Toll样受体⁃4抑制剂TAK⁃242对大鼠重度牙周炎骨吸收的影响

目的探讨Toll样受体⁃4(Toll like receptor⁃4,TLR⁃4)抑制剂TAK⁃242对大鼠重度牙周炎骨质吸收的影响,为重度牙周炎寻找辅助治疗手段提供实验基础。方法18只3周龄雄性Wistar大鼠随机分为3组(n=6),其中1组为正常对照组,另外2组以含有牙龈卟啉单胞菌(P.gingivalis)ATCC33277的5⁃0丝线结扎大鼠双侧上颌磨牙行重度牙周炎建模,分为牙周炎组、TAK⁃242组;TAK⁃242组从丝线结扎第1天起,通过尾静脉隔天注射1次溶于DMSO的TAK⁃242(2 mg/kg),另外两组注射相同体质量比例的DMSO溶剂,连续8周;第8周末处死3组大鼠,获取大鼠上颌骨标本,采用micro⁃CT扫描后三维重建,测量特定位点釉牙骨质界⁃牙槽嵴顶的距离评估骨丧失量,并对牙槽骨骨质相关参数和骨质微结构进行分析;组织学切片苏木精⁃伊(HE)染色观察牙周组织病理改变;甲基绿染色观察牙槽骨吸收情况;抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞分布情况。结果Micro⁃CT定量分析显示:牙周炎组与TAK⁃242组牙槽骨吸收显著高于对照组;与牙周炎组相比,TAK⁃242组大鼠上颌第一磨牙近、远中根吸收位点的骨丧失均显著减轻(P<0.001),骨密度(P<0.05)与骨体积/总体积分数(P<0.01)显著增高,骨小梁数目与骨小梁厚度(P<0.01)相对增多,骨小梁分离度(P<0.01)和骨小梁结构模式指数显著降低。牙周炎组骨质呈现疏松多孔的蜂窝状结构,骨小梁结构恶化,向杆状结构转变;TAK⁃242组骨质微结构改善,骨量改善,骨小梁分布相对更致密,骨小梁结构与对照组更相似。HE染色发现牙周炎组与TAK⁃242组牙周附着丧失与牙槽骨吸收较对照组显著;与牙周炎组相比,甲基绿染色表明TAK⁃242组骨吸收减轻,TRAP染色显示破骨细胞浸润减少(P<0.001)。结论TLR⁃4抑制剂TAK⁃242能缓解大鼠重度牙周炎骨吸收,改善其多孔、稀疏、排列紊乱的炎症性骨小梁结构。

Effect of remifentanil on toll-like receptor 4, NF-κB and IL-6 in rabbit myocardial ischemia/reperfusion model

Objective： To investigate whether remifentanil induced cardioprotecting effect is associated with expression of toll-like receptor 4 （TLR4）, nuclear factor rB （NF-r.B） and serum interleukin -6 （IL-6）. Methods： Fifty rabbits were randomly divided into 5 groups （n=10） according to the treatment： sham operation group （group A）, ischemla-reperfusion group （group B）, low-dose remifentanil group （group C）, mediate-dose remifentanil group （group D）, and high-dose remlfentanil group （group E） Myocardial TLR4 mRNA levels, NF-r.B protein expression and serum levels of IL-6 were observed in 120 min after reperfusion. Results： The myocardial expressions of TLR4 mRNA, NF-rd3 protein and IL-6 level in sera of groups B, C, D and E were elevated compared with group A. However, remifentanil significantly reduced the levels of TLR4 mRNA, NF- r.B protein expression and serum IL-6 in groups C, D and E compared with group B. There were remarkable differences between the groups （P〈O.O1）. Conclusion： Intravenous remifentanil has protective effect against rabbit myocardial ischemia/reperfusion injury. This effect may be associated with TLR4, NF-r.B expressions on myocytes and serum level of IL-6 in a dose-dependent manner

LBP and CD14 polymorphisms correlate with increased colorectal carcinoma risk in Han Chinese

