Objective:To study the correlation of the changes of Toll-like receptor 4 (TLR4) / nuclear factorκB (NF-κB) pathway function in intrauterine adhesion (IUAs) tissue with the characteristics of cytokine secretion and ...Objective:To study the correlation of the changes of Toll-like receptor 4 (TLR4) / nuclear factorκB (NF-κB) pathway function in intrauterine adhesion (IUAs) tissue with the characteristics of cytokine secretion and collagen metabolism.Methods:The patients with IUAs who were treated in our hospital between February 2015 and March 2018 were selected as the IUAs group, and the patients who underwent hysteroscopy due to infertility and were pathologically confirmed to have normal endometrium during the same period were selected as the control group. The expression levels of TLR4/NF-κB pathway molecules and collagen metabolism genes as well as the contents of cytokines and collagen metabolism markers in the adhesion tissues of IUAs group and the normal endometrial tissue of control group were measured.Results: TLR4, NF-κB, a disintegrin and metalloproteinase 15 (ADAM15), ADAM17, matrix metalloproteinase 9 (MMP9) and plasminogen activator inhibitor 1 (PAI-1) mRNA expression as well as transforming growth factor-β1 (TGF-β1), Smad2/3, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), basic fibroblast growth factor (bFGF), periostin/osteoblast-specific factor 2 (Postn), type I collagen (Col-I) and actin-α (α-SMA) contents in the adhesion tissues of IUAs group were significantly higher than those of control group while urokinase-type plasminogen activator (uPA) mRNA expression was significantly lower than that of control group;TLR4 and NF-κB mRNA expression were positively correlated with TGF-β1, Smad2/3, IGF-1, IGF-1R, bFGF, Postn, Col-I,α-SMA, ADAM15, ADAM17, MMP9 and PAI-1, and negatively correlated with uPA.Conclusion:The excessive activation of TLR4/NF-κB pathway in IUAs is associated with the cytokine secretion and collagen metabolism abnormalities.展开更多
Objective:To investigate the mechanism of the effects of Radix isatidis on lung inflammation in tuberculosis rats through TLR4-NF-κB pathway.Methods:Forty-five SD rats were randomly divided into three groups(n=15 in ...Objective:To investigate the mechanism of the effects of Radix isatidis on lung inflammation in tuberculosis rats through TLR4-NF-κB pathway.Methods:Forty-five SD rats were randomly divided into three groups(n=15 in each group):control group,model group,and isatis root polysaccharide group.Through the tail vein injection of H37RV Mycobacterium tuberculosis model,the rats in the isatis root polysaccharide group received the intervention of Radix isatidis polysaccharides on the second day of infection.The serum inflammatory factors and the expression levels of TLR4-NF-κB pathway and inflammatory factors in lung tissue of each group were detected and compared.Results:A large number of Mycobacterium tuberculosis infection in the lung tissue of the model group,and clustered outside the cell.In the isatis root polysaccharide group,the CFU level was significantly lower compared with the model group,and the number of Mycobacterium tuberculosis was less than that of the model group(P<0.05),mainly distributed in tuberculous nodules and giant cells.The level of serum inflammatory factors in the model group was significantly higher than that in the control group(P<0.05).The levels of IL-1β,IL-6 and IFN-γin the serum of the isatis root polysaccharide group were significantly lower than those in the model group and significantly higher than those in the control group(P<0.05).The TLR4-NF-κB pathway and inflammatory factor mRNA levels in the lung tissue of the model group were significantly higher than those of the control group(P<0.05).The levels of TLR4,NF-κB,IL-1β,IL-6 and IFN-γin the lung tissue of the isatis root polysaccharide group were significantly lower than those of the model group and significantly higher than those of the control group(P<0.05).The expression level of TLR4-NF-κB pathway was significantly up-regulated in the lung tissue of model rats(P<0.05).The levels of TLR4 and NF-κB in the lung tissue of the isatis root polysaccharide group were significantly lower than those of the model group and significantly higher than those of the control group(P<0.05).Conclusion:Radix isatidis can alleviate lung inflammation caused by Mycobacterium tuberculosis infection by regulating TLR4-NF-κB pathway.展开更多
