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Objective CARD9 can activate several pathways involved in immunity, such as NF-ΚB, MAPK, etc. However the mechanism of this process has not yet been elucidated. For conducting relevant experiments in vitro, a prokaryotic expression vector of CARD9-MBP fusion protein has to been construct, and the fusion protein need to be expressed and purified. Methods The coding sequence of CARD9 and MBP genes were amplified by PCR and the recombinant plasmid was correctly inserted into the pET-30a(+) vector. The recombinant plasmid was transformed into E.coli DH5α competent cells and proceeded PCR identification, restriction analysis and gene sequencing. The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The target protein was induced to express by IPTG under different conditions. Relative molecular weight of the target protein was detected by SDS-PAGE electrophoresis. The CARD9-MBP fusion protein was purified by MBP maltose chromatography column and gel filtration chromatography column, and identificated by MALDI-TOF mass spectrometry after MBP-tag to be removed by HRV3C enzyme. Results The CARD9-MBP fusion protein was successfully constructed and confirmed by PCR and restriction analysis. The result of gene sequencing was consistent with the target sequence. The SDS-PAGE electrophoresis showed that the target protein with molecular mass (MR) about 105 000 was successfully induced to express in E.coli BL21 (DE3). A quite pure CARD9-MBP fusion protein was obtained by purification of MBP maltose chromatography column. Identification by MALDI-TOF mass spectrometry demonstrated that the target protein after MBP-tag to be removed by HRV3C enzyme is CARD9 protein. In the later stage, gel filtration chromatography column was used to obtain further pure CARD9-MBP fusion protein. Conclusion The prokaryotic expression vector of CARD9-MBP fusion protein was successfully constructed and a large number of soluble protein expressed. The purified target protein can be obtained by purification with MBP maltose chromatography column and gel filtration chromatography column.
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Objective To investigate the effects of caspase recruitment domain-containing protein 9 (CARD9)expression in peritoneal macrophages on severe acute pancreatitis(SAP)in rats and its mechanism.Methods A total of 60 male Sprague Dawley rats were divided into control group(n=6), SAP group(n=18),small interfering RNA(siRNA)control group(n=18)and siRNA CARD9 group (n=18).SAP rat models were established.At three,six and twelve hours after the models were established,ascites was collected,peritoneum was lavaged and peritoneal macrophages were isolated and cultured.The expressions of CARD9,nuclear factor-kappaB(NF-κB),p38 mitogen-activated protein kinase(p38MA PK)at mRNA level in peritoneal macrophages was measured by real-time polymerase chain reaction(RT-PCR).The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6 in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA).LSD or Tamhane′s T2 methods were performed for statistical analysis.Results At three,six and twelve hours after the models were established,CA RD9 mRNA levels of peritoneal macrophages in SAP group were 1.63 ± 0.05,1.68 ± 0.24 and 2.61 ± 0.02,respectively,which were all higher than that of control group(1.01 ± 0.23),and the differences were statistically significant(t=25.97,6.86 and 131.59;all P<0.05);the levels of CA RD9 mRNA of siRNA CARD9 group were 1.45 ± 0.02,1.24 ± 0.03 and 1.63 ± 0.03,respectively,which were lower than that of SAP group at the same time points,and the differences were statistically significant(t=-7.81,-4.46 and -62.13;all P< 0.05).At three,six and twelve hours after the models were established,the mRNA levels of NF-κB and p38MA PK of peritoneal macrophages of rats in SAP group were 1.51 ± 0.08,1.81 ± 0.10,2.30 ± 0.05 and 1.37 ± 0.13,1.69 ± 0.18,2.42 ± 0.23,respectively, which were higher than those of control group(1.00 ± 0.01,1.03 ± 0.08),and the differences were statistically significant(tNF-κB=15.10,19.95 and 60.36;tp38MAPK=5.37,8.34 and 14.11;all P<0.05);the levels of N F-κB mRNA in siRNA CARD9 group were 1.38 ± 0.05,1.57 ± 0.06 and 1.76 ± 0.09, respectively,which were lower than that of SAP group at the same time points,and the differences were statistically significant(t= -3.32,-5.07 and -12.70;all P<0.05).At six and twelve hours after the models were established,the p38MA PK mRNA levels of siRNA CARD9 group were 1.50 ± 0.10 and 2.00 ± 0.09,respectively,which were lower than that of SAP group,and the differences were statistically significantly(t= -2.30 and -4.17,both P< 0.05).At three,six and twelve hours after the models were established,the levels of TNF-α,IL-1β and IL-6 in peripheral blood of SAP group were(53.49 ± 21.64)pg/mL,(108.62 ± 22.76)pg/mL and(139.00 ± 15.35)pg/mL;(43.86 ± 18.30)pg/mL, (87.51 ± 17.10)pg/mL and(117.27 ± 14.57)pg/mL;(78.38 ± 32.70)pg/mL,(156.39 ± 30.56)pg/mL and(209.56 ± 26.09)pg/mL,respectively,which were higher than those of control group((2.79 ± 1.17),(7.13 ± 4.52),(12.73 ± 8.08)pg/mL),and the differences were statistically significant(tTNF-α=5.73,11.37 and 21.69;tIL-1β=4.77,11.13 and 17.68;tIL-6=4.77,11.32 and 17.68;all P<0.05).At six and twelve hours after the models were established,the levels of TNF-α,IL-1β and IL-6 of siRNA CARD9 group were(75.73 ± 16.93)pg/mL,(108.23 ± 14.02)pg/mL;(63.05 ± 11.98)pg/mL, (91.56 ± 14.28)pg/mL and(112.67 ± 21.40)pg/mL,(163.62 ± 25.51)pg/mL,respectively,which were lower than those of SAP group,and the differences were statistically significant(tTNF-α= -2.84,-3.63;tIL-1β= -2.88,-3.09;tIL-6= -2.88,-3.09;all P< 0.05).Conclusions There are CARD9-related NF-κB and p38MAPK pathways in peritoneal macrophages of SAP rats.Intervention of the expression of CARD9 in peritoneal macrophages,especially at early stage of SAP may relieve the inflammation reaction and pancreatic injury,which may provide a new method for SAP treatment.
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Caspase recruitment domain-containing protein 9 (Card9) is located upstream of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) inflammatory pathways. This study investigated the therapeutic effect and potential mechanism of pioglitazone in rats with severe acute pancreatitis (SAP). SAP was induced by a retrograde infusion of 5.0% sodium taurocholate into the biliopancreatic duct of Sprague Dawley rats (n=54), which were then treated with pioglitazone. Blood and pancreatic tissues were harvested at 3, 6, and 12 h after SAP induction. Pancreatic pathological damage was evaluated by hematoxylin and eosin staining. Serum amylase, serum pro-inflammatory cytokines, and pancreatic myeloperoxidase (MPO) activities were determined by enzyme-linked immunosorbent assay. The expression of Card9 mRNA and protein in pancreatic tissues was detected by real-time polymerase chain reaction and western blotting. Pioglitazone had a therapeutic effect in treating rats with SAP by decreasing the level of amylase activity, ameliorating pancreatic histological damage, decreasing serum pro-inflammatory cytokine levels and tissue MPO activity, and downregulating the expression of NF-κB, p38MAPK, and Card9 mRNAs and proteins (P<0.05). The present study demonstrated that the inhibition of Card9 expression could reduce the severity of SAP. Card9 has a role in the pathogenic mechanism of SAP.