AIM: To explore the associations of polymorphisms of lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR-4), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) with the colorectal carcinoma (CRC) risk in Han Chinese. METHODS: Polymorphisms of LBP (rs1739654, rs223 2596, rs2232618), CD14 (rs77083413, rs4914), TLR-4 (rs5030719), IL-6 (rs13306435) and TNF-α (rs35131721) were genotyped in 479 cases of sporadic colorectal carcinoma and 486 healthy controls of Han Chinese in a case-control study. Single-nucleotide polymorphisms (SNPs) between cases and controls were analyzed by unconditional logistic regression. RESULTS: GA and GG genotypes of LBP rs2232596 were associated with a significantly increased risk ofCRC [odds ratio (OR) = 1.51, 95% confidence interval (CI) 1.15-1.99, P = 0.003; OR = 2.49, 95% CI 1.16-5.38, P = 0.016, respectively]. A similar association was also observed for the CG genotype of CD14 rs4914 (OR= 1.69, 95% CI 1.20-2.36, P = 0.002). In addition, a combination of polymorphisms in LBP rs2232596 and CD14 rs4914 led to a 3.4-fold increased risk of CRC (OR = 3.44, 95% CI 1.94-6.10, P = 0.000). CONCLUSION: This study highlights the LBP rs2232596 and CD14 rs4914 polymorphisms as biomarkers for elevated CRC susceptibility in the Chinese Han population.

Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population

AIM: To study the polymorphisms of toll-like receptor 4 (TLR4) gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp and susceptibility to inflammatory bowel disease (IBD) in the Zhuang population from Guangxi, China. METHODS: A case-control study was performed from February 2007 to October 2011 which included 146 Zhuang patients with IBD in the experimental group and 164 healthy Zhuang subjects who acted as the control group. All patients and healthy subjects were from the Guangxi Zhuang Autonomous Region of China. Genomic DNA was extracted from intestinal tissue by the phenol chloroform method. TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp were amplified by polymerase chain reaction (PCR), and then detected by PCR-restriction fragment length polymorphism (RFLP). RESULTS: The TLR4 gene Asp299Gly was digested using Nco Ⅰ restriction enzyme, and a single band of 249 bp was observed which showed that it was a wild type (AA). The TLR4 gene Thr399Ile was digested using Hinf Ⅰrestriction enzyme and only the wild type (CC) was detected. In addition, the TLR2 gene Arg-677Trp was digested using Aci Ⅰ restriction enzyme and only the wild type (CC) was detected. The TLR2 gene Arg753Gln was digested using Pst Ⅰ restriction enzyme. Only the wild type (GG) as a single band of 254 bp was observed during RFLP. Overall, no heterozygous or homozygous single nucleotide polymorphism mutations were found in patients with Crohn's disease and ulcerative colitis both in the TLR4 gene Asp299Gly, Thr399Ile and the TLR2 gene Arg677Trp, Arg753Gln in the Zhuang population from the Guangxi Zhuang Autonomous Region of China. CONCLUSION: The TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China.

CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer

AIM:To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor(MIF)/Toll-like receptor 4(TLR4) in gastric cancer.METHODS:CD74,MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining.Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting.MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8(CCK8) assay and MIF concentration in the culture medium was detected by enzymelinked immunosorbent assay.Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide(LPS) was measured by flow cytometry.MIF,CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation.RESULTS:CD74,MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage,and were also associated with lymph node metastasis.Correlation analysis revealed that CD74 was positively correlated with MIF(r = 0.2367,P < 0.01) and both proteins were also associated with TLR4(r = 0.4414,r = 0.5001,respectively,P < 0.01).LPS can significantly promote MKN-45 cell proliferation(3.027 ± 0.388 vs 4.201 ± 0.092,P < 0.05),induce MIF production(54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL,P < 0.01) and cell surface expression of CD74(75.6% ± 4.046%vs 9.4% ± 0.964%,P < 0.01) at LPS concentration of 1 μg/mL compared to medium control.Knockdown of CD74 or using antiCD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation(4.201 ± 0.092 vs 3.337 ± 0.087,4.534 ± 0.222 vs 3.368 ± 0.290,4.058 ± 0.292vs 2.934 ± 0.197,respectively,P < 0.01).MIF,CD74 and TLR4 could co-localize in the MKN-45 cell line.CONCLUSION:Upregulation of MIF,CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.