The syndrome of dampness stagnancy due to spleen deficiency(DSSD)is relatively common globally.Although the pathogenesis of DSSD remains unclear,evidence has suggested that the gut microbiota might play a significant ...The syndrome of dampness stagnancy due to spleen deficiency(DSSD)is relatively common globally.Although the pathogenesis of DSSD remains unclear,evidence has suggested that the gut microbiota might play a significant role.Radix Astragali,used as both medicine and food,exerts the effects of tonifying spleen and qi.Astragalus polysaccharide(APS)comprises a macromolecule substance extracted from the dried root of Radix Astragali,which has many pharmacological functions.However,whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown.Here,we used DSSD rats induced by high-fat and low-protein(HFLP)diet plus exhaustive swimming,and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes,decreased the levels of interleukin-1β(IL-1β),IL-6,and endotoxin,and suppressed the Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)pathway.Moreover,a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size(LEfSe).APS increased the diversity of the gut microbiota and changed its composition,such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella,and increasing that of Parasutterella,Parabacteroides,Clostridium XIVb,Oscillibacter,Butyricicoccus,and Dorea.APS also elevated the contents of short-chain fatty acids(SCFAs).Furthermore,the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes.In general,our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota,especially for some bacteria involving immune and inflammatory response and SCFA production,as well as the TLR4/NF-κB pathway.This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD.展开更多
AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance....AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance.METHODS:The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues,and expression of TLR4,NF-κB p65 and HIF-1α protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues.The relationship between TLR4 or HIF-1α and pathologic features,as well as the association between TLR4 and HIF-1α,were also analyzed.Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic cancer.RESULTS:The relative quantif ication of TLR4 and HIF-1α mRNA in tumor tissues was 0.81±0.10 and 0.87±0.11,respectively,signif icantly higher than that in adjacent tissues(0.81±0.10 vs 0.70±0.16,P=0.002;0.87±0.11 vs 0.68±0.13,P=0.000).The protein expression of TLR4,NF-κB p65 and HIF-1α in tumor tissues was 69.20%,66.15% and 70.80%,respectively,being signif icantly higher than that in adjacent normal tissues(69.20% vs 39.50%,P=0.003;66.15% vs 31.58%,P=0.001;70.80% vs 36.80%,P=0.001).There was no signif icant correlation between TLR4 or HIF-1α expression and the age,gender,tumor location,the degree of tumor differentiation in the patients(P>0.05).However,there was signif icant correlation between the expression of TLR4 or HIF-1α and tumor size,lymph node metastasis,venous invasion and clinical staging(P<0.05).The expression of TLR4 and HIF-1α had a signif icant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4,NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma,TLR4 may be partly involved in up-regulating HIF-1α,and both synergestically promote development of pancreatic adenocarcinoma.展开更多
This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA se...This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-2,protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA,real-time quantitative PCR and Western blotting,respectively.The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h.The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1,2 groups than in the TRAF6-shRNA3,4 groups.Therefore,pGCsi-TRAF6-shRNA1,2 were selected for the subsequent experiments.Our results still showed that pGCsi-TRAF6-shRNA1,2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α,IL-1β,IL-6 and COX-2,and inhibit NF-κB nuclear translocation.Moreover,pGCsi-TRAF6-shRNA1,2 could suppress the release of TGF-β1 at the protein level.It was concluded that the recombinant plasmid pTRAF6-shRNA can,to some extent,inhibit inflammatory response stimulated by LPS at the initial phase.TRAF6 may become the potential therapeutic target of many inflammation-related diseases.展开更多
