| Objective:1. To induce the expression of recombinant CC16 protein and purify the recombinant CC16 protein.2. To observe the effect of recombinant proteins r CC16 on the expression of inflammatory mediators in macrophages induced by lipopolysaccharide(LPS)3. To discuss the effect of recombinant r CC16 protein on lung inflammatory response in COPD mice model and the expression of IL-6, TNF-a, IL-8, IL-1β in bronchoalveolar lavage fluid.4. To discuss effect of recombinant r CC16 protein on the expression of CC16, NF-κB,ICAM-1,TIMP-1and MMP-9 in lung tissue of COPD mice Methods:1. The recombinant plasmid of p ET-30a(+)-r CC16 was successfully transferred into the competent cell BL21, which was induced by IPTG, purified by Ni+-NTA resin affinity, analysis the recombinant r CC16 proteins by western blotting. The recombinant CC16 protein was obtained by removal of endotoxin.2. The biological activity of r CC16 was examined by detecting the activity of phospholipase A2.RAW264.7 cells were cultured with the different concentrations of recombinant r CC16 at 0, 0.5, 1,2.5 μg/m L for 1,2,3,4,5 days.To detect the proliferation of cells by Cell Counting Kit-8 assay and defined the toxicological effects of recombinant r CC16 on RAW264.7 cells.3. RAW264.7 cells were intervented through different concentrations of recombinant r CC16(0,0.5,1,2.5 μg/m L) for 2 hours,then RAW264.7 cells were stimulated by LPS(0.1 VII μg/m L) for 24 hours. The levels of IL-1β, IL-6, IL-8 and TNF-α protein in supernatants were tested using an ELISA assay.4. Copy the COPD model mice by cigarette smoke exposure and intratracheal instillation of lipopolysaccharide(LPS). The experimental mices were intervented by giving different concentrations of recombinant r CC16(low dose and high dose) The pathological changes of lung tissue were observed by H&E staining after 1 month. Collecting bronchoalveolar lavage fluid and serum, the levels of IL-6、IL-8、IL-1β and TNF-α were measured by ELISA assay to further clarify the role of recombinant r CC16 treatment on inflammation of COPD.5. The expression of CC16, NF-κB, MMP-9, TIMP-1, ICAM-1 which were expressed in COPD mouse lung tissues and r CC16-treated COPD mouse tissues were detected by immunohistochemical method. Results:1. SDS-PAGE and Western blotting showed that the recombinant protein r CC16 was successfully obtained.2.(1) r CC16 can inhibit the activity of PLA2 at the concentration of 0.5 μg/m L and the inhibiting effects was increased with the increased dosage of r CC16..It showed that the recombinant protein r CC16 had bioactivity.(2) r CC16 had no effect on proliferation and viability of RAW264.7 cells at the concentration less than 5 μg/m L, suggesting that r CC16 had no cytotoxicity on RAW264.7 cells at suitable dosage.3. RAW264.7 cells were intervened with r CC16 at the concentration 0, 0.5, 1, and 2.5 μg/m L in serum free media for 2 hours, then stimulated with LPS(0.1 μg/m L) in serum free media for 24 hours, supernatants were collected.ELISA were used to determine the levels of inflammatory factors(1) RAW264.7 cells were stimulated by LPS for 24 hours. The cell supernatant was collected and the levels of TNF-α were measured: compared with the control group, the content of TNF-α was significantly increased in LPS group(control group: 85.76+13.41 ng /L,LPS group:443.77+20.7 ng/L)(P<0.01). Pretreatment with different concentrations of recombinant r CC16 significantly decreased the levels of TNF-α compared with the LPS group( 1.0 μg/m L r CC16 group : 372.73±25.38 ng/L, 2.0 μg/m L r CC16 group :357.02±24.84 ng/L),(P<0.01).(2) RAW264.7 cells were stimulated by LPS for 24 hours. The cell supernatant was collected, the levels of IL-8 were determined. Compared with the control group, the content of IL-8 was significantly increased in LPS group(control group: 23.55±8.55 pg/m L, LPS group: 82.66±9.76 pg/m L)(P <0.01). The levels of IL-8 in 2 μg/m L r CC16 group were statistically decreased compared with the LPS group(60.30±8.09 pg/m L)(P <0.01). While the levels of IL-8 in other groups were not statistically decreased(0.5 μg/m L r CC16 group : 76.34±8.34 pg/m L, 1.0 μg/m L r CC16 group : 69.03±10.45 pg/m L)(P >0.05).