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ObjectiveTo investigate the effect of macrophages (MCs) on the differentiation of mouse induced pluripotent stem cells (iPSCs) into hepatic progenitor cells (HPCs). MethodsA total of 24 C57BL/6N mice were used to obtain MCs by peritoneal irrigation, and the supernatant was collected to obtain the conditioned medium of MCs (MC-CDM). Activin A, bone morphogenetic protein 4, and fibroblast growth factor were used to induce the differentiation of mouse iPSCs into HPCs. The differentiation of HPCs were randomly divided into control group (normal medium) and experimental group (MC group; use of MC-CDM medium on day 5 of induction). Morphology, immunofluorescence assay, and Western blot were used to compare the morphology of HPCs and the expression of related proteins between the control group and the MC group. The t-test was used for comparison of continuous data between two groups. ResultsHPCs derived from iPSCs were established in vitro, and HPCs had the potential to differentiate into hepatocytes. Immunofluorescence assay showed that compared with the D12 control group, the D12 MC group had a significant increase in the protein expression of the HPC-specific protein CK19 (0.901±0.072 vs 0.686±0.097, t=-3.093, P＜0.05). Western blot showed that compared with the D12 control group, the D12 MC group had a significant increase in the protein expression of the HPC-related protein CK19 (1.922±0.103 vs 1.448±0.012, t =-7.881, P ＜005), as well as a significant increase in the protein expression of the autophagy-related protein LC3 (1.392±0.042 vs 1.101±0048, t =-5.978, P＜005). ConclusionMCs can promote the differentiation of mouse iPSCs into HPCs, possibly by increasing the autophagy level of HPCs.
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Background:Tryptophan(TRP)is an essential amino acid,and can produce 5-hydroxytryptamine(5-HT)via 5-HT signal pathway and kynurenine( KYN)metabolic pathway under the catalysis of enzyme,thereby participating in the pathogenesis of visceral hypersensitivity in diarrhea-predominant irritable bowel syndrome(IBS-D). Aims:To investigate the relationship between visceral sensitivity and TRP metabolic pathway in patients with IBS-D. Methods:Thirty patients with IBS-D and 30 healthy controls from June 2012 to January 2014 at Guangdong Provincial TCM Hospital were enrolled. Score of gastrointestinal symptom rating scale( GSRS)was evaluated. Visceral sensitivity was measured by anorectal manometry. RT-PCR and Western blotting were used to detect mRNA and protein expressions of colon mucosal IDo, respectively. Serum 5-HT,5-HIAA,TRP,IDo,KYN,KYNA concentrations and IDo activity,KAT activity were determined by high performance liquid chromatography assay. Results:Compared with control group,GSRS score was significantly increased(P < 0. 05),initial perception threshold,defecation threshold,pain threshold were significantly decreased(P < 0. 05),anorectal constriction pressure was significantly increased( P < 0. 05),serum 5-HT,5-HIAA concentrations were significantly increased(P < 0. 05),mRNA and protein expressions of IDo were significantly increased (P < 0. 05),serum KYN was significantly increased(P < 0. 05),KYNA was significantly decreased(P < 0. 01),IDo activity was significantly incseased(P < 0. 01),and KAT activity was significantly decreased in IBS-D group(P < 0. 01). Correlation analysis showed that initial perception threshold,defecation threshold,pain threshold and anorectal constriction pressure were correlated with 5-HT,5-HIAA,TRP,KYN,KYNA,IDo activity and KAT activity in patients with IBS-D (P < 0. 05). Conclusions:TRP metabolic pathway is correlated with visceral hypersensitivity in patients with IBS-D.
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Objective To investigate the cytokines levels in peripheral blood of ulcerative colitis (UC) patients at active stage, and to observe the effect of baicalin at various concentrations on the cytokines. Methods Quantitative polymerase chain reaction (Q-PCR) assay was used for the detection of interleukin 4 receptor (IL4R), IL6R, IL23R and RAR-related orphan receptor C (RORC) expression in single peripheral mononuclear cell of UC patients. Enzyme-linked immunosorbent assay (ELISA) was used to examine the serum levels of interferon gamma (IFN-γ), IL-4, IL-5, IL-6, IL-10, and transforming growth factor beta (TGF-β) levels in UC patients, and was applied for in-vitro detection of these indicators after baicalin intervention. Results Q-PCR results showed that IL4R, IL6R, IL23R, RORC gene expression levels in UC patients were increased to various degrees as compared to diarrhea-predominant irritable bowel syndrome (IBS-D) patients and the healthy volunteers, and then the levels were decreased to various degrees after intervention by baicalin at different concentrations, in particular at 20 and 40μmol/L. The results of ELISA showed that serum levels of cytokines of IFN-γ, IL-5, IL-6 in UC patients were increased to various degrees while IL-4, IL-10 and TGF-β1 was decreased as compared to IBS-D patients and the healthy volunteers. Baicalin at the concentrations of 20 and 40 μmol/L had an effect on decreasing IFN-γ, IL-5, IL-6 and on increasing IL-4 and IL-10. Conclusion Higher concentrations of baicalin show obvious effect on inhibiting RORC and IL23R expression, on decreasing IFN-γ, IL-5, IL-6 levels, and on increasing IL-4, IL-10 and TGF-β1 levels, which indicated that the therapeutic mechanism of baicalin in relieving ulcerative colonic inflammation is related with the regulation of immune function.