Chaihu Longgu Muli Decoction relieving temporal lobe epilepsy in rats by inhibiting TLR4 signaling pathway through miR-146a-3p and miR-146a-5p

Objective To explore the effect and mechanism of Chaihu Longgu Muli Decoction(柴胡龙骨牡蛎汤,CHLGMLD)in rats with temporal lobe epilepsy(TLE).Methods A total of 80 Sprague-Dawley(SD)male rats were randomized into control(CON),model(MOD),carbamazepine(CBZ,0.1 g/kg),CHLGMLD low dose(CHLGMLD-L,12.5 g/kg),and high dose(CHLGMLD-H,25 g/kg)groups,with 16 rats in each group.TLE rat models were established in the four groups with the use of lithium-pilocarpine except for the CON group.After the successful establishment of TLE models,all drugs were administered through gavage,and distilled water was given to rats in the CON and MOD groups for four weeks.The frequency and duration of seizures before and after treatment were recorded for the evaluation of the alleviation degree.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-146a-3p and miR-146a-5p.The expression levels of toll-like receptor 4(TLR4),interleukin-1 receptor-associated kinase 1(IRAK1),tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6),TAK1-binding protein(TAB),nuclear factor-kappa B(NF-κB),and interleukin-1 beta(IL-1β)in hippocampus were tested by immunofluorescence assay.Correlation analysis between the above factors and expressions of miR-146a-3p and miR-146a-5p were performed separately.Results CHLGMLD decreased the frequency(P<0.05)and duration(P<0.01)of seizures in rats.CHLGMLD down-regulated the expression levels of miR-146a-5p and miR-146a-3p(P<0.05),and inhibited the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β(P<0.01).The correlation analysis revealed that the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β were positively correlated with the expression levels of miR-146a-3p and miR-146a-5p detected by qRT-PCR,respectively(P<0.01).Conclusion CHLGMLD can inhibite the TLR4 signaling pathway by lowering the expression levels of miR-146a-3p and miR-146a-5p to alleviate hippocampal dentate gyrus inflammation in TLE rats,thus relieving seizures.

TLR4 -2242 T→C variant increases transcriptional activity of its promoter

Objective:To investigate the effects of -2242,-1892 and -1837 single nucleotide polymorphisms(SNPs) on toll-like receptor 4(TLR4) promoter activity.Methods:Polymerase chain reaction(PCR) and site direct mutation technology were used to construct TLR4 basic promoter and -2242C,-1892A and -1837G mutate promoter plasmids.Dual-Luciferase Reporter assay system was used to detect the activity of constructed promoter following human embryonic kidney(HEK) 293 cells were transiently cotransfected with the constructed plasmids and the control plasmid pRL-CMV.Results:In HEK293 cells,the activity of -2242C mutate promoter was higher than -2242T promoter,and there was no significant difference when both -1892A and -1837G mutate promoter compared with -1892G and -1837A promoter,respectively.Conclusion:It is implied that -2242T→C base variation can enhance the activity of TLR4 promoter,while -1892 and -1837 SNPs have no effect on TLR4 promoter activity.

Vigorous,but differential mononuclear cell response of cirrhotic patients to bacterial ligands

AIM： To study the role of gram-positive and gram-negative bacteria in the pathogenesis of liver injury, specifically the activation of inflammatory mediators. METHODS： Peripheral blood mononuclear cells of 20 out-patients were studied, 10 of them with cirrhosis,Peripheral blood mononuclear cells were isolated and exposed to lipopolysaccharide or lipoteichoic acid. CD14, Toll-like receptor 2 and 4 expression was determined by flow cytometry, and tumor necrosis factor （TNF） α, interleukin （1L）-1β, IL-6, IL-12 and IL-10 secretion in su- pernatants was determined by ELISA. RESULTS： Higher CD14, Toll-like receptor 2 and 4 expression was observed in peripheral blood mononudear cells from cirrhotic patients, （P 〈 0.01, P 〈 0.006, P 〈 0.111） respectively. Lipopolysaccharide and lipoteichoic acid induced a further increase in CD14 expression （P 〈 0.111 lipopolysaccharide, P 〈 0.013 lipoteichoic acid）, and a decrease in Toll-like receptor 2 （P 〈 0.008 lipopolysaccharide, P 〈 0.008 lipoteichoic acid） and Toll-like receptor 4 （P 〈 0.008 lipopolysaccharide, P 〈 0.028 li- poteichoic acid） expression. With the exception of TNFα, absolute cytokine secretion of peripheral blood mononuclear cells was lower in cirrhotic patients under nonexposure conditions （P 〈 0.070 IL-6, P 〈 0.009 IL-1]5, P 〈 0.022 IL-12）. Once exposed to lipopolysaccharide or lipoteichoic acid, absolute cytokine secretion of peripheral blood mononuclear cells was similar in cirrhotic and non-cirrhotic patients, determining a more vigorous response in the former （P 〈 0.005 TNFα, IL-1β, IL-6, IL-2 and IL-10 lipopolysaccharide; P 〈 0.037 TNFα; P 〈 0.006 IL-113; P 〈 0.005 IL-6; P 〈 0.007 IL-12; P 〈 0.014 IL-10 lipoteichoic acid）. Response of peripheral blood mononuclear cells was more intense after lipopolysaccharide than after lipoteichoic acid exposure. CONCLUSION： Peripheral blood mononuclear cells of cirrhotic patients are able to respond to a sudden bacterial ligand exposure, particularly lipopolysaccharide, suggesting that immune regulation mechanisms are still present.

