| Objective Receptor interacting protein140(RIP140) may recruit NF-κ B and activate the release of inflammatory cytokines. The present study is to assess RIP140expression level and its correlation with inflammatory cytokines and free fatty acids.Methods Patients were divided into two groups:control group (30healthy volunteers) and diabetic group (24new-diagnosed diabetes). The two groups were matched in terms of gender and age. Subjects’ height and weight were measured, and the levels of free fatty acids (FFA),TG, TC, HDL-C. LDL-C, fasting blood insulin (FIN) and fasting blood glucose (FBG) from fasting plasma were measured. The insulin resistance index was calculated using the homeostasis model assessment (HOMA)=FIN (μU/ml)×FBG (mmol/L)/22.5. mRNA abundance of RIP140and TNF-a from isolated peri-pheral blood mononuclear cells (PBMCs) were measured by reverse transcription-polymerase chain reaction (RT-PCR), and the concentrations of TNF-a and IL-6in plasma were evaluated by enzyme-linked immunosorbent assay (ELISA). Cultured PBMCs isolated from healthy volunteers were separately treated with either0μmol/L or500u.mol/L palmitate for24h, RIP140, TNF-a and IL-6mRNA levels were analyzed by RT-PCR, RIP140protein level was analyzed by western blotting, and the concentrations of TNF-a and IL-6in the supernatant were measured by ELISA. The correlation between RIP140and other factors were analyzed by SPSS17.0.Results There were no significant difference in age, gende, height, weight, TC, LDL-C,ALT between two groups (all P>0.05). HDL-C of diabetic group was lower than that of control group (P<0.01), FIN, FBG, TG, HOMA and FFA of diabetic group were higher than those of control group (all P<0.01), and expression levels of RIP140, TNF-a and IL-6in diabetic group were significantly higher than those in control group (all P<0.01). R1P140mRNA level in diabetic group was positive correlation with FFA (r=0.667, P<0.001), TNF-a mRNA (r=0.714, P＜0.001) and protein (r=0.872,P<0.001), IL-6protein (r=0.943, P<0.001), HDL-c (r=0.466, P=0.022), FIN(r=0.421,P=0.040) and FBG (r=0.412, P=0.045). RIP140, TNF-a and IL-6mRNA and protein in500μmol/L treated PBMCs were higher than those in untreated cells (IL-6mRNA P<0.05, the others P<0.01).Conclusion R1P140expression level is upregulated in new-diagnosed type2diabetes, and its expression level is closely correlation with TNF-α, IL-6as well as FIN. FBG. HOMA-IR and FFA levels. Objective To elaborate the effects of RIP140expression change on inflammatory cytokines and whether curcumin could modulate its expression level.Methods The optimum treatment time and concentration of curcumin on RAW264.7cells were determined by MTT assay. RAW264.7cells were treated with0,250,500μmol/L palmitate for24h, the morphology of cells were observed under inverted microscope, and RIP140, TNF-a and IL-6mRNA levels were determined by RT-PCR and concentrations of TNF-a and IL-6in supernatant were tested by ELISA. Cells were separately treated with20μmol/L curcumin and the same volume of dimethyl sulfoxide (DMSO). After1h intervention, both groups were treated with500μmol/L palmitate for24h, and the morphology of cells were observed under inverted microscope, and the mRNA abundance of RIP140, TNF-a and IL-6and protein levels of TNF-a, IL-6in supernatant were detected. Data analysis was assessed by one-way ANOWA and t test.Results The optimum treatment time of curcumin on RAW264.7cells was1h, and the optimum concentration was20μmol/L. After intervening with500μmol/L palmitate for24h, the mRNA level of RIP140was higher than that in0μmol/L palmitate group (3.40±0.51vs.1.01±0.21, t=7.436, P<0.01). After intervening with0,250,500μmol/L palmitate for24h, TNF-a mRNA levels and protein levels were significantly increased in a dose-dependent manner [TNF-a mRNA1.00±0.03,1.79±0.12,2.16±0.13, F=99.308,P<0.01; TNF-a protein levels (196.86±25.13),(371.34±9.82),(484.64±16.99)pg/ml, F=187.049, P<0.01; IL-6mRNA levels1.00±0.51,2.55±0.15,2.59±0.17, F=152.958, P<0.01; IL-6protein levels (952.97±65.93),(1080.73±36.05),(1182.42±17.83)pg/ml, F=26.594, P<0.01]; Compared with control group, RIP140mRNA abundance (0.63±0.01vs.1.00±0.05, t=-13.79, P<0.01), TNF-a mRNA abundance (0.64±0.11vs.1.00±0.07, t=-4.532, P<0.05) and protein concentration [(322.33±11.67) vs.