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OBJECTIVE: To explore the expression of miR-199a in ulcerative colitis (UC) rats induced by 2, 4, 6-trinitrobenzene sulfonic (TNBS)/ethanol and the study on the effect of TWP on them. METHODS: Through injecting TNBS/ethyl alcohol acid liquid into the anus of the rats to establish the UC rat model. The colitics commom morphous damage and grade the histopathological score (CMDI) of colon mucosa injury were evaluated. Chip analysis and Real-time PCR were used to verify the expression of miR-199a in each colon mucosa tissue. Based on the expression profile, the downstream target genes mRNA in milwalk database was selected, then the expression of target genes mRNA by Real-time PCR in each group was veritied, at last the relevant signal pathway in the DAVID database was analyzed. Doing these to analyse the target gene mRNA regulated by the miR-199a in the inflammatory activity of UC. RESULTS: Compared with the model group, TWP high dose group was significantly lower on gross morphological damage score and histopathological injury score(P < 0.01). Chip analysis showed that in model group, the expression of miR-199a was significantly higher than the normal group(P < 0.01), and expression of the AZA group was significantly lower than the model group(P < 0.01, P < 0.05). The expression of miR-199a-3p in medium dose group and the expression of miR-199a-5p in high dose group were significantly lower than the model group(P < 0.05). The results of Real-time PCR showed that expression of miR-199a in the model group was significantly increased than that in the normal group(P < 0.01). The expression of miR-199a-3p in TWP medium dose group, high dose group and AZA group were decreased than that in model group(P < 0.05). Meanwhile, the expression of miR-199a-5p in TWP medium dose group was decreased than that in model group(P < 0.05). The gene expression profile showed that FASL was the target gene of miR-199a. In the model group, the expression of FASL was higher than that in the normal group. The expression of FASL in AZA group was significantly decreased than that in the model group(P < 0.01). The results by the Real-time PCR of the target gene FASL showed that in the model group, the expression of FASL was higher than that in the normal group (P < 0.01). The expression of FASL in medium dose group, high dose group and AZA group were significantly decreased than that in the model group (P < 0.01). CONCLUSION: miR-199a is up-regulated in TNBS/Ethanol UC rats, and FASL is the downstream target gene of miR-199a. TWP can reduce the UC's inflammatory effectively and decrease the up-regulated miR-199a in UC. FASL is up-regulated in UC's inflammatory activity. TWP can reduce downstream target gene FASL of miR-199a.
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Objective: To study the regulatory effect of Tripterygium wilfordii Polycoride Tadlet (TWPT) towards miR-146a, miR-146b, and TLR4/MyD88 dependent signaling pathway in TNBS/ethanol ulcerative colitis (UC) rat model. Methods: TNBS enema was adopted to build TNBS/ethanol UC rat model. After the modeling procedure, 90 male Wistar rats were divided into six groups, including normal, model, low-, mid-, high-dose TWPT, and azathioprine (AZA) groups, and each for 15 rats. All rats in each group were administered with corresponding medicines for 14 d. After 14 d administration, corresponding colon tissues were taken to undergo general and microscopic evaluation. qPCR was adopted to test the expression of miR-146a and miR-146b. Western blotting analysis and RT-PCR were adopted to test the mRNA and protein expression levels of TLR4/MyD88 dependent signaling pathway related molecular, including TLR4, MyD88, TRAF-6, NF-κB, TNF-α, and IL-1β. Results: DAI, general and microscopic evaluation all showed that TNBS/ethanol UC rat model was successfully established. TWPT could improve UC-related clinical manifestation and promote the colonic mucosa healing procedure and such effect was equal to AZA. qRT-PCR showed that the expression of miR-146a and miR-146b in model group was significantly superior to that in normal group (P 0.05). Conclusion: In TNBS/ethanol UC rat model, TWPT could inhibit the expression of miR-146a, miR-146b, and TLR4/MyD88 dependent signaling pathway. The inhibitory effect of TWPT towards pathway and inflammatory cytokines shows a dose-dependent manner.