Saturated Fatty Acid Induces Insulin Resistance Partially Through Nucleotide-binding Oligomerization Domain 1 Signaling Pathway in Adipocytes

Objective To investigate the potential role of nucleotide-binding oligomerization domain 1 （NOD1）, a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes. Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB （NF-κB） activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[SH] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA （siRNA） targeting NOD 1 upon fatty acids treatment were analyzed. Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-R, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD 1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake. Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

Lipopolysaccharide/Toll-like receptor 4 signaling pathway involved Qingdu decoction treating severe liver injury merging with endotoxemia

OBJECTIVE: To investigate the protective effects and underlying mechanism of Qingdu decoction(QDD) on experimental rats with severe liver injury induced by thioacetamide(TAA).METHODS: A total of 40 Wistar rats were randomly divided into normal group(n = 10) and experimental group(n = 30). Rats were administrated the same content of saline in normal group. The rats inthe experimental group were pretreated with TAA at dose of 12 mg/kg lasting 8 weeks, and from 9th week to 12 th week, with TAA at concentration of 36mg/kg. During the 9th week to 12 th week period,the rats were randomly divided into three subgroups(n = 10 each) simultaneously based on the treatment categories: model group, lactulose(LA,3.5 m L/kg) group and QDD(5.95 g/kg) group, orally once per day respectively. At the 12 th week, the content of serum alanine transaminase(ALT), aspartate transaminase(AST), total bilirubin(TBIL), endotoxin(ET) and tumor necrosis factor α(TNF-α) was detected by automatic biochemical analyzer. The plasma prothrombin time(PT), prothrombin time-international normalized ratio(PTR) and prothrombin time activity(PTA) were measured by automatic coagulation analyzer. The level of lipopolysaccharide(LPS)-binding protein(LBP), cluster differentiation 14(CD14) and Toll-like receptor 4(TLR4) expressions was measured by both western blot(WB) and real-time polymerase chain reaction(real-time PCR).RESULTS: Compared with the model group, hepatic morphology in the QDD group was improved under light microscope and transmission electron microscope; at the same time, the contents of serum ALT, AST, TBIL, ET and TNF-α, and level of LBP, CD14 and TLR4 expressions in liver tissues were significantly decreased compared with the model group(P < 0.05), while PTA in the QDD group was enhanced(P < 0.05).CONCLUSION: QDD has the functional effect on improving the injured liver through inhibiting the LPS/TLR4 signaling pathway thus decreasing the level of the inflammatory medicators.

Catalpol ameliorates LPS-induced endometritis by inhibiting inflammation and TLR4/NF-κB signaling

Catalpol is the main active ingredient of an extract from Radix rehmanniae,which in a previous study showed a protective effect against various types of tissue injury.However,a protective effect of catalpol on uterine inflammation has not been reported.In this study,to investigate the protective mechanism of catalpol on lipopolysaccharide(LPS)-induced bovine endometrial epithelial cells(bEECs)and mouse endometritis,in vitro and in vivo inflammation models were established.The Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qRT-PCR),western blot(WB),and immunofluorescence techniques.The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factorα(TNF-α),interleukin(IL)-1β,and IL-6,and chemokines such as C-X-C motif chemokine ligand 8(CXCL8)and CXCL5,both in bEECs and in uterine tissue.From the experimental results of WB,qRT-PCR,and immunofluorescence,the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group.The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase(MPO)activity.The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.

