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Objective@# To investigate the transcriptomic changes in primary human oral keratinocytes (pHOKs) after coculture with Prevotella melaninogenica (P.m) and to verify the changes in human oral keratinocyte (HOK) cell lines.@*Methods@# pHOK was isolated and cocultured with P.m for 0, 4 and 24 h. Total RNA was extracted, a gene library was constructed, transcriptional sequencing was performed, differentially expressed genes (DEGs) were analyzed, gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and the validation of DEGs was performed by qRT-PCR and Western Blot in the HOK and P.m coculture cell model. @*Results @#1 788 DEGs were detected between the 4 h group and control group, including upregulated DEGs such as lymphocyte cytosolic protein 1(LCP1), keratin 7 (KRT7) and Cilia and flagella associated protein 251(CFAP251) and downregulated DEGs such as FERM, ARH/RhoGEF and Pleckstrin domain protein 1 (FARP1), WW domain containing transcription regulator 1(WWTR1) and Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2). 1 832 DEGs were detected between the 24 h group and control group, including upregulated DEGs such as LCP1, complement C1s(C1S), kynureninase (KYNU) and downregulated DEGs such as phosphoserine aminotransferase 1 (PSAT1), FARP1 and FKBP prolyl isomerase 10 (FKBP10). There were 1 090 common differentially expressed genes (cDEGs) in the 4 h and 24 h groups, including LCP1, KYNU and long intergenic nonprotein coding RNA 958 (LINC00958). The GO pathways were mainly enriched in response to lipopolysaccharide and the molecules of bacterial origin and apical part of the cell. KEGG pathway analysis revealed enrichment in the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor (TLR) pathway, etc. We verified the expression of a cDEG, Myosin1B (MYO1B), and qRT-PCR and Western Blot analysis showed that MYO1B expression was significantly upregulated between the control group and the P.m cocultured group (P<0.001), and its expression followed a time-dependent and concentration-dependent manner. @*Conclusion@#P.m played an important role in the transcriptome of oral keratinocytes.
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Objective:Sodium urate was used to induce acute gouty arthritis rat model, and to observe the inflammatory response of rats and the intervention effect of diclofenac sodium on the expression of Toll-like receptor-related (TLR) protein of ankle joint.Methods:Thirty males specific pathogen free (SPF) grade Wistar rats were used to develop the models. Random number table method was used to divide the rats into normal saline control group, model group, and drug group (diclofenac sodium t 1.35 mg/g body weight), 10 rats in each group. After fully grinding the sodium urate crystals, an appropriate amount of saline and Tween-80 (9∶1) was added to make a suspension, and the sodium urate crystals (25 mg/ml) were injected to the right posterior ankle of the rats in the model and drug groups. The solution was 0.2 ml, and rats in the sham group were injected with 0.2 ml of normal saline at the same location. After the model was established, drug and equal volume of purified water were administrated intragastrically once a day for 7 days. The toe volume device was used to measure the joint swelling of the rat (at 4 h, 8 h, 24 h, 48 h, 72 h) , and blood was taken from the abdominal aorta after anesthesia to determine the rat kidney function, interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) content, the rat ankle joint TLR4, myeloid differentiation factor (MyD88), NF-κBp65 protein expression were determined using Western blot and immunohistochemical methods. Multiple comparisons were carried out using single factor analysis of variance (ANOVA), comparing the two groups by using LSD- t, the comparison of different times using repetitive measure analysis of variance (repeated measures). Results:After the models were established, the rat's right ankle joint showed various degrees of redness, slow walking, and unresponsiveness. Compared with the normal saline control group, under the light microscope, the ankle synovial cells of the model group proliferated, with localized degeneration and necrosis, and many inflammatory cell infiltration. The rat serum inflammatory factors IL-1β, IL-6, TNF-α in the diclofenac sodium group [(24.6±3.3) pg/ml, (151±21) pg/ml, (61±16) pg/ml] were significantly reduce compared with model group [(28.4±4.3) pg/ml, (173±26) pg/ml, (81±5) pg/ml] ( t=2.296, P<0.01; t=2.909, P<0.01; t=2.352, P<0.01). Compared with normal saline group, variance analysis showed that the NF-κBp65, MyD88, TLR4 protein expression of ankle joint detected by Western bolt method and immunohistochemistry method was significantly increased in the model group. Compared with the model group, diclofenac sodium the ankle tissue protein expression of NF-κBp65, MyD88, and TLR4 was significantly inhibited. There were statistical significances in three groups ( P<0.05 or P<0.001). Conclusion:The level of inflammatory factors in acute gout arthritis rats model induced by sodium urate crystals is increased, and the expression of TLR4/MyD88/NF-КBp65 proteins in ankle joint tissue is increased, which affects the TLR signaling pathway. Diclofenic has inhibitory and relieving effects.
