Inhibition of Toll-like receptor 4 pathway by TAK242 protects the liver in sepsis / 中华危重病急救医学

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.

Protective effect of TAK242 blocking Toll-like receptor 4 pathway on septic myocardial injury and cardiac dysfunction / 中华危重病急救医学

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.

Protective effects of TAK-242 against coronary microembolization in rat associated with involvement of TLR4/NF-κB signaling pathway / 中华急诊医学杂志

Objective To investigate the role of TLR4/NF-κB signaling pathway under the action of TAK-242 in the cardiomyocyte apoptosis after coronary micro-embolism (CME) in rats.Methods Fortyfive rats were randomized (random number) into three groups:sham operation,CME and CME plus TAK242 groups (n =15 per group).CME was induced by injecting polyethylene microspheres (42 μm) into the left ventricle except the sham group.CME plus TAK-242 group was treated with TAK-242 (2 mg/kg) via the tail vein of mice 30 min before CME modeling.Cardiac function was evaluated 6 h after operation.Tissue biopsy was stained with HBFP to measure the size of infarction area.TUNEL assay was used to detect cardiomyocyte apoptosis.Western blot and qPCR were used to evaluate the protein levels and mRNA expressions of TLR4,NF-κB p65 and cleaved caspase-3,respectively.Statistical analysis was performed using one-way analysis of variance followed by LSD-t test.Results Compared with the sham group,left ventricular ejection fraction (LVEF) in the CME group was significantly decreased [(68.91 ± 4.12) ％ vs.(84.80 ± 2.51) ％,P ＜ 0.05],and the infarction area (P ＜ 0.05),the apoptosis index [(3.36 ± 0.63) ％ vs.(0.19 ± 0.08) ％,P ＜0.05],the mRNA expressions of TLR4,NF-κB p65 and cleaved caspase-3 in CME group were increased significantly (all P ＜ 0.05).Compared with CME group,LVEF in the CME plus TAK-242 group was significantly improved [(75.58 ± 5.01) ％ vs.(68.91 ± 4.12) ％,P＜0.05],and the infarction area [(8.58 ± 2.12) ％ vs.(14.65 ± 4.23) ％,P＜0.05],the apoptosis index [(1.43 ± 0.51) ％ vs.(3.36 ± 0.63) ％,P ＜ 0.05],the mRNA expressions of TLR4,NF-κB p65 and cleaved caspase-3 in CME + TAK-242 group were decreased significantly (all P ＜ 0.05).Conclusions TAK-242 effectively improved CME-induced cardiac dysfunction by regulating TLR4/NF-κB signaling pathway and then reducing the cardiomyocyte apoptosis.

TAK-242,an antagonist for Toll-like receptor 4,protects against myocardial ischemia/reperfusion injury in C57BL/6 mice / 国际药学研究杂志

Objective To investigate the effect of TAK-242,an antagonist for Toll-like receptor 4,against myocardial isch?emia/reperfusion injury(I/R)in C57BL/6 mice along with the underlying mechanism. Methods C57BL/6Mice(n=36)were random?ized into three groups:sham group,I(30 min)/R 24 h model group and I/R+TAK-242(3 mg/kg)treatment group. At 24 h after reper?fusion,cardiac function and myocardial infarct size were evaluated with echocardiography and triphenyltetrazolium chloride(TTC), myocardial pathological pattern in mice was detected by HE staining,the mRNA and protein levels of TLR4 were determined by real time PCR and Western blot respectively,and the serum levels of IL-6,TNF-α,IL-10 and HMGB1 were detected by ELISA. Re?sults Our results showed that left ventricular systolic diameters(LVID)were shortened(P<0.01),left ventricular ejection fraction (LVEF)and left ventricular short axis shortening fraction(LVFS)were both decreased significantly(P<0.01)in following I/R mice. Myocardial infarction size was large and myocardial inflammatory cell infiltration was severe in I/R mice. The mRNA and protein levels of TLR4 were elevated(P<0.01),the serum levels of IL-6,TNF-α,IL-10 and HMGB1 in I/R mice were significantly increased com?pared with the sham group(P<0.01). We found that TAK-242 significantly extended LVID(P<0.05),increased LVEF and LVFS (P<0.05),reduced myocardial infarction and improved myocardial inflammatory cell infiltration,inhibited the mRNA and protein levels of TLR4(P<0.05),down-regulated the expression of IL-6 and TNF-α(P<0.01),and up-regulated IL-10 and HMGB1 com?pared with I/R group(P<0. 01). Conclusion Treatment with TAK-242 can significantly reduce myocardial ischemia/reperfusion in?jury partly via regulating inflammation.

TAK-242, an antagonist for toll-like receptor 4, protects against myocardial ischemia/reperfusion injury in C57BL/6 mice / 国际药学研究杂志

Objective To investigate the effect of TAK-242, an antagonist for Toll-like receptor 4, against myocardial ischemia/reperfusion injury (I/R) in C57BL/6 mice along with the underlying mechanism. Methods C57BL/6 Mice (n=36)were randomized into three groups: sham group, I (30 min)/R 24 h model group and I/R+TAK-242 (3 mg/kg) treatment group. At 24 h after reperfusion, cardiac function and myocardial infarct size were evaluated with echocardiography and triphenyltetrazolium chloride (TTC), myocardial pathological pattern in mice was detected by HE staining, the mRNA and protein levels of TLR4 were determined by real time PCR and Western blot respectively, and the serum levels of IL- 6, TNF- α, IL- 10 and HMGB1 were detected by ELISA. Results Our results showed that left ventricular systolic diameters (LVID) were shortened (P<0.01), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening fraction (LVFS) were both decreased significantly (P<0.01)in following I/R mice. Myocardial infarction size was large and myocardial inflammatory cell infiltration was severe in I/R mice. The mRNA and protein levels of TLR4 were elevated(P<0.01), the serum levels of IL-6, TNF-α, IL-10 and HMGB1 in I/R mice were significantly increased compared with the sham group (P<0.01). We found that TAK-242 significantly extended LVID (P<0.05), increased LVEF and LVFS (P<0.05), reduced myocardial infarction and improved myocardial inflammatory cell infiltration, inhibited the mRNA and protein levels of TLR4 (P<0.05), down-regulated the expression of IL-6 and TNF-α (P<0.01), and up-regulated IL-10 and HMGB1 compared with I/R group (P<0. 01). Conclusion Treatment with TAK-242 can significantly reduce myocardial ischemia/reperfusion injury partly via regulating inflammation.