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In order to study the regulatory effect of Tripterygium wilfordii polycoride (TWP) towards TLR4/MyD88 independent pathway in TNBS/ethanol ulcerative colitis (UC) rat model, TNBS/ethanol enema was adopted to build TNBS/ethanol UC rat model. After the successful modeling procedure, 90 male Wistar rats are were divided into 6 groups, including namely normal group, model group, TWP low, middle, high dose groups (3, 6, 12 mg•kg⁻¹)and azathioprine (AZA) group (6 g•kg⁻¹), with 15 rats in each group. All rats in each group were administrated with corresponding medicines for 14 days. After 14 days of administration, corresponding colon tissues were taken for general and microscopic evaluation. Western blotting analysis and RT-PCR were adopted to test the mRNA and protein expressions of TLR4/MyD88 independent pathway-related molecules, namely TLR4, TRAM, TRIF, NF-κB and IFN-γ. The results showed that DAI, general and microscopic evaluations all indicated that TNBS/ethanol UC rat model was successful. TWP can improve UC-related clinical manifestation and heal colonic mucosa, which was equal to AZA. RT-PCR and WB results showed that the expression of TLR4/MyD88 independent pathway-related molecules in model group were significantly superior to that in normal group at either mRNA or protein level (P<0.01). Compared with model group, TWP can inhibit the expression of each node in TLR4/MyD88 independent pathway in a dose-dependent manner. The inhibitory effect of TWP with high dose towards the above molecules was inferior to that in model group at either mRNA or protein level (P<0.05). The inhibitory effect of TWP with high dose towards upstream molecules of TLR4/MyD88 independent pathway (TLR4, TRAM, TRIF, NF-κB) was slightly superior to AZA group at either mRNA or protein level. However, such inhibitory effect towards terminal inflammatory cytokines (IFN-γ) was inferior to AZA group at either mRNA or protein level. All the above differences had no statistical significance. Therefore, in TNBS/ethanol UC rat model, TLR4/MyD88 independent pathway took part in regulating inflammation. TWP exerted its anti-inflammation effect by inhibiting the expression of TLR4/MyD88 independent pathway in a dose-dependent manner.
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Objective: To investigate the inhibitory effect of Tripterygium wilfordii polycoride (TWP) towards the pro-inflammatory factors (TNF-α and IL-1β) on the inflammatory reaction in macrophages induced by LPS and its regulatory effect and influence on the inflammation via TLR4/NF-kB. Methods: The MTT method was adopted to test the effect of drugs, TWP, dexamethasone (DXM) and azathioprine (AZA) on cell growth and to select the appropriate concentration. LPS was used to induce the inflammatory reaction in RAW264.7 cell line of mice. Elisa kit was adopted to test the levels of TNF-α and IL-1β. Western blotting was adopted to test the protein expression of TNF-α and IL-1β. RT-PCR was adopted to test the expression of TLR4 and NF-κB. Results: The inhibiting effect of TWP on the release of TNF-α and IL-1β in a dose dependent manner. The inhibitory effect of three different TWP dose groups is weaker than that in DXM group. However, TWP in high dose is better than AZA on TNF-α and is as strong as AZA on IL-1β. The dose dependent manner also exits in the effect on the expression of TLR4 and NF-κB, the effect is not weaker, but even stronger than that of DXM and AZA. Conclusion: The research shows that down regulation of TLR4 and NF-kB p65 may be one of the mechanisms about the TWP inhibitory effect on TNF-α and IL-1β.
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To investigate the effect of Tripterygium wilfordii polycoride (TWP) on LPS-induced macrophage inflammatory response, particularly the inhibitory effect on inflammatory factors TNF-α and IL-1β and the regulatory effect on inflammation via TLR4/NF-κB. The MTT method was adopted to test the effects of tested drugs, TWP, dexamethasone (DXM) and azathioprine (AZA) on cell growth to define the appropriate concentration. LPS was used to induce the inflammatory reaction in mouse RAW264. 7 cell lines. The Elisa kit was adopted to test the release level of TNF-α and IL-1β. The Western blotting was applied to test the protein expressions of TNF-α and IL-1β. The RT-PCR was adopted to test the expressions of TLR4 and NF-κB. According to the results, TWP could inhibit the release of macrophage inflammatory factors TNF-α and IL-1β in a dose dependent manner. All of TWP groups showed a weaker efficacy than that of the DXM group. But the TWP high dose group revealed a better effect on TNF-α and equal effect on IL-1β compared with the AZA group. TWP show an equal or better effect in down-regulating TLR4 and NF-κB p65 expressions in a dose dependent manner than DXM and AZA. In conclusion, TWP could inhibit TLR4 and NF-κB p65, which may be related to the down-regulation of TLR4 and NF-κB p65 receptor expressions.