(3) RAW264.7 cells were stimulated by LPS for 24 hours. The cell supernatant was collected, the levels of IL-6 were determined. Compared with the control group, the content of IL-6 was significantly increased in LPS group(control group: 21.02±6.00 pg/m L, LPS group: 128.96±5.94 pg/m L)(P <0.01). The levels of IL-6 were decreased statistically in r CC16-treated groups compared with the LPS group,(0.5 μg/m L r CC16 group: 111.27±6.37 pg/m L, 1.0 μg/m L r CC16 group: 95.22±10.63 pg/m L, 2.0 μg/m L group: 57.38±14.26 pg/m L)(P <0.01).(4) RAW264.7 cells were stimulated by LPS for 24 hours. The cell supernatant was collected, the levels of IL-1β were determined. The data showed that r CC16 had no effect on expression of IL-1β(control group: 17.46±1.09 ng/L, LPS group: 21.08±4.16 ng/L, 0.5 μg/m L group: 16.44±1.73 ng/L, 1.0 μg/m L r CC16 group: 16.33±1.41 ng/L, 2.0 r CC16 μg/m L group: 15.36±1.79 ng/L).4. Copy the COPD model mice by cigarette smoke exposure and intratracheal instillation of lipopolysaccharide(LPS).The recombinant r CC16(low dose and high dose) was given to the COPD mice via intranasal route and PBS group served as controls:(1) H&E staining showed that the complete structure of mouse alveolar was destroyed, then gathered together into larger alveolar, airway mucosal folds increased, cilia exfoliated and strictured. While the above symptoms were improved after treatment by applying the recombinant r CC16, especially, the intervention effect was more obvious in high dosage group than that in low dosage group.(2) The expressions of IL-6、IL-8、IL-1β and TNF-α in serum and bronchoalveolar lavage fluid(BALF) which were collected from COPD mice group and r CC16-treated groups were measured by ELISA assay. Compared with the control group, the contents of TNF-α in COPD group were significantly higher in serum and lavage(serum of control group: 61.12±13.37 ng/L, BALF of control group: 67.57±18.03 ng/L, serum of COPD group: 116.04±12.10 ng/L, BALF of COPD group: 138.69±19.74 ng/L)(P <0.05). After administration of different concentrations of recombinant r CC16 intervention, the contents of TNF-α in r CC16-treatment groups were significantly reduced with a dose-dependent manner(BALF of low dose group.: 109.05±10.94 ng/L, BALF of high dose group: 97.55±17.76 ng/L)(P <0.01) and the content of TNF-α was significantly reduced by r CC16 in the high dose group in serum(serum of high dose group:66.37±18.50 ng/L)(P <0.01);and the normal control group comparison of COPD, The contents of IL-6 from normal mice were significantly increased in serum and lavage(serum of control group: 69.12±5.21 pg/m L, BALF of control group: 20.70±5.23 pg/m L, serum of COPD group: 179.90±9.46 pg /m L, BALF of COPD group: 69.04±9.64 pg/m L)(P <0.01). After administration of different concentrations of recombinant r CC16, the contents of IL-6 in treatment groups were significantly reduced with a dose-dependent manner(P <0.05).(BALF of low dose group: 51.28±11.94 pg/m L, BALF of high dose group: 43.04±8.63 pg/m L, serum of low dose group: 146.80±8.58 pg/m L, serum of high dose group: 106.64±5.26 pg/m L). The contents of IL-8 in COPD group were significantly higher in serum and lavage than that in normal mice group(serum of control group: 96.35±9.70 pg/m L, BALF of control group: 63.54±7.69 pg/m L, serum of COPD: 157.60±19.31pg/m L, BALF of COPD: 133.25±7.29 pg/m L)(P <0.01). After administration of different concentrations of recombinant r CC16, the contents of IL-8 in treatment groups were significantly reduced with a dose-dependent manner( BALF of high dose group:89.24±9.25 pg/m L, BALF of high dose group: 