(484.64±16.99) pg/ml, t=-13.577,P<0.01], IL-6mRNA abundance (0.57±0.05vs.1.00±0.02, t=-14.167. P<0.01) and protein concentration [(241.15±46.76) vs.1182.42±17.83pg/ml, t=-44.810, P<0.01] of curcumin group were significantly lower. After treated with palmitate, cells became shrank, rounded, or even disintegrated, whereas cell morphology was not impaired and cell junction still existed after pre-treated with curcumin. Conclusions Palmitate upregulates RIP140. TNF-a and IL-6levels. Curcumin represses palmitate-induced inflammatory cytokines by decreasing RIP140expression to alleviate inflammation. Objective Elevated levels of free fatty acids promote macrophages infiltration, which disturb insulin signaling and contribute to insulin resistance and β cells dysfunctions. RIP140could activate NF-κB pathways to trigger TNF-a and IL-6transcription. The aim of this study is to elaborate the role of RIP140in macrophages infiltration to islet p cells under500μmol/L palmitate treatment.Methods Macrophages were divided into:BSA group (without transfection, cells were subjected to equal volume of BSA for24h), PA group (without transfection, cells were cultured with500μmol/L palmitate for24h), pEGFP-N1+PA group (cells were transfected with pEGFP-N1plasmid before treated with500μmol/L palmitate for24h), pEGFP-N1-RIP140+PA group (gells were transfected with pEGFP-N1-RIP140plasmid before treated with500μmol/L palmitate for24h), scramble siRNA+PA group (cells were transfected with scramble siRNA before treated with500μmol/L palmitate for24h), RIP140siRNA+PA group (cells were transfected with RIP140siRNA before treated with500μmol/L palmitate for24h), and supernatant was collected for TNF-a and IL-6measurement, the mRNA levels of TNF-a and IL-6were measured by RT-PCR. Fresh media were added to cells for another24h incubation, then conditional media were collected for stimulating MIN6cells. MIN6cells were incubated with conditional media for24h, then cell viability, early apoptotic cells, insulin secretion of MIN6cells were analyzed, and PGC-1α, UCP2, iNOS1, PDX-1, PEPCK and PCNA mRNA were measured by RT-PCR, and JNK and ERK1/2pathways were evaluated by western blotting. MIN6cells were treated as RAW264.7cells, then supernatant was collected for Chemotaxis assays of RAW264.7cells and primary peritoneal macrophages.Results1) The release of TNF-a and IL-6in pEGFP-N1-RIP140+PA group were increased compared with those in pEGFP-N1+PA group (P<0.01), whereas the release of TNF-a and IL-6in RIP140siRNA+PA group were decreased compared with those in scramble siRNA+PA group (P<0.01).2) Compared with conditional media from pEGFP-N1+PA group, conditional media from pEGFP-N1-RIP140+PA group repressed MIN6cell viability (P<0.01), increased early apoptotic cells (P<0.01). decreased insulin secretion (P<0.01) and PGC-la. PEPCK and PCNA mRNA levels (P<0.05, P<0.05. P<0.01, respectively), and elevated UCP2, iNOS and PDX-1mRNA levels (P<0.01, P<0.01, P<0.05. respectively) and activated JNKand ERK1/2pathways (both P<0.01). Compared with conditional media from scramble siRNA+PA group, conditional media from RIP140siRNA+PA group promoted MIN6cells viability (P<0.01), decreased early apoptotic cells (P<0.01), increased insulin secretion of MIN6cells (P<0.01) and PGC-la, PEPCK and PCNA mRNA levels (P<0.01, P<0.05, P<0.01, respectively), decreased UCP2, iNOS and PDX-1mRNA levels (P<0.01, P<0.01, P<0.05, respectively) and decreased activated JNK and ERK1/2levels (both P<0.01).3) overexpressing RIP140under the condition of500μmol/L palmitate in MIN6cells, chemotaxis of RAW264.7cells and primary peritoneal macrophages to β cells were increased, whereas knowdown of RIP140by siRNA in the presence of500μmol/L palmitate in MIN6cells, chemotaxis of RAW264.7cells and primary peritoneal macrophages to β cells were decreased.Conclusions Under high levels of fatty acids, RIP140of macrophages mediates inflammatory infiltration to β cells and impairs βcells function through modulating the transcription of inflammatory cytokines, which might be achieved by activating JNK and ERK1/2pathways and promoting specific genes transcription. Under high levels of fatty acids, RIP140of islet β cells increases macrophage chemotaxis through TNF-α and IL-6and triggers local low-grade inflammation. |
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