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The aim of this paper is to study the effect of astragaloside Ⅳ on renal fibrosis mice with ischemia-reperfusion injury (IRI) and discuss the mechanism. Male C57BL/6 50 mice were randomly divided into four groups, namely Sham-operated group, model group, AS-Ⅳ prevention group and AS-Ⅳ treatment group. Since the day of surgery, the mice in astragaloside Ⅳ prevention group were treated with astragaloside Ⅳ by gavage for 30 days at the dose of 30 mg·kg⁻¹·d⁻¹. At the 60th day after surgery, the mice in astragaloside Ⅳ treatment group were treated with astragaloside Ⅳ 100 by gavage for 30 days at the dose of 30 mg·kg⁻¹·d⁻¹. The mice in Sham-operated group and model group were treated with double distilled water containing 0.1% ethanol instead of astragaloside Ⅳ. Serum creatinine and blood urea nitrogen were detected by chemical methods. Histopathological changes and collagen deposition of affected kidneys were observed under optical microscope by HE and Masson staining. The expression levels of Toll like receptor pathway related molecules (TLR4,MyD88,TRAF6,TRAM,TRIF,NF-κB,TNF-α,IL-6, IFN-) in affected kidneys were observed by immunohistochemistry, Western blot methods and reverse transcription-PCR atprotein and mRNA levels in each group. The results showed that the degrees of fibrosis and histopathological damage of affected kidneys of mice in model group were the most obvious. And the expression levels of TLR4/MyD88 dependent signaling pathway-related molecules (TLR4 and MyD88, TRAF6 and NF-κB) in affected kidneys of mice in model group were the highest. At the same time, there was no difference in the expression levels of TLR4/MyD88 independent signaling pathway-related molecules（TRAM, TRIF）among sham-operated group, model group, astragaloside IV prevention group and astragaloside Ⅳ treatment group. In astragaloside Ⅳ prevention group and astragaloside Ⅳ treatment group, the injury of affected kidney was obviously reduced, and the protein expression levels of TLR4/MyD88 dependent signaling pathway-related molecules were also correspondingly reduced; at the same time, the expressions of terminal inflammatory cytokines (TNF-α,IL-6, IFN-) were suppressed. Therefore, astragaloside Ⅳ may improve renal interstitial fibrosis in mice after IRI by inhibiting the expression of TLR4/MyD88 dependent signaling pathway and the release of inflammatory cytokines (TNF-α,IL-6, IFN-), while the TLR4/MyD88 independent signaling pathway may not be involved in the process of renal fibrosis after ischemia-reperfusion injury.
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Objective To study the effects of modified Danggui Beimu Kushen Pills on the expressions of TLR2, TLR4, TLR6, TRAF6 and MyD88 in tumor tissues on MFC gastric cancer bearing mice; To discuss relevant mechanism of action. Methods MFC gastric cancer bearing mice were employed to perform anti-tumor experiment in vivo in this study. A total of eligible 48 mice were randomly divided into model group, DDP positive control group, modified Danggui Beimu Kushen Pills high-dose and low-dose groups, modified Danggui Beimu Kushen Pills high-dose and low-dose combined with DDP groups. The treatment was conducted once a day, and lasted for 14 continuous days. After the last administration of gavage orally treatment, all mice were anaesthetized and killed by cervical dislocation method to obtain tumor tissue completely for further HE staining measure and detection of TLR2, TLR4, TLR6, TRAF6 and MyD88 in tumor tissue with the method of RT-qPCR and immunohistochemistry. Meanwhile, the tumor growth was observed and the general conditions of mice were recorded. Results The model group was rich in tumor cells; the sizes of cells were different; the volume was large; the nucleus was deeply stained and the heterotypic shape was obvious, and the small focal necrosis was seen. The number of tumor cells in each administration group was reduced; the arrangement was loose; the cell volume was significantly reduced, and the nuclear pyknosis was reduced. Cell necrosis significantly increased; the number of interstitial blood vessels decreased; collagen fibers increased, especially in modified Danggui Beimu Kushen Pills high-dose and low-dose combined with DDP groups. Compared with the model group, the expressions of TLR2, TLR4, TLR6, TRAF6 and MyD88 mRNA and protein decreased in each administration group. TLR2, TLR4, TLR6, TRAF6 and MyD88 were lighter and weakly positive expressed in modified Danggui Beimu Kushen Pillshigh-dose and low-dose combined with DDP groups, the protein changes were more obvious Compared with DDP positive control group, modified Danggui Beimu Kushen Pills high-dose and low-dose groups. Conclusion Modified Danggui Beimu Kushen Pills can down-regulate TLR2, TLR4, TLR6, TRAF6 and MyD88 expression of tumor tissue in MFC gastric cancer bearing mice at both mRNA and protein levels to play anti-tumor pharmacology action.
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Objective To investigate the relationship between genetic variants in the Toll-like receptor (TLR) pathway genes and susceptibility of gastric cancer (GC) and esophageal squamous cell carcinoma (ESCC).Methods The data of whole genome association studies of the high-risk population of GC and ESCC in China were analyzed by adaptive rank-truncated product (ARTP) method in pathway and gene level.The associations between single nucleotide polymorphism (SNP) and susceptibility of GC and ESCC were analyzed with additive model of unconditional Logistic regressions.PLINK 1.07 and SPSS 19.0 software were performed for statistical analyses,and ARTP package in R3.0.2 was used for pathway and gene level analysis.Results In gene-level analyses,eight genes were found to be associated with susceptibility of GC (P ＜0.05) and six genes were associated with susceptibility of ESCC (P ＜ 0.05).In single SNP-level analyses,21 SNPs were statistically correlated with susceptibility of GC (P ＜ 0.01),and 11 SNPs were statistically correlated with susceptibility of ESCC (P ＜0.01).Conclusions Some genetic variants in TLR pathway are associated with risk of GC and ESCC.The potential molecular mechanisms need further investigation.