8 ±0.5)pm and (2.0 ±0.8) × 108/m2,(2.1 ±0.7) × 108/m2,(6.2 ± 1.3) × 108/m2,(4.6 ± 1.4) × 108/m2,with statistically significant differences among the 4 groups (F =98.130,56.780,P ＜ 0.05).There were statistically significant differences in the aperture size and distribution density of the peritoneal lymphatic stomata between the intra-abdominal infection group and control group or intra-abdominal infection drug intervention group (t =11.586,8.573,3.854,3.098,P ＜ 0.05) and no statistically significant differences between the control group and sham operation group (t =1.281,0.514,P ＞0.05).(2) The concentrations of NO in the peritoneal tissues in the control group,sham operation group,intra-abdominal infection group and intra-abdominal infection drug intervention group were respectively (0.380 ± 0.024) μmol/gprot,(0.450 ±0.020) μmol/gprot,(1.253 ±0.033) μmol/gprot and (0.579 ±0.035) μmol/gprot,with a statistically significant difference among the 4 groups (F =52.725,P ＜ 0.05).There were statistically significant differences in the concentration of NO between the intra-abdominal infection group and control group or intra-abdominal infection drug intervention group (t =10.536,67.798,P ＜ 0.05) and no statistically significant difference in the concentration of NO between the control group and sham operation group (t =2.007,P ＞ 0.05).(3) The results of Western blot showed that the expressions of eNOS and P-eNOS in the control group,sham operation group,intra-abdominal infection group and intra-abdominal infection drug intervention group were respectively (0.591 ± 0.028)U/mg,(0.603 ± 0.007) U/mg,(0.615 ± 0.027) U/mg,(0.626 ±0.026) U/mg and (0.578 ±0.003)U/mg,(0.603 ± 0.071) U/mg,(0.773 ± 0.033) U/mg,(0.710 ± 0.012) U/mg,with no statistically significant difference in the expression of eNOS among the 4 groups (F =0.902,P ＞ 0.05) and with a statistically significant difference in the expression of P-eNOS among the 4 groups (F =205.062,P ＜ 0.05).There were statistically significant differences in the expression of P-eNOS between the control group and sham operation group or intra-abdominal infection group (t =7.678,13.322,P ＜ 0.05) and between the intra-abdominal infection group and intraabdominal infection drug intervention group (t =4.035,P ＜0.05).(4) The results of flow cytometry showed that Ca2+ concentration in the control group,sham operation group,intra-abdominal infection group and intraabdominal infection drug intervention group were respectively 82.200％ ± 0.060％,81.730％ ± 0.052％,21.980％ ± 0.010％,29.500％ ± 0.004％,showing a statistically significant difference between the 4 groups (F =21 271.030,P ＜ 0.05).There were statistically significant differences in the Ca2+ concentration between the intra-abdominal infection group and control group (t =164.750,P ＜ 0.05) and between the intra-abdominal infection group and intra-abdominal infection drug intervention group (t =21.338,P ＜ 0.05),and no statistically significant difference between the control group and sham operation group (t =1.861,P ＞ 0.05).Conclusion The intra-abdominal infection could increase aperture size and distribution density of peritoneal lymphatic stomata,and PD123319 may be through inhibiting the activation of NO synthase to decrease the concentration of NO,enhance the concentration of Ca2+ in peritoneal mesothelial cells and reduce the opening of peritoneal lymphatic stomata.
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Aim To investigate the anticancer activity and the mechanism of the apoptosis induced by Ama-ranthus spinosus L. extract ( ASE ) in human hepatic carcinoma cell line HepG2 . Methods Alamar blue assay was used for detecting the influence of ASE on the proliferation of the cancer cells. The morphological changes of cells were observed under inverted micro-scope and Hoechst 33258 stainning. The apoptosis of HepG2 cells was detected by flow cytometry. Western blot and caspase-3 activity kit were used to detect the protein expression in HepG2 cells. The specific inhibi-tor of caspase-9 and caspase-3 ( Z-LEHD-FMK and Ac-DEVD-CHO) was used to validate the signal transduc-tion pathyway. Results The results indicated that the cell proliferation was inhibited by ASE,especicially the HepG2 cells. The HepG2 cells showed obvious apop-totic characteristics. Flow cytometry analysis further validated the apoptosis of HepG2 cells. The expression of Bcl-2 and survivin was downreagulated in HepG2 cells treated with ASE, and Bax, caspase-9, caspase-3, Apaf-1 and PARP were upregualted. Besides, the caspase-3 activity was also increased. Z-LEHD-FMK and Ac-DEVD-CHO significantly increased the cell vi-abilty of HepG2 cells induced by ASE. Conclusion These results confirm that ASE induces the apoptosis of HepG2 through mitochondria-mediated pathway.
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Thelephoric acid is an antioxidant produced by the hydrolysis of polyozellin, which is isolated from Polyozellus multiplex. In the present study, the inhibitory effects of polyozellin and thelephoric acid on 9 cytochrome P450 (CYP) family members (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were examined in pooled human liver microsomes (HLMs) using a cocktail probe assay. Polyozellin exhibited weak inhibitory effects on the activities of all 9 CYPs examined, whereas thelephoric acid exhibited dose- and time-dependent inhibition of all 9 CYP isoforms (IC50 values, 3.2-33.7 muM). Dixon plots of CYP inhibition indicated that thelephoric acid was a competitive inhibitor of CYP1A2 and CYP3A4. In contrast, thelephoric acid was a noncompetitive inhibitor of CYP2D6. Our findings indicate that thelephoric acid may be a novel, non-specific CYP inhibitor, suggesting that it could replace SKF-525A in inhibitory studies designed to investigate the effects of CYP enzymes on the metabolism of given compounds.
Subject(s)
Humans , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP2D6 , Cytochrome P-450 CYP2E1 , Cytochrome P-450 Enzyme System , Hydrolysis , Metabolism , Microsomes, Liver , Proadifen , Protein IsoformsABSTRACT
OBJECTIVE: New nonsteroidal anti-inflammatory drugs (NSAIDs) with highly selective cyclooxygenase-2 (COX-2) inhibition afford protection against gastropathy, but their acute and long-term effects on the central nervous system are unclear. Our aim was to investigate the influence of COX-2 specific inhibitor (celecoxib) on cognitive function. METHODS: Within the context of a randomized controlled parallel trial of NSAIDs for osteoarthitis (OA), we performed a battery of neuropsychological tests in consecutive 10 osteoarthritis patients with celecoxib (200 mg/day) and 13 osteoarthritis patients with diclofenac (100 mg/day) before and after 4 weeks by clinical psychologists who were not involoved in the study and unaware of study protocols and treatment allocation. The tests were performed randomly in sequence in order to minimize learning effect. The examed cognitive domains included memory, reasoning/problem solving, simple and complex attention, visual-spatial processing, and psychomotor speed. RESULTS: Demographic characteristcs (age, sex, disease duration, functional status measured by patient's and physician's global assessment and KWOMAC, CES depression score, education level) were not significantly different between both treatment groups. In all cognitive domains, we did not find out significant cognitive decline before and after treatments either with celecoxib or diclofenac. There was no difference in the change of cognitive function between both treatment groups. CONCLUSION: The short-term use of COX-2 specific inhibitor as well as conventional NSAID may not impair cognitive function. The long-term follow up study using large number of patients is in progress.