Objective:To study the therapeutic effect of crocin on diabetic retinopathy(DR)in rats based on toll-like receptor 4(TLR4)/myeloid differentiation factor 88(My D88)/nuclear factor-κB(NF-κB)pathway.Methods:Thirty SPF...Objective:To study the therapeutic effect of crocin on diabetic retinopathy(DR)in rats based on toll-like receptor 4(TLR4)/myeloid differentiation factor 88(My D88)/nuclear factor-κB(NF-κB)pathway.Methods:Thirty SPF SD rats were used in the experiment,which were randomly divided into DR group,control group and crocin group,with 10 rats in each group.The DR rat model was established by feeding the rats in both the DR group and crocin group with a high glucose and high fat diet,along with intraperitoneal injection(IP)of streptozotocin.Crocin IP was administered to the rats in the crocin group,whereas the rats in the DR group and control group received an equivalent dosage of saline IP for 12 weeks.A comparison was made among the three groups regarding retinal thickness,vascular permeability,expression of TLR4/My D88/NF-κB pathway protein,levels of inflammatory factors,and levels of Bcl-2,Bax,and Bcl-2/Bax.Results:The DR group and crocins group exhibited a lower retinal thickness compared to the control group,while the crocins group displayed a higher thickness than the DR group.The DR group and crocins group had higher retinal vascular permeability than the control group,and the crocins group had lower retinal vascular permeability than the DR group(P<0.05).TLR4,My D88,and P-NF-κB relative expressions were higher in the DR and crocin groups than in the control group,whereas TLR4,My D88,and P-NF-κB relative expressions were lower in the crocin group than in the DR group(P<0.05).The DR group and crocin group exhibited elevated levels of inflammatory cytokines compared to the control group,while the crocin group displayed decreased levels in comparison to the DR group(P<0.05).The DR group and crocin group exhibited lower levels of Bcl-2 and Bcl-2/Bax compared to the control group,whereas the control group displayed higher levels of Bax.The crocin group exhibited elevated levels of Bcl-2 and Bcl-2/Bax compared to the DR group,whereas the DR group displayed diminished levels of Bax(P<0.05).Conclusion:Crocin has the potential to enhance the retinal thickness and vascular permeability of DR rats,and the inhibition of the TLR4/My D88/NF-κB pathway by crocin could play a crucial role in impeding the advancement of DR.展开更多
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...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.展开更多
Objective: To investigate the possible mechanism of San-Cao Granule(SCG, 三草颗粒) mediating antiliver fibrosis. Methods: A total of 60 male Sprague-Dawley rats were randomly divided into the normal control group,...Objective: To investigate the possible mechanism of San-Cao Granule(SCG, 三草颗粒) mediating antiliver fibrosis. Methods: A total of 60 male Sprague-Dawley rats were randomly divided into the normal control group, porcine serum-treated group, ursodesoxycholic acid(UDCA, 60 mg/kg), SCG(3.6 g/kg) group, SCG(1.8 g/kg) group and SCG(0.9 g/kg) group, with 10 rats in each group. Liver fibrosis was induced with porcine serum by intraperitoneal injection for 8 weeks, except for the normal control group. Then, the rats in the three SCG-treated groups and UDCA group were administered SCG and UDCA respectively for 4 weeks. The serum levels of alanine transaminase(ALT), aspartate transaminase(AST), albumin(ALB), total bilirubin(TBIL), hyaluronic acid(HA), laminin(LN), and type Ⅳcollagen(ⅣC) were examined using commercial kits and hepatic histopathology was examined with hematoxylin and eosin and Masson staining. Moreover, the protein expression levels of high mobility group box-1 protein(HMGB1), transforming growth factor β1(TGF-β1), phosphorylated mothers against decapentaplegic homolog 3(p-Smad3), Smad7, toll-like receptor 4(TLR4), myeloid differentiation factor 88(My D88), nuclear factor-kappa B(NF-κB) and α-smooth muscle actin(α-SMA) were determined by western blot, immunohistochemistry and real time quantitativereverse transcription polymerase. Results: Both SCG(3.6 and 1.8 g/kg) and UDCA significantly ameliorated the liver fibrosis induced by porcine serum as indicated by retarding the serum levels increasing of ALT, AST, TBIL, HA, LN and ⅣC and preventing the serum level reducing of ALB compared with the model group(all P〈0.01). Meanwhile, the collagen deposition was attenuated by SCG and UDCA treatment. Furthermore, SCG markedly reduced the expressions of HMGB1, TGF-β1, p-Smad3, TLR4, My D88, NF-κB and α-SMA, and enhanced the expression of the Smad7 compared with the model group(all P〈0.01). Conclusion: SCG ameliorates hepatic fibrosis possibly through inhibiting HMGB1, TLR4/NF-κB and TGF-β1/Smad signaling pathway.展开更多
