RESUMO
Aim To investigate the possible mechanism of paeonol inhibiting the inflammatory response of fibroblast synovial cells (RA-FLSS) in rheumatoid arthritis. Methods CCK-8 assay was used to detect Paeonol's inhibitory level on the abnormal proliferation of arthritis human fibroblast synovial cells (RA-FLSs). The levels of endoplasmic reticulum stress-related proteins MANF and ATF6 were detected by Western blot. Cell localization of transcription factor p65 and Mesencephalic Astrocyte Derived Neurotrophic Factor (MANF) was detected by immunofluorescence. RT-qPCR detected the changes of p65 target genes. Results Paeonol could significantly inhibit the abnormal proliferation of RA-FLSS cells. Paeonol activates ATF6 and increases the expression of MANF. Paeonol promoted the nuclear transfer of MANF protein and inhibited the transcriptional activity of p65. Conclusion Paeonol promotes the expression of MANF and nuclear transfer through the endoplasmic reticulum stress pathway and affects the progression of RA by inhibiting the transcriptional activity of p65.
RESUMO
Aim To construct colorectal cancer Caco-2 cell line with overexpression of mesencephalic astrocyte-derived neurotrophic factor (MANF) and to investigate the effects of MANF on the proliferation, migration, invasion and apoptosis of human colorectal cancer Caco-2 cells. Methods Caco-2 cells were transfected with MANF-GFP plasmid and GFP empty vector plas-midrespectively. Real time polymerase chain reaction (RT-PCR) and Westernblot (WB) were used to detect the the expression level of MANF. Transwell was used to detect the effect of MANF on the migration and invasion ability of Caco-2 cells. CCK8 was used to dectet the influence of MANF on the proliferation of Caco-2 cells. WB and flow cytometrywere used to detect the activation ofcaspase-3andthe apoptosis rateof Caco-2 cells. Luciferase experiment was used to verify the effect of MANF on NF-κB pathway. Results MANF-GFP plasmid was successfully expressed in Caco-2 cells. The migration and invasion ability of Caco-2 cells were effectively reduced (P <0. 01), and the a-bility of the cell proliferation was downregulated (P < 0.01) in the MANF overexpression group; the expression of cleaved caspase-3 protein and apoptosis of Caco-2 cells were upregulated; compared with the control group, the transfection of MANF plasmid significantly reduced the transcription activity of NF-κB (P < 0. 01). Conclusions MANF can inhibit proliferation, migration, invasion of Caco-2 cells, while promote its apoptosis by negatively regulating NF-κB signal pathway.
RESUMO
Aim To observe the expression of mesen-cephalic astrocyte-derived neurotrophic factor(MANF) in synovial membrane and serum of rats with adjuvant arthritis (AA) and to analyse the relationship between MANF expression and arthritis. Methods AA models were prepared by injecting Freund complete adjuvant (FCA) into SD rats. The swelling of the secondary joint was measured by foot volume measurement. The severity of AA was recorded by arthritis index (AI). Synovial pathological changes were observed by HE staining. The protein and mRNA levels of MANF,BiP and CHOP extracted from synovial tissues in different periods of AA rats were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The levels of MANF, C-reactive protein (CRP), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in serum were detected by enzyme-linked immunosorbent assay (ELISA) and then the relationship between MANF level and inflam-matory factors were explored. Results AA rat model was established successfully. The expression of BiP significantly increased in synovial tissue on d 2 after CFA injection,and decreased until d 28. The expres-sion of MANF slightly increased on d 2,then remained stable,and significantly increased on d 14, and then decreased gradually. The expression of CHOP kept to rise slowly at a low level. The level of MANF in serum markedly increased on d 14,then gradually decreased, but it was still higher than the normal level on d 28. The level of CRP exhibited similar trend with MANF. Correlation analysis showed that MANF had a negative correlation with arthritis symptoms, IL-1β and TNF-α in the secondary inflammatory period of AA rats. Con-clusions Arthritis induces the expression and secre-tion of MANF,and the level of MANF is closely relat-ed to the progression and severity of arthritis.
RESUMO
Aim To investigate the protective effects of MANF on human neuroblastoma SH-SY5 Y cells suf-fering from oxygen-glucose deprivation/reperfusion ( OGD/R) and the underlying mechanism. Methods SH-SY5Y cells were treated with OGD for 6 h, fol-lowed by reperfusion for 12 h. Meanwhile, the cells were incubated with 2 μmol · L-1 recombinant human protein MANF for 12 h during reperfusion. The cell morphology was observed under an optical microscope. The cell viability was determined by MTT assay. PI staining was performed to detect the number of dead cells. Western blot was performed to determine the protein levels of endogenous MANF, glucose-related protein 78 ( GRP78/BiP) , phosphorylated inositol re-quiring enzyme 1 ( p-IRE1 ) , phosphorylated eukaryot-ic translation initiator factor 2α ( p-eIF2α) , cleaved caspase-3, and C/EBP-homologous protein (CHOP). Results The cells exposed to OGD/R became smaller and round, and the neurites of the cells were shortened or disappeared . Recombinant human protein MANF improved the survival rate ( P and cleaved caspase-3 in SH-SY5 Y cells induced by OGD/R were significantly suppressed by MANF. Con-clusion OGD/R up-regulates the ER stress-associated proteins and causes apoptosis. MANF inhibits OGD/R-induced cell death, which may be related to attenua-ting ER stress-induced apoptosis.