62.07±6.75 pg/m L)(P <0.01); compared with the COPD group, the serum IL-8 from high dose group r CC16-treated group was reduced significantly(113.73±5.75 pg/m L). The levels of IL-1β were significantly increased in serum from COPD group than those in normal group(control group: 91.02±9.82 pg/m L, COPD group: 118.49±15.18 pg/m L)(P <0.01), but there was no statistically significant between COPD group and the r CC16-treatment group in both serum and BALF( serum of low dose group:116.96±8.42 ng/L,serum of high dose group:109.90±6.79 ng/L,BALF of low dose group 1.0 μg/g:127.99±18.47 ng/L,BALF of high dose group:118.15±10.96 ng/L)(P> 0.05).5. The immunohistochemical results:the expression level of CC16, MMP-9, TIMP-1, ICAM-1 in the airway epithelium of mice lung tissue was expressed by the gray value. The greater the gray value, the lower the level of expressions. The expression of NF-κB p65 in the lung tissue of mice was expressed by the percentage of the positive cells and the percentage of the total cells. the greater the percentage, the stronger the expression.The content of CC16 in the lung tissue of COPD group was significantly lower than that in the normal group(the average gray value of normal group :178.61±1.58, the average gray value of COPD group: 196.05±2.30)(P <0.05). The expression of MMP-9, TIMP-1 and ICAM-1 in the lung tissue of COPD group was higher than that in the normal group. Differences were statistically significant(MMP-9 in the normal group the average gray value: 201.08 ±3.03, MMP-9 in COPD group average gray values: 148.17±2.12; average grey value of the normal group of TIMP-1: 206.09± 1.93, TIMP-1 in COPD group average gray values: 176.27±1.79; average grey value of the normal group of ICAM-1:193.67±2.88, average gray values of the COPD group of ICAM-1: 176.35±1.94)(P <0.05). With the intervention of the r CC16, the lung tissue of CC16 content was increased in high dose intervention group,(high dose group the average gray value:181.59±3.12, P<0.01), The expression of MMP-9 was decreased with the increase concentration of r CC16 in a dose dependent manner(low dose group: 164.93±1.92, high dose group: 178.77±2.38, P <0.05); The expression of ICAM-1and TIMP-1 was decreased with the intervention of COPD compared with the COPD group, but there was no difference between the high dose group and the low dose group(TIMP-1 of low dose group: 197.43±2.04, TIMP-1 of high dose group: 195.03±2.21; ICAM-1 of low dose group:190.82±3.87, ICAM-1 of high dose group:186.69±3.12, P >0.05); Compared with the normal phase, the p65 nuclear transfer, nuclear transfer was significantly increased in the lung tissue of group COPD mice(Normal group: 20.32±1.98, COPD group: 48.02±4.70, P <0.05) With r CC16 intervention, the content of NF-κB p65 in the nucleus of lung tissue in intervention group was decreased, and there is a certain dose dependent relationship, the difference was statistically significan(low dose group: 38.92±3.27; high dose group: 31.79±2.02, P <0.05). Conclusion:1. The purified recombinant CC16 protein was of biological activity, and it has no cytotoxicity on RAW264.7 cells when the concentration was less than 5 ug/m L.2. r CC16 can inhibit the secretion of TNF-α, IL-6 and IL-8 induced by LPS in RAW264.7 cells, but it has no effect on the secretion of IL-1β.3.(1) We have prepared the COPD model mice via cigarette smoke exposure and intratracheal instillation of lipopolysaccharide(LPS). r CC16 treatment could improve the pathological changes of lung tissue in COPD mice.(2) r CC16 can inhibit the expression of TNF-α, IL-6 and IL-8 in both lavage fluid and serum which were from COPD mice. The results showed that r CC16 has an antiinflammatory effect.(3) r CC16 could inhibit the expression of TIMP-1, MMP-9, ICAM-1 in lung tissue of COPD mice and these effects may be involved in NF-κB pathway. |
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