文摘Objective:To study the correlation of the changes of Toll-like receptor 4 (TLR4) / nuclear factorκB (NF-κB) pathway function in intrauterine adhesion (IUAs) tissue with the characteristics of cytokine secretion and collagen metabolism.Methods:The patients with IUAs who were treated in our hospital between February 2015 and March 2018 were selected as the IUAs group, and the patients who underwent hysteroscopy due to infertility and were pathologically confirmed to have normal endometrium during the same period were selected as the control group. The expression levels of TLR4/NF-κB pathway molecules and collagen metabolism genes as well as the contents of cytokines and collagen metabolism markers in the adhesion tissues of IUAs group and the normal endometrial tissue of control group were measured.Results: TLR4, NF-κB, a disintegrin and metalloproteinase 15 (ADAM15), ADAM17, matrix metalloproteinase 9 (MMP9) and plasminogen activator inhibitor 1 (PAI-1) mRNA expression as well as transforming growth factor-β1 (TGF-β1), Smad2/3, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), basic fibroblast growth factor (bFGF), periostin/osteoblast-specific factor 2 (Postn), type I collagen (Col-I) and actin-α (α-SMA) contents in the adhesion tissues of IUAs group were significantly higher than those of control group while urokinase-type plasminogen activator (uPA) mRNA expression was significantly lower than that of control group;TLR4 and NF-κB mRNA expression were positively correlated with TGF-β1, Smad2/3, IGF-1, IGF-1R, bFGF, Postn, Col-I,α-SMA, ADAM15, ADAM17, MMP9 and PAI-1, and negatively correlated with uPA.Conclusion:The excessive activation of TLR4/NF-κB pathway in IUAs is associated with the cytokine secretion and collagen metabolism abnormalities.
基金This study was supported by Chongqing Municipal Health and Family Planning Commission(Grant No.ZY201703067).
文摘Objective:To investigate the mechanism of the effects of Radix isatidis on lung inflammation in tuberculosis rats through TLR4-NF-κB pathway.Methods:Forty-five SD rats were randomly divided into three groups(n=15 in each group):control group,model group,and isatis root polysaccharide group.Through the tail vein injection of H37RV Mycobacterium tuberculosis model,the rats in the isatis root polysaccharide group received the intervention of Radix isatidis polysaccharides on the second day of infection.The serum inflammatory factors and the expression levels of TLR4-NF-κB pathway and inflammatory factors in lung tissue of each group were detected and compared.Results:A large number of Mycobacterium tuberculosis infection in the lung tissue of the model group,and clustered outside the cell.In the isatis root polysaccharide group,the CFU level was significantly lower compared with the model group,and the number of Mycobacterium tuberculosis was less than that of the model group(P<0.05),mainly distributed in tuberculous nodules and giant cells.The level of serum inflammatory factors in the model group was significantly higher than that in the control group(P<0.05).The levels of IL-1β,IL-6 and IFN-γin the serum of the isatis root polysaccharide group were significantly lower than those in the model group and significantly higher than those in the control group(P<0.05).The TLR4-NF-κB pathway and inflammatory factor mRNA levels in the lung tissue of the model group were significantly higher than those of the control group(P<0.05).The levels of TLR4,NF-κB,IL-1β,IL-6 and IFN-γin the lung tissue of the isatis root polysaccharide group were significantly lower than those of the model group and significantly higher than those of the control group(P<0.05).The expression level of TLR4-NF-κB pathway was significantly up-regulated in the lung tissue of model rats(P<0.05).The levels of TLR4 and NF-κB in the lung tissue of the isatis root polysaccharide group were significantly lower than those of the model group and significantly higher than those of the control group(P<0.05).Conclusion:Radix isatidis can alleviate lung inflammation caused by Mycobacterium tuberculosis infection by regulating TLR4-NF-κB pathway.
