Preliminary Study On The Effect Of IL-18 And TNF-α On The 3T3-L1 Adipocytes,RAW264.7 Macrophages In Chronic Inflammation

Objective:To explore the effect of interleukin-18(IL-18) and tumor necrosis factor alpha(TNF-α)on chronic inflammation.To preliminary explore the IL-18 inflammatory pathways in the intensity of the role of adipocytes and macrophages and the possible mechanism.Methods:(1)Different levels of IL-18( 0,0.01ng/ml,0.1ng/ml,1ng/ml,10ng/ml,50ng/ml,100ng/ml)processed the 3T3-L1 preadipocytes 24 h,The effect of IL-18 on 3T3-L1 preadipocytes proliferation was detected by CCK-8 after being processed by different levels of IL-18.(2)The expression of adiponectin, peroxisome proliferator-activated receptor gamma(PPAR-γ) and TOLL-like receptor 4( TLR-4) m RNA was determined by reverse transcription polymerase chain reaction(RT-PCR) in 3T3-L1 adipocytes after being processed by different levels of IL-18 and TNF-α( 0,0.01ng/ml,0.1ng/ml,1ng/ml,10ng/ml,100ng/ml).(3)The expression of IL-18Rβ was determined by RT-PCR and Western blotting in both 3T3-L1 adipocytes and RAW264.7 macrophages after being processed by different levels of IL-18 and TNF-α.(4)The concentrations of IL-6 and TNF-α in 3T3-L1 adipocytes and RAW264.7macrophages culture medium was detected by enzyme-linked immuno sorbent assay(ELISA) after being processed by different levels of IL-18.(5)The concentrations of IL-6 and IL-18 in 3T3-L1 adipocytes and RAW264.7macrophages culture medium was detected by ELISA after being processed by different levels of TNF-α.Results:The results of the first part(1) Compared with the control group,IL-18 inhibited the proliferation of 3T3-L1 preadipocytes.When IL-18 concentrations went to 50ng/ml, 100ng/ml the proliferation of 3T3-L1 preadipocytes was significantly inhibited( 0.57 ± 0.09 vs 0.45± 0.03,0.41 ± 0.04; P <0.01).(2) The adiponectin m RNA was not affected by IL-18 in 3T3-L1 adipocytes,the difference was not statistically significant(P> 0.05).The adiponectin m RNA was significantly inhibited after treatment with 10 ng/ml,100 ng/ml TNF-α in 3T3-L1adipocytes(1 ± 0 vs 0.66 ± 0.07,0.15 ± 0.04,P <0.01).(3) The PPAR-γ m RNA was not affected by IL-18 in 3T3-L1 adipocytes,the difference was not statistically significant(P> 0.05).The PPAR-γ m RNA was significantly inhibited after treatment with 10 ng/ml,100 ng/ml TNF-α in 3T3-L1adipocytes(1 ± 0 vs 0.67 ± 0.07,0.4 ± 0.07, P <0.01).(4) The TLR-4 m RNA expression was not influenced by IL-18 and TNF-α in 3T3-L1adipocytes;the difference was not statistically significant(P> 0.05).The results of the second part(1) The IL-18Rβ m RNA and protein expression was not influenced by IL-18 in3T3-L1 adipocytes, the difference was not statistically significant(P> 0.05).IL-18Rβm RNA and protein were significantly increased after treatment with 10 ng/ml,100ng/ml TNF-α in 3T3-L1 adipocytes(1 ± 0 vs 4.66 ± 1.62,5.91 ± 2.29;P <0.01)(1 ± 0vs 4.40 ± 1.54,5.87 ± 1.98; P <0.01).(2) The IL-18Rβ m RNA and protein were also increased after treatment with 10ng/ml,100 ng/m L IL-18 in RAW264.7 macrophages(1±0 vs 4.06±1.24,5.82±1.71;P<0.01)(1±0 vs 4.32±1.34,5.93±1.84; P<0.01). The IL-18Rβ m RNA and protein expression was not influenced by TNF-α in RAW264.7 macrophages, the difference was not statistically significant(P> 0.05).The results of the third part(1) IL-18 had no effect on IL-6 and TNF-α secreted by 3T3-L1 adipocytes, the difference was not statistically significant(P> 0.05).(2) The level of IL-6 secreted by 3T3-L1 adipocytes increased with the concentrations of TNF-α, and the difference was statistically significant(692.80±69.37pg/ml vs1151.47±176.48pg/ml,1375.20±443.59pg/ml,1690.07±431.80pg/ml,2660.27±95.54pg/ml,3391.20±128.01pg/ml;P<0.01). TNF-α had no effect on IL-18 secreted by 3T3-L1 adipocytes, and there was no significant difference(P>0.05).(3) IL-18 had no effect on RAW264.7 macrophages secretion of IL-6, the difference was not statistically significant(P>0.05); when IL-18 concentrations rised to100ng/ml, the secretion of TNF-α level was significantly increased,and the difference was statistically significant(141.93±23.85pg/ml vs 1863.86±746.37pg/ml; P<0.01).(4) TNF-α had no effect on IL-6 and IL-18 secreted by RAW264.7 macrophages, the difference was not statistically significant(P>0.05).Conclusions:1.IL-18 inhibit the proliferation of 3T3-L1 preadipocytes.2. The IL-18Rβ m RNA and protein are increased after treatment with IL-18 in macrophages.3. The secretion of TNF-α level is increased in macrophages after treatment with IL-18.4. The expression of adiponectin and PPAR-γ is inhibited after treatment with TNF-αin adipocytes.5. IL-18Rβ m RNA and protein are increased after treatment with TNF-α in adipocytes.6. The level of IL-6 secreted by adipocytes increases with the concentrations of TNF-α.