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Objective:To study the anti-inflammatory effects of low， middle， and high doses of Anchang decoction on ulcerative colitis in SD rats， and also explore the possible mechanism of Anchang decoction in the prevention and treatment of ulcerative colitis through the effect of different doses on miRNA-146a/non-receptor tyrosine protein kinase（JAK）/signal transduction and activator of transcription 3（STAT3）/cytokine signaling protein-3（SOCS-3） signal pathway and its downstream proteins. Method:The experimental rats were divided into control group ， model group ， mesalazine group（1 g·kg<sup>-1</sup>） and Anchang decoction low（6 g·kg<sup>-1</sup>）， middle（12 g·kg<sup>-1</sup>）and high dose groups（24 g·kg<sup>-1</sup>）， with 10 rats in each group. Except for the control group， 2，4，6-trinitrobenzenesulfonic acid （TNBS）/ethanol enema was used in all the other groups to establish a rat model of ulcerative colitis for 14 days respectively. The general changes of the mental state， stool traits， hair and other general conditions of the rats were observed， and score was graded with reference to the disease activity index （DAI） table. The pathological changes of colon tissue of rats in each group were observed by hematoxylin-eosin （HE） staining. The levels of serum tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）， interleukin-10（IL-10）， interleukin-17（IL-17）， interleukin-1<italic>β</italic>（IL-1<italic>β</italic>）and interleukin-6（IL-6）were detected by enzyme linked immunosorbent assay （ELISA）. The expression levels of JAK2， phosphorylation STAT3 （p-STAT3）， STAT3 and inhibitor of SOCS-3 in colon tissue were detected by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the expression of JAK2， p-STAT3， STAT3， SOCS-3 mRNA in rat colon and miRNA-146a in rat plasma. Result:Compared with the normal group， the expression of JAK2， p-STAT3， STAT3 protein and the expression of JAK2， p-STAT3 and STAT3 mRNA in the model group increased （<italic>P</italic><0.05）， and the relative expression of miRNA-146a， SOCS-3 mRNA and SOCS-3 protein decreased in the model group （<italic>P</italic><0.05）. Compared with the model group， the mental state， food intake， coat color， etc. of rats in the administration groups were significantly improved， the DAI score was significantly reduced （<italic>P</italic><0.05）， the colonic ulcer tissues of rats in the administration groups were improved significantly， the expression levels of JAK2， p-STAT3， STAT3 protein and the expression of JAK2， p-STAT3 and STAT3 mRNA in the colon tissue of the administration groups were decreased （<italic>P</italic><0.05）， and the relative expression levels of miRNA-146a， SOCS-3 mRNA and SOCS-3 protein were increased in the administration groups （<italic>P</italic><0.05）. Conclusion:Anchang decoction can alleviate ulcerative colitis and reduce the activation of inflammatory factors by affecting the expression of genes and proteins related to miRNA-146a/JAK/STAT/SOCS-3 signal transduction pathway.
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Objective To lay the foundation for studying the possible pathogenesis of epilepsy and the anti-epileptic mechanism of Banxia Baizhu Tianma Decoction through the bioinformatic analysis of target gene prediction and signal pathway of miRNA-146a-5p in hippocampus of epileptic rats. Methods Lithium-pilocarpine was used to induce seizures in rat models. The experiment rats were randomly divided into normal control group, model group, Banxia Baizhu Tianma Decoction group, with 20 rats in each group. The method of miRNA expression profiling was used to observe the miRNA differential expression of hippocampus neuron cell of rats. The expression level of miRNA-146a-5p was detected by real-time quantitative PCR. MiRDB was used for target gene prediction of miRNA-146a-5p, and miRTarBase and DAVID were used for enrichment analysis on the GO function and KEGG signaling pathway. Results The attack times and grades of the rats in Banxia Baizhu Tianma Decoction group were significantly lower than those in the model group from behavioral observation. MiRNA microarray analysis showed that the expression level of miRNA-146a-5p in model group was 2.107 times normal control group (P＜0.05), and the expression level decreased to 1.377 times after treatment with Banxia Baizhu Tianma Decoction (P＜0.05). The results of RT-PCR was consistent with that of miRNA microarray, with statistical significance (P＜0.05). MiRNA-146a-5p target gene prediction results had 140 target genes by GO, and there were 14 annotation information of biological process (P＜0.05), 9 annotation information of cellular component (P＜0.05), 11 annotation information of molecular function (P＜0.05). Enrichment analysis of KEGG biological pathway showed that 140 target genes of miRNA-146a-5p were enriched in EB virus infection signal pathway and thyroid hormone signaling pathway (P＜0.05). Conclusion miRNA-146a-5p is closely related to the inflammatory reaction after epilepsy, and Banxia Baizhu Tianma Decoction can control epilepsy possibly by controlling the inflammatory reaction after epilepsy.
