| Weaning piglets are easily infected by bacteria and pathogens,resulting in the immune stress to the pig breeding. Piglets which in immune stress caused the excessive inflammatory reaction that seriously affect the pig production. Lipopolysaccharides(LPS) is one of representative immune inducing agent,often used to construct the animal immune stress model. Our project team previous work have succeeded in using LPS to establish immunological stress inflammation model in piglets. The study main research different concentration of baicalin to alleviate piglet peripheral blood mononuclear cell release immune stress inflammatory cytokines,in order to explore the role of NF-κB and NLRP3 signaling pathway on baicalin alleviate piglet peripheral blood mononuclear cells immune stress inflammation.1. The effect of baicalin on LPS induced by piglets peripheral blood mononuclear cells of NF-κB signaling pathwayThe model of piglets peripheral blood mononuclear cell immune stress disease was established in vitro. Method : Take the piglets precaval vein anticoagulated blood, in accordance with the cell separation method, isolated peripheral blood mononuclear cells of piglets, the piglets PBMCs were divided into six groups respectively:control group,LPS-induced group,baicalin treatment group(12.5,25,50 and 100μg/m L),incubation with baicalin for 1 h,later LPS(the final concentration was 1 μg/m L) were added and continued to incubate for 3,16, 20 h. The ratio of NF-κB p65 nuclear protein and cytoplasmic protein was determined by ELISA after 3h LPS-induced; The content of TNF-α(3h) and IL-6(16, 20h) in the cell culture supernatant also determined by ELISA; The m RNA expression of TNF-α and IL-6 in the 3h after LPS-stimulated monocytes was determined by RT-PCR.Results:① 3h after LPS-induced,the ratio of nuclear protein and cytoplasmic protein NF-κB p65 was significantly increased(P<0.01) in piglets PBMCs,baicalin in 12.5μg/m L and 100μg/m L were significantly(P<0.05 or P<0.01) inhibition NF-κB p65 protein translocates to the nucleus by LPS-induced; Other concentration of baicalin inhibited NF-κB p65 protein translocates to the nucleus in a certain extent, but did not reach significant level.② 3h after LPS-induced,the content of TNF-α in piglets PBMCs cell culture medium was significantly increased(P<0.01). Baicalin in 12.5μg/m L were significantly(P<0.05) inhibition the release of TNF-α,baicalin in 25,50 and 100μg/m L were significantly(P<0.01) reducing on the secretion of TNF-α.③ 16,20 h after LPS stimulation,the content of IL-6 in piglets PBMCs cell culture medium was significantly increased(P<0.01). Baicalin in 12.5μg/m L could significantly(P<0.05) inhibition the release of IL-6 in 16 h and 20h;and the same time baicalin in 25,50 and 100μg/m L were significantly(P<0.01) down regulation the content of IL-6 in piglets PBMCs cell culture medium with 16 h and 20 h after LPS-stimulated.④ 3h after LPS stimulation, the m RNA expression of TNF-α and IL-6 in piglets PBMCs was significantly up-regulation(P<0.01). Baicalin in 25, 50 and 100μg/m L were significantly(P<0.01) inhibition the up-regulation expression of TNF-α m RNA by LPS-induced, baicalin in 12.5μg/m L also significantly(P<0.05) down regulation the m RNA expression of TNF-α. Baicalin in 50μg/m L and 100μg/m L were significantly(P<0.01) inhibition the m RNA expression of IL-6, baicalin in 12.5μg/m L and 25μg/m L were significantly(P<0.05) inhibition the expression of IL-6 m RNA.2. The effect of baicalin on LPS induced by piglets peripheral blood mononuclear cells of NLRP3 inflammasome signaling pathwayThe model of piglets peripheral blood mononuclear cell immune stress disease was established in vitro. Method: Peripheral blood mononuclear cells which was isolation and culture are respectively arranged in control group, LPS model group,baicalin treatment group(12.5,25,50 and 100μg/m L),the medicine group incubation with baicalin for 1 h, then LPS(the final concentration was 1 μg/m L) were added and continued to incubate for 3,16 and 20 h.The content of ROS was intracellular by fluorescence microscope after 3h LPS-stimulated; The protein expression levels of IL-1β and IL-18(16h and 20h) in the cell culture supernatant was determined by ELISA; The m RNA expression of NLRP3, ASC,Caspase-1, IL-1β and IL-18 in the 3h LPS-stimulated piglets peripheral blood mononuclear cell were determined by RT-PCR. The content of cell late apoptosis was intracellular by fluorescence microscope after 3h and 6h LPS-stimulated.The protein expression of Cleaved Caspase-1 p20 in the 10 h LPS-stimulated piglets peripheral blood mononuclear cell were determined by Western blot method.Results:① 3h after LPS-stimulated, the content of ROS in piglets PBMCs was significantly increased(P<0.01). Baicalin in all concentration group(12.5,25, 50 and 100μg/m L) were significantly(P<0.01) inhibition cells express of ROS.② 16, 20 h after LPS-induced, the protein expression levels of IL-1β and IL-18 which from peripheral blood of piglets supernatant were significantly increased(P<0.01). Baicalin in 50μg/m L was significantly(P<0.05) inhibition protein secretion of IL-1β and IL-18 in supernatant with 16 h and 20 h after LPS-stimulated,baicalin in 100μg/m L was significantly(P<0.01) cut down protein secretion of IL-1β and IL-18 in supernatant(16h and 20 h after LPS-induced).③ 3h after LPS-induced, the m RNA expression of NLRP3, IL-1β and IL-18 in piglets PBMCs were significantly increased(P<0.01), but the m RNA expression of ASC and Caspase-1 were not significantly difference.Baicalin in 25,50 and 100μg/m L were significantly(P<0.01) reduce the m RNA expression of NLRP3, baicalin in 12.5μg/m L were significantly(P<0.01) cut down the expression in NLRP3 gene.Baicalin in 50,100μg/m L were significantly(P<0.05) inhibition the m RNA expression of IL-1β, other concentration of baicalin have some extent to cut down the expression in IL-1β gene, but did not reach significant level. Baicalin in 50,100μg/m L were significantly(P<0.01) inhibition the m RNA expression of IL-18,baicalin in 25μg/m L were significantly(P<0.05) cut down the m RNA expression of IL-18 gene.④ 3,6h after LPS-induced,the fluorescence intensity values of cell late apoptosis were significantly(P<0.01) increased in piglets PBMCs.Baicalin in all group(12.5,25,50 and 100μg/m L) were able to significantly(P<0.01) reduce the number and density of cells in late apoptosis, and relief the level of apoptosis.⑤ 10 h after LPS-induced,the protein expression of Cleaved Caspase-1 p20 in piglets PBMCs were significantly increased(P<0.01), baicalin in 25, 50 and 100μg/m L were significantly(P<0.01) inhibition protein expression of Cleaved Caspase-1 p20.The results of this experiment showed that,baicalin could relief inflammatory reaction by LPS-induced in piglet peripheral blood mononuclear cell, the mechanism of action was closely related with NF-κB and NLRP3 signaling pathway. |
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