基金supported by the National Natural Science Foundation of China(No.81903947)the Key Research and Development Project of Shandong Province(No.2019GSF108209),China.
文摘The syndrome of dampness stagnancy due to spleen deficiency(DSSD)is relatively common globally.Although the pathogenesis of DSSD remains unclear,evidence has suggested that the gut microbiota might play a significant role.Radix Astragali,used as both medicine and food,exerts the effects of tonifying spleen and qi.Astragalus polysaccharide(APS)comprises a macromolecule substance extracted from the dried root of Radix Astragali,which has many pharmacological functions.However,whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown.Here,we used DSSD rats induced by high-fat and low-protein(HFLP)diet plus exhaustive swimming,and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes,decreased the levels of interleukin-1β(IL-1β),IL-6,and endotoxin,and suppressed the Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)pathway.Moreover,a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size(LEfSe).APS increased the diversity of the gut microbiota and changed its composition,such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella,and increasing that of Parasutterella,Parabacteroides,Clostridium XIVb,Oscillibacter,Butyricicoccus,and Dorea.APS also elevated the contents of short-chain fatty acids(SCFAs).Furthermore,the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes.In general,our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota,especially for some bacteria involving immune and inflammatory response and SCFA production,as well as the TLR4/NF-κB pathway.This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD.
基金Supported by Grants from the National Natural Science Foundation of China, No 30972898
文摘AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance.METHODS:The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues,and expression of TLR4,NF-κB p65 and HIF-1α protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues.The relationship between TLR4 or HIF-1α and pathologic features,as well as the association between TLR4 and HIF-1α,were also analyzed.Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic cancer.RESULTS:The relative quantif ication of TLR4 and HIF-1α mRNA in tumor tissues was 0.81±0.10 and 0.87±0.11,respectively,signif icantly higher than that in adjacent tissues(0.81±0.10 vs 0.70±0.16,P=0.002;0.87±0.11 vs 0.68±0.13,P=0.000).The protein expression of TLR4,NF-κB p65 and HIF-1α in tumor tissues was 69.20%,66.15% and 70.80%,respectively,being signif icantly higher than that in adjacent normal tissues(69.20% vs 39.50%,P=0.003;66.15% vs 31.58%,P=0.001;70.80% vs 36.80%,P=0.001).There was no signif icant correlation between TLR4 or HIF-1α expression and the age,gender,tumor location,the degree of tumor differentiation in the patients(P>0.05).However,there was signif icant correlation between the expression of TLR4 or HIF-1α and tumor size,lymph node metastasis,venous invasion and clinical staging(P<0.05).The expression of TLR4 and HIF-1α had a signif icant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4,NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma,TLR4 may be partly involved in up-regulating HIF-1α,and both synergestically promote development of pancreatic adenocarcinoma.
文摘This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-2,protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA,real-time quantitative PCR and Western blotting,respectively.The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h.The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1,2 groups than in the TRAF6-shRNA3,4 groups.Therefore,pGCsi-TRAF6-shRNA1,2 were selected for the subsequent experiments.Our results still showed that pGCsi-TRAF6-shRNA1,2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α,IL-1β,IL-6 and COX-2,and inhibit NF-κB nuclear translocation.Moreover,pGCsi-TRAF6-shRNA1,2 could suppress the release of TGF-β1 at the protein level.It was concluded that the recombinant plasmid pTRAF6-shRNA can,to some extent,inhibit inflammatory response stimulated by LPS at the initial phase.TRAF6 may become the potential therapeutic target of many inflammation-related diseases.