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Objective To observe the effects of Ganlu Xiaodu Dan and its incomplete prescription on expressions of MiR-146a and TLR4 mRNA in RD cells infected by EV71. Methods With technique of cell culturing, Ganlu Xiaodu Dan therapy group, incomplete Qing prescription therapy group, incomplete Li prescription therapy group, normal cells control group, model control group and ribavirin control group were set, and tests for virus toxicity and medicine toxicity in cells were taken, then expressions of miRNA-146a and TLR4 mRNA in these RD cells 24 hours after intervention with medicine were detected. Results Compared with normal cells control group, miR-146a in mRNA model control group decreased and TLR4 mRNA increased. Compared with model control group, miR-146a mRNA in Ganlu Xiaodu Dan therapy group, incomplete Qing prescription therapy group and incomplete Li prescription therapy group all increased while TLR4 mRNA decreased, and differences between ribavirin control group and model control group were not significant. Compared with Ganlu Xiaodu Dan therapy group, both expressions of miR-146a and TLR4 mRNA in incomplete Qing prescription therapy group were lower; miR-146a increased and TLR4 mRNA decreased in incomplete Li prescription therapy group. Compared with incomplete Qing prescription therapy group, miR-146a mRNA in incomplete Li prescription therapy group increased, but expression of TLR4mRNA between them was not significant. Conclusion Ganlu Xiaodu Dan can regulate the immune reactions caused by infection of EV71 by increasing expression of miR-146a mRNA and reducing expression of TLR4 mRNA. There may be antagonism effect between incomplete Qing prescription and incomplete Li prescription.
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Objective To investigate the expression level of miRNA-146a (miR-146a) in retinal pigment epithelial (RPE) cells aging and age-related macular degeneration (AMD),and discuss its regulation mechanism of AMD by repressing VEGF-A.Methods The expressions of miR-146 and VEGF-A were examined by qRT-PCR in RPE cells in mice aged 2 months,8 months,12 months,18 months or 24 months,and in RPE cells from 75 years old AMD patients.The protein level of VEGF-A was also detected by Western Blotting.Finally,the effects of overexpression of miR-146a in APRE-19 cell line on expression of VEGF-A was checked.Results MiR-146 was up-regulated to 8 times or 24 times at 18 months or 24 months aged mice,and the expression of VEGFA was down-regulated in aging RPE from 1.5 times to 0.8 times.However,the expression of miR-146 decreased to 14.5 times and VEGF-A increased in RPE cells of AMD.In cultured cells,overexpression of miR-146a inhibited the expression of VEGF-A.Conclusion Up-regulation of miR-146a in aging RPE cells and its down-regulation in AMD suggest a potential of miR-146a as molecular marker.MiR-146a overexpression inhibits the expression of VEGF-A,supporting a potential clinical treatment of miR-146 in AMD.
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@#AIM: To study serum soluble intercellular adhesion molecules -1(sICAM-1), soluble vascular cell adhesion molecule -1(sVCAM-1)and expression of miRNA-146a in peripheral blood mononuclear cells(PBMC)and its significance in patients with thyroid associated ophthalmopathy(TAO). <p>METHODS: From June 2014 to December 2015 in our hospital, 37 patients with TAO(TAO group), 40 patients with hyperthyroidism without TAO(non eye disease group)and 30 healthy people(control group)were enrolled and the serum concentrations of sICAM-1, sVCAM-1 and expression of miRNA-146a in PBMC were detected. <p>RESULTS: The serum sICAM-1 and sVCAM-1 of TAO group were 366.14±67.28g/L, 211.07±27.45g/L level was significantly higher than those of non eye disease group(286.62±51.09μg/L, 179.83±25.09μg/L)and healthy group(234.51±38.969μg/L, 164.51±22.57μg/L)(<i>P</i><0.05). In TAO group, miRNA-146a(0.071±0.016)in PBMC was lower than that in non eye disease group(0.381±0.084)and healthy group(1.105±0.216)(<i>P</i><0.05). The serum levels of sICAM-1 and sVCAM-1 were significantly higher in non eye disease group than in healthy group(<i>P</i><0.05), and the expression of RNA-146a in PBMC was lower in the non eye disease group than in the healthy group(<i>P</i><0.05). The serum levels of sICAM-1 and sVCAM-1 in the mild TAO group were significantly lower than those in the moderate-severe groups and extremely severe group(<i>P</i><0.05), and the miRNA-146a expression in the mild TAO group was higher than those in the moderate-severe group and extremely severe group(<i>P</i><0.05). The serum levels of sICAM-1 and sVCAM-1 were significantly lower in the moderate-severe group than those in the extremely severe group(<i>P</i><0.05), and the expression of miRNA-146a in the moderate -severe group was significantly higher than that in the extremely severe group(<i>P</i><0.05). <p>CONCLUSION: Serum sICAM-1, sVCAM-1 in TAO patients is with high expression, miRNA-146a in PBMC in TAO patients with low expression, and related to the degree of patient's condition.