Xiao-Xu-Ming decoction extract alleviates LPS-induced neuroinflammation associated with down-regulating TLR4/MyD88 signaling pathway in vitro and in vivo

In the present study, we aimed to investigate the effects of Xiao-Xu-Ming decoction extract(XXM) on lipopolysaccaride(LPS)-induced neuroinflammation in vitro and in vivo. In vitro, the microglia BV2 cells were treated with 200 ng/mL LPS for 24 h to induce inflammatory responses. In vivo, mice were treated with 5 mg/kg LPS to induce inflammatory responses. The NO level was determined by Griess Reagents. The levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by ELISA. The expressions of Iba-1, TLR4 and MyD88 at the protein levels were determined by Western blotting analysis. The mRNA levels of TLR4 and MyD88 were determined by real-time PCR. In vitro, XXM significantly reduced the levels of various pro-inflammatory factors, including NO, IL-1β, IL-6 and TNF-α, induced by LPS in the supernatant of BV2 cells and suppressed expressions of inflammatory proteins TLR4 and MyD88 induced by LPS in BV2 cells. In vivo, XXM significantly inhibited microglia activation, attenuated LPS-induced inflammatory factors and chemokine production, such as IL-1β, IL-6, TNF-α and MCP-1, and inhibited the expressions of inflammatory proteins including TLR4 and MyD88, in the cortex of LPS-induced mice. Our findings suggested that XXM could attenuate LPS-induced neuroinflammation via down-regulating TLR4/MyD88 signaling pathway.

Heat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways

Mesenchymal stem cell （MSC） transplantation has shown a therapeutic potential to repair the ischemic and infracted myocardium, but the effects are limited by the apoptosis and loss of donor cells in host cardiac microenvironment. The aim of this study is to explore the cytoprotection of heat shock protein 90 （Hsp90） against hypoxia and serum deprivation-induced apoptosis and the possible mechanisms in rat MSCs. Cell viability was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Apoptosis was assessed by Hoechst 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of Toll-like receptor-4 （TLR-4） and V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 （ErbB2） was detected by real-time poly- merase chain reaction （PCR）. The protein levels of cleaved caspase-3, Bcl-2, Bcl-xL, Bax, totaI-ERK, phospho-ERK, totaI-Akt, phospho-Akt, and Hsp90 were detected by Western blot. The production of nitric oxide was measured by spectrophotometric assay. Hsp90 improves MSC viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates Bcl-2/Bax and Bcl-xL/Bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then ac- tivating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs. These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.

Protective effects of Yindanxinnaotong capsule in a rat model of myocardial ischemia/reperfusion injury

OBJECTIVE: To investigate the effects of Yindanxinnaotong capsule(YDXNTC) and main components compatibility and ratios on myocardium against ischemia/reperfusion injury and the effect's underlying mechanism.METHODS: Myocardial ischemia/reperfusion injury(MIRI) was induced by ischemia for 30 min and reperfusion for 30 min. Electrocardiogram data and coronary flow were recorded, and superoxide dismutase(SOD), malondialdehyde(MDA), lactate dehydrogenase, creatine kinase-MB, cardiac troponin T and I(cT nT, cT n I) and interleukin-1β, interleukin-8,interleukin-18(IL-1β, IL-8, IL-18) in myocardium were measured. Hypoxia/reoxygenation and hydrogen peroxide(H2O2) injury were induced by hypoxia for 3 h/reoxygenation for 2 h, and 100 μM H2O2 for 1 h, respectively, in vitro rat myocardial cells(H9c2). Cell viability, SOD, MDA, cT nT and inflamma-tory factors(IL-1β, IL-8 and IL-18) were determined,and Toll-like receptor 4(TLR-4) expression was measured by western blotting.RESULTS: In the isolated heart experiment, elevated heart function, coronary flow and SOD levels,and decreased MDA levels and inflammatory factors were noted in the YDXNTC, main components and main components compatibility groups. Ventricular tachycardia/ventricular fibrillation occurrence decreased in the ginkgo biloba extract(GBE),and GBE and salvia miltiorrhiza ethanol extract compatibility(SM-E, GSEC) groups. Lactic dehydrogenase levels decreased in the YDXNTC and aqueous extract of salvia miltiorrhiza(SM-H) groups. Creatine kinase-MB decreased with GBE, SM-E, SM-H and GSEC treatment, and cT n I and cT nT levels decreased with GSEC. In the in vitro cell study,YDXNTC and main components ratios improved cell viability and SOD levels, and suppressed MDA,cT nT and inflammatory factors. TLR-4 expression was down-regulated.CONCLUSION: YDXNTC and main components compatibility showed protective effects on MIRI in this rat model and in vitro study. Regulating the Toll-like receptor signaling pathway may affect the mechanism.