Subject(s)
Animals , Humans , Mice , Anti-Inflammatory Agents , Pharmacology , Cell Proliferation , Down-Regulation , Drugs, Chinese Herbal , Pharmacology , Inflammation , Drug Therapy , Genetics , Allergy and Immunology , Interleukin-1beta , Genetics , Allergy and Immunology , Macrophages , Allergy and Immunology , NF-kappa B , Genetics , Allergy and Immunology , Toll-Like Receptor 4 , Genetics , Allergy and Immunology , Transcription Factor RelA , Genetics , Allergy and Immunology , Tripterygium , ChemistryABSTRACT
<p><b>OBJECTIVE</b>To study the effect of Modified Dachengqi Decoction (MDD) as whole course therapy on mediators of inflammation in severe acute pancreatitis (SAP) model rats, and to compare interventional advantages over intestinal mucosal barrier (IMB) of SAP rats between whole course therapy of MDD and early stage therapy of MDD.</p><p><b>METHODS</b>Totally 190 SD rats were divided into five groups according to random digit table, i.e., the sham-operation group, the model group, the octreotide (OT) group, the early stage MDD treatment group, the whole course MDD treatment group, 38 in each group. SAP models were established with retrograde injection of 5% sodium taurocholate into the pancreaticobiliary duct. Three hours after modeling normal saline (NS) was administered to rats in the sham-operation group and the model group by gastrogavage, once per 12 h.1.35 µg/100 g OT was subcutaneously injected to rats in the OT group, once every 8 h. 0.4 mL/100 g MDD was administered to rats in the early stage MDD treatment group, and 6 h later changed to NS (once per 12 h).0.4 mL/100 g MDD was administered to rats in the whole course MDD treatment group, once every 12 h. The accumulative survival rate and morphological manifestations of pancreas and small intestine were observed under microscope 48 h after modeling. Pathologic scores of the pancreas and small intestine were conducted at 4, 6, 24, and 48 h after modeling. Contents of serum amylase (AMY), alanine transaminase (ALT), and TNF-α were also detected. The expression of high mobility group box protein 1 (HMGB1) in the small intestine tissue was also detected by Western blot. The positive rate of bacterial translocation in mesenteric lymph nodes (MLNs) was observed within 48 h. Correlations between serum TNF-α or HMGB1 in small intestinal tissue and pathological scores of the pancreas or the small intestine were analyzed.</p><p><b>RESULTS</b>The accumulative survival rate was 100. 0% in the sham-operation group, 79. 2% in the whole course MDD treatment group, 70. 8% in the OT group, 45. 8% in the early stage MDD treatment group, and 37.5% in the model group. At 6 h after modeling, pathological scores decreased more in the whole course MDD treatment group, the early stage MDD treatment group, the OT group than in the model group (P < 0.05). At 24 and 48 h after modeling, pathological scores of the pancreas and the small intestine decreased more in the whole course MDD treatment group and the OT group than in the early stage MDD treatment group (P <0. 05). At 6, 24, and 48 h after modeling, serum contents of AMY and ALT both decreased more in the whole course MDD treatment group, the early stage MDD treatment group, the OT group than in the model group (P < 0.05). At 48 h after modeling serum contents of AMY and ALT both decreased more in the whole course MDD treatment group and the OT group than in the early stage MDD treatment group (P < 0.05). At 6 h after modeling serum TNF-α levels decreased more in the whole course MDD treatment group, the early stage MDD treatment group, the OT group than in the model group (P < 0.05). At 6, 24, and 48 h after modeling the level of HMGB1 in the small intestinal tissue decreased more in the whole course MDD treatment group, the early stage MDD treatment group, the OT group than in the model group (P < 0.05). Of them, HMGB1 levels at 24 and 48 h were lower in the whole course MDD treatment group and the OT group than in the early stage MDD treatment group (P < 0.05). The number of MLNs bacterial translocation at 48 h after modeling was lower in the whole course MDD treatment group and the OT group than in the early stage MDD treatment group and the model group (P < 0.05). Serum TNF-α contents within 6 h were positively correlated with pathological scores of pancreas (r = 0.579, P < 0.01). ROC curve showed that serum TNF-α contents could predict the severity of SAP (ROC = 0.990, 95% Cl: 0.971 to 1.000). HMGB1 in the small intestine was positively correlated with pathological scores of the small intestine (r = 0.620, P < 0.01).</p><p><b>CONCLUSIONS</b>Early stage use of MDD could effectively reduce the release of TNF-α, while whole course use of MDD could effectively inhibit the expression of HMGB1. The latter could preferably attenuate injuries of the pancreas and the small intestine, lower MLNs bacterial translocation, and elevate the survival rate.</p>