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Object To study the expression of miRNA-146a in sepsis-induced acute lung injury (ALI) patients and its effect on the inflammation in mouse models.Methods miRNA-146a expression in peripheral blood was determined in sepsisinduced ALI patients and healthy volunteers by qRT-PCR.Sepsis-induced ALI mouse model were reproduced by LPS treatment and miRNA-146a mimic,miRNA-146a NC and miRNA-146a inhibitors were injected through trachea.The expressions of miRNA-146a,TNF-α,IL-1β and COX-2 mRNA were determined by qRT-PCR 24h after the intervention.Results The miRNA-146a expression in peripheral blood significantly increased in severe sepsis with ALI patients,compared with the control subjects.For mouse model,the expressions of TNF-o,IL-1β and COX-2 mRNA significantly decreased in miRNA-146a group,while increased in miRNA-146a inhibitor group compared with miRNA-146a NC group (P＜0.05).The TNF-α and IL-1β expressions significantly decreased in miRNA-146a inhibitor+COX-2 inhibitor group compared with miRNA-146a inhibitor group (P＜0.05).Conclusion MiR-146a treatment can effectively alleviate the lung inflammation in sepsis-induced ALI mice.
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Objective:To detect and verifica the gene profile difference of microRNA-146a (miR-146a) and its role in the pro-liferation of vascular smooth muscle cells (VSMCs) by gene chip technology. Methods: Artificially synthesized miR-146a mimics(50 nmol/L) ,miR-146 inhibitor ( 50 nmol/L ) , scramble ( 50 nmol/L ) and PBS were transfected into cultured primary rat VSMCs in vitro. After transfection,Real time PCR was used to measure the levels of miR-146a and the cell counting kit 8(CCK8) was employed to investigate the proliferation of VSMCs. The VSMCs interfered by miR-146a inhibitor or miR-146a control were examined by gene chips and the profile of gene were analyzed by bioinformatics technology to detect the different genes and signal transduction pathway. The changes in mRNAs and proteins were accessed separately by Real time PCR and Western blot. Results: Compared with sham and control VSMCs,miR-146a expression level was significantly decreased in treatment with miR-146a inhibitor(P0. 05),however,the mRNA and protein expression levels of cyclin D1 significantly increased in treatment with miR-146a mimics VSMCs group and decreased in miR-146a inhibitor VSMCs group ( compared with sham VSMCs group, both P<0. 05 ) . Conclusion: Our data indicated that miR-146a may promote the proliferation of rat VSMCs by up-regulating cyclin D1 expression.
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Objective To detect the effects of bifidobacterium or bifidobacterium cultured supernatant on the mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),glycogen synthase kinase-3β (GSK-3β) and the miRNA-146a in rat small intestinal epithelial cell(IEC-6) induced by lipopolysaccharide (LPS).Methods IEC-6 in logarithmic phase were randomly divided into LPS group,cultured supernatant group and inactivated bacteria group.All the 3 groups were exposed to 5 mg/L LPS for 5 hours,and then 1 mL sterile saline was added in LPS group and culturing continued for 24 hours ; 1 mL bifidobacterium cultured supernatant was added in cultured supernatant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 x 1010 CFU/L added in inactivated bacteria group and continued culturing for 24 hours.The mRNA expressions of TRAF6,GSK-3 β and miRNA-146a were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The level of TRAF6,GSK-3 β of culture supematant group (0.72 ± 0.05,0.46 ± 0.14) were all lower than LPS group (1.01 ± 0.14,1.02 ± 0.25),but the level of miRNA-146a(3.05 ± 0.40) was higher than that in LPS group(1.01 ± 0.12),and there were significant differences between them (t =5.278,6.316,13.218,P =0.000).The level of GSK-3 β of inactivated bacteria group(0.59 ±0.13) was significantly lower than that in LPS group(t =4.837,P =0.000).The levels of TRAF6 and miRNA-146a of inactivated bacteria group(1.05 ±0.11,0.78 ±0.22) had no significant differences with LPS group (t =0.732,1.463,P ＞ 0.05).The level of TRAF6 of cultured supernatant group was lower than that in inactivated bacteria group,and the level of miRNA-146a was higher than that in inactivated bacteria group,and there were significant differences between 2 groups (t =6.009,14.687,P =0.000).Conclusions Bifidobacterium cultured supernatant and inactivated bacteria both have certain protective effect on the IEC-6 induced by LPS.One of the protective mechanisms of bifidobacterium cultured supernatant may be achieved by elevating the expression of miRNA-146a,and decreasing the levels of inflammation related factor TRAF6 and damage related factor GSK-3β.The protective effects of inactivated bifidobacterium may be achieved by decreasing the level of damage related factor GSK-3β.