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BACKGROUND:Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells.It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line(SH-SY5Y)was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells. OBJECTIVE:To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemic-hypoxic SH-SY5Y and their associated epigenetic mechanisms. METHODS:Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury,and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture,respectively,with mesenchymal stem cells,as well as lentiviral-transfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture.Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification,and joint analysis was conducted with RNA-sequencing.Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells.qRT-PCR and western blot assay were performed to detect the expression level of REST.The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression difference of H3K36me3 group proteins was detected by western blot assay,and transcriptome sequencing was performed to analyze the differentially expressed genes. RESULTS AND CONCLUSION:(1)Compared with the control group,monocyte chemotactic protein 1,interleukin-6,interleukin-18,and interleukin-1 beta,interferon α2,and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy(P<0.01).(2)After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia,the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced.(3)Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1,interleukin-6 related regulatory proteins CXCL1 and BGN.(4)Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated,and functional enrichment of histone modifications,and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group.(5)Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3.(6)Stable knockdown REST in SH-SY5Y cells was successfully constructed;the transcript levels of REST mRNA and protein expression were both decreased.(7)After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells,apoptosis was significantly increased and H3K36me3 expression was significantly decreased.Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level(P<0.01).(8)It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3,which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2,and facilitated neuronal survival.
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Objective:To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the maturation and differentiation of dendritic cells (DCs) and the mechanism involved in the regulation of inflammation in patients with severe acute pancreatitis (SAP).Methods:The full-term fetal umbilical cords(about 4-5 cm) were collected from Zhengzhou Central Hospital Affiliated to Zhengzhou University after cesarean section. hUC-MSCs were isolated and cultured in primary culture. Flow cytometry was used for phenotype identification, adipogenic and osteogenic staining. 20 ml peripheral blood samples from 5 SAP patients were collected, and monocytes were isolated using lymphocyte separation solution and then induced by adding granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4 and tumor necrosis factor (TNF)-α and cultured as DCs. According to different culture methods, DCs were divided into DCs group, hUC-MSCs+ DCs group and hUC-MSCs+ DCs+ NS398 group (NS398 was a specific inhibitor of COX-2, a downstream regulatory gene of NF-κB). The phenotype of DCs was detected by flow cytometry, and the levels of IL-1β, IL-lα, IL-2, IL-6 and IL-10 in the supernatant of cell culture for 24 hours were determined. The expression of toll like receptor (TLR)-4, IKKα and NF-κB-p65 were detected by Western blot.Results:The hUC-MSCs were successfully cultured, and their surface markers CD 90, CD 105 and CD 73 were positively expressed, and they could differentiate into adipocytes and bone cells. With the prolongation of culture time, DCs differentiated from immature to mature cells. Compared with the DCs group, the proportion of regulatory DCs (regDCs) was increased in the hUC-MSCs+ DCs group, and the marker CD 11b was significantly up regulated [(14.26±1.25)% vs (4. 87±0.58)%], CD 1a and CD 11c were significantly down regulated [(2.81±0.34)% vs (13.62±1.52)%, (3.88±0.5)% vs (11. 8±1.22)%]. All the difference were statistically significant ( P<0.05). The expression of IL-1β, INF-γ and IL-6 in culture supernatant were down regulated, but the difference was not statistically significant; The pro-inflammatory factor IL-1α was significantly decreased [(14.91±2.58)ng/L vs (30.19±7.75)ng/L], and the anti-inflammatory factor IL-10 was significantly increased [ (17.03±4.69)ng/L vs (1.83±0.14)ng/L]. The expression levels of NF-κB-p65 and TLR4 were significantly down regulated (0.74±0.02 vs 0.97±0.01, 0.89±0.01 vs 1.72±0.01), and the expression of IKKα protein was significantly up regulated (1.12±0.01 vs 0.21±0.01) in hUC-MSCs-DCs group. All the differences were statistically significant (all P value<0.05). Compared with DCs group and hUC-MSCs+ DCs group, the expression levels of NF-κB-p65 and TLR4 were significantly down regulated (0.34±0.01 vs 0.97±0.01, 0.74±0.02 and 0.14±0.01 vs 1.72±0.01, 0.89±0.01), while the expression of IKKα protein was significantly up regulated (1.68±0.01 vs 0.21±0.01, 1.12±0.01) in hUC-MSCs+ DCS+ NS398 group. All the differences were statistically significant (all P value<0.05). Conclusions:In SAP patients, hUC-MSCs can inhibit the maturation and differentiation of DCs, and induce CD 11bhigh CD 1alow CD 11clowrregDCs to participate in immune regulation, which may play an anti-inflammatory role by inhibiting the inflammatory cascade through TLR4/IKKα/NF-κB/COX-2 pathway.
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Objective:Understanding arthritis condition of rural residents of five countries in Liaoning province,analyzing their epidemiological factors,to further improve the prevention and treatment of arthritis and provide the basis for the promotion of Chinese medicine appropriate technologies.Methods:The cluster sampling method was used,at each of five counties in Liaoning we had surveyed 40 villages,and each village,we had spot-checked 50 families,the total amount was 2000.Each family member was a survey subject.Results:Among the five counties of Liaoning province,the incidence of arthritis in rural population was that women was higher than men,and 50-59 year-old age group had a higher proportion in arthritis,followed by 40-49 age group. Conclusion:Age and sex were also important factors for arthritic incidence of the five counties in Liaoning province.