RESUMO
Objective·To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods?·?Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results?·?The titer of adenovirus was 2.2×1011PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion?·?Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.
RESUMO
Objective • To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods • Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results • The titer of adenovirus was 2.2×1011 PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion • Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.
RESUMO
Objective To observe the effects of Jiuxieling Granules on the expressions of MyD88 and IRAK1 in ulcerative colitis model rats with spleen-kidney yang deficiency; To discuss its mechanism of action. Methods Animal models were established by compound methods. 90 Wistar rats were randomly divided into blank group, model group, positive medicine group, and Jiuxieling Granules high-, medium-, and low-dose groups. Each administration group was given relevant medicine for gavage. RT-qPCR, SP immunohistochemistry and Western blot were used to detect mRNA and proteins of MyD88 and IRAK1 in colon tissues. Results Compared with the blank group, mRNA and proteins of MyD88 and IRAK1 in the model group increased (P<0.01). Compared with the model group, mRNA and proteins of MyD88 and IRAK1 in each administration group decreased (P<0.01), especially in Jiuxieling Granules high-dose group. Conclusion Jiuxieling Granules can reduce the expressions of MyD88 and IRAK1, and then influence the transmission of MyD88 signaling pathway and block the release of downstream inflammatory factors to achieve the result of treating ulcerative colitis with spleen-kidney yang deficiency.