Effect of penehyclidine hydrochloride on patients with acute lung injury and its mechanisms

Objective： To assess the effects of penehyclidine hydrochloride on patients with acute lung injury （ALI）, to observe the expression of Toll-like receptor 4 （TLR4） on the peripheral monocytes of ALI patients and changes of inflammatory ＆ anti-inflammatory cytokines and to investigate the mechanism of TLR4 in ALI.Methods： Forty-five patients with ALI were randomly divided into penehyclidine hydrochloride treatment group （P group, n=21） and conventional treatment group （control group, C group, n=24）. Patients in both groups received conventional treatment, including active treatment of the primary disease, respiratory support, nutritional support and fluid management therapy, while those in P group were given penehyclidine hydrochloride （1 mg, im, q. 12 h） in addition.The TLR4 expression of 20 healthy volunteers were detected.The clinical effect, average length of stay in ICU and hospital,values of PaO2 and PaO2/FiO2, expression of TLR4 on the surface of peripheral blood mononuclear cells and some serum cytokines were evaluated for 48 h.Results： The general conditions of the two groups were improved gradually and PaO2 increased progressively.Compared with 0 h, PaO2 and PaO2/FiO2 at 6, 12, 24 and 48 h after treatment were significantly increased （P＜0.05）. The improvement in P group was obviously greater than that in C group （P＜0.05）. The average length of hospitalization showed no difference between the two groups, but penehyclidine hydrochloride significantly decreased the average length of stay in ICU （t=3.485, P＜0.01）. The expression of TLR4 in two groups were both obviously higher than that of healthy volunteers （P＜0.01）. It decreased significantly at 24 h （t=2.032, P＜0.05） and 48 h （t=3.620, P＜0.01）and was lower in P group than in C group. The patients who showed a higher level of TLR4 expression in early stage had a worse prognosis and most of them developed acute respiratory distress syndrome （ARDS）. The incidence of ARDS was 23.8% in P group and 29.17% in C group at 24 h.Until148 h, there were other two patients developing ARDS in control group. Serum IL-l, IL-8 and TNF-α expressions reduced after 24 h in both groups. The reduction in P group was more obvious than that in C group （P＜0.05）. IL-13 increased gradually from 0 h to 24 h, and decreased slightly at 48 h, which showed no difference between two groups （t=1.028, P＞0.05）.Conclusions： Penehyclidine hydrochloride improves the arterial oxygen pressure, down-regulates the expression of TLR4 and restrains the inflammatory cytokines in the downstream of TLR4 signaling pathway. It prevents the development of ALI and can be considered as an important drug in ALI treatment.

Effect of glycyrrhizin on traumatic brain injury in rats and its mechanism

Objective： To investigate the neuropro- tective effects of glycyrrhizin （Gly） as well as its effect on expression of high-mobility group box 1 （HMGB1） in rats after traumatic brain injury （TBI）. Methods： Male Sprague-Dawley rats were randomly divided into three groups： sham group, TBI group, and TBI＋Gly group （n=36 per group）. Rat TBI model was made by using the modified Feeney＇s method. In TBI＋Gly group, Gly was administered intravenously at a dosage of l0 mg/kg 30 min after TBI. At 24 h after TBI, motor function and brain water content were evaluated. Meanwhile, HMGB 1/HMGB 1 receptors including toll-like receptor 4 （TLR4） and receptor for advanced glycation end products （RAGE）/nuclear fac- tor-κ B（NF- κ B） signaling pathway and inflammatory cytokines in the injured brain tissues were detected using quantitative real-time polymerase chain reaction, western blot, electrophoretic mobility shift assay and enzyme-linked immunosorbent assay. Furthermore, HMGB l, RAGE and TLR4 immunohistochemistry and apoptosis were analyzed. Results： Beam walking performance impairment and brain edema were significantly reduced in TBI＋Gly group compared with TBI group; meanwhile, the over-expressions of HMGB 1PHMGB 1 receptors （TLR4 and RAGE）/NF- κB DNA-binding activity and inflammatory cytokines were inhibited. The percentages of HMGB 1, RAGE and TLR4- positive cells and apoptotic cells were respectively 58.37%±5.06%, 54.15%±4.65%, 65.50%± 4.83%, 52.02%±4.63% in TBI group and 39.99%±4.99%, 34.87%±5.02%, 43.33%±4.54%, 37.84%±5.16% in TBI＋Gly group （all P〈0.01 compared with TBI group）. Conclusion： Gly can reduce secondary brain injury and improve outcomes in rat following TBI by down-regula- tion of HMGB 1/HMGB 1 receptors （TLR4 and RAGE）/NF- κB - mediated inflammatory responses in the injured rat brain.