文摘Objective:To study the therapeutic effect of crocin on diabetic retinopathy(DR)in rats based on toll-like receptor 4(TLR4)/myeloid differentiation factor 88(My D88)/nuclear factor-κB(NF-κB)pathway.Methods:Thirty SPF SD rats were used in the experiment,which were randomly divided into DR group,control group and crocin group,with 10 rats in each group.The DR rat model was established by feeding the rats in both the DR group and crocin group with a high glucose and high fat diet,along with intraperitoneal injection(IP)of streptozotocin.Crocin IP was administered to the rats in the crocin group,whereas the rats in the DR group and control group received an equivalent dosage of saline IP for 12 weeks.A comparison was made among the three groups regarding retinal thickness,vascular permeability,expression of TLR4/My D88/NF-κB pathway protein,levels of inflammatory factors,and levels of Bcl-2,Bax,and Bcl-2/Bax.Results:The DR group and crocins group exhibited a lower retinal thickness compared to the control group,while the crocins group displayed a higher thickness than the DR group.The DR group and crocins group had higher retinal vascular permeability than the control group,and the crocins group had lower retinal vascular permeability than the DR group(P<0.05).TLR4,My D88,and P-NF-κB relative expressions were higher in the DR and crocin groups than in the control group,whereas TLR4,My D88,and P-NF-κB relative expressions were lower in the crocin group than in the DR group(P<0.05).The DR group and crocin group exhibited elevated levels of inflammatory cytokines compared to the control group,while the crocin group displayed decreased levels in comparison to the DR group(P<0.05).The DR group and crocin group exhibited lower levels of Bcl-2 and Bcl-2/Bax compared to the control group,whereas the control group displayed higher levels of Bax.The crocin group exhibited elevated levels of Bcl-2 and Bcl-2/Bax compared to the DR group,whereas the DR group displayed diminished levels of Bax(P<0.05).Conclusion:Crocin has the potential to enhance the retinal thickness and vascular permeability of DR rats,and the inhibition of the TLR4/My D88/NF-κB pathway by crocin could play a crucial role in impeding the advancement of DR.
基金Project supported by the National Natural Science Foundation of China(No.31472254)
文摘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.
基金Supported by the Major Projects of the National Science and Technology(No.2012ZX10005010-002-002)the National Natural Science Foundation of China(No.81303120 and No.81173571)
文摘Objective: To investigate the possible mechanism of San-Cao Granule(SCG, 三草颗粒) mediating antiliver fibrosis. Methods: A total of 60 male Sprague-Dawley rats were randomly divided into the normal control group, porcine serum-treated group, ursodesoxycholic acid(UDCA, 60 mg/kg), SCG(3.6 g/kg) group, SCG(1.8 g/kg) group and SCG(0.9 g/kg) group, with 10 rats in each group. Liver fibrosis was induced with porcine serum by intraperitoneal injection for 8 weeks, except for the normal control group. Then, the rats in the three SCG-treated groups and UDCA group were administered SCG and UDCA respectively for 4 weeks. The serum levels of alanine transaminase(ALT), aspartate transaminase(AST), albumin(ALB), total bilirubin(TBIL), hyaluronic acid(HA), laminin(LN), and type Ⅳcollagen(ⅣC) were examined using commercial kits and hepatic histopathology was examined with hematoxylin and eosin and Masson staining. Moreover, the protein expression levels of high mobility group box-1 protein(HMGB1), transforming growth factor β1(TGF-β1), phosphorylated mothers against decapentaplegic homolog 3(p-Smad3), Smad7, toll-like receptor 4(TLR4), myeloid differentiation factor 88(My D88), nuclear factor-kappa B(NF-κB) and α-smooth muscle actin(α-SMA) were determined by western blot, immunohistochemistry and real time quantitativereverse transcription polymerase. Results: Both SCG(3.6 and 1.8 g/kg) and UDCA significantly ameliorated the liver fibrosis induced by porcine serum as indicated by retarding the serum levels increasing of ALT, AST, TBIL, HA, LN and ⅣC and preventing the serum level reducing of ALB compared with the model group(all P〈0.01). Meanwhile, the collagen deposition was attenuated by SCG and UDCA treatment. Furthermore, SCG markedly reduced the expressions of HMGB1, TGF-β1, p-Smad3, TLR4, My D88, NF-κB and α-SMA, and enhanced the expression of the Smad7 compared with the model group(all P〈0.01). Conclusion: SCG ameliorates hepatic fibrosis possibly through inhibiting HMGB1, TLR4/NF-κB and TGF-β1/Smad signaling pathway.