Study On The Mechanism Of Action Of In The Shenling Baizhu Powder Treatment Of UC By Regulating

Purpose: In this study,LPS induced IEC-6 cells were used to establish cell injury model and DSS induced ulcerative colitis model,and the therapeutic effect of Shenling Baizhu Powder on IEC-6 cells inflammatory injury and ulcerative colitis was observed and the relationship between the regulation of autophagy-NLRP3 inflammatory corpuscle pathway was studied.Method: In vivo experiment: After 3 days of adaptive feeding,70 male BALB/c mice were randomly divided into 7 groups: Normal group(N),model group(3%DSS,DSS group),Shenling Baizhu powder low-dose,medium-dose and high-dose groups(3,6,12 g/kg/d,DSS+SLBZSL,M,H groups),NLRP3 inhibitor MCC950 group(0.1g/kg/d,DSS+MCC950 group)and Mesalazine group(0.4 g/kg/d,DSS+5-ASA group)had 10 birds in each group.During the experiment,mice in the normal group were given free water and feed,while mice in the other groups were given free water containing 3%DSS to induce the establishment of acute UC model.The feed dosage was the same as that in the normal group,and 3%DSS was prepared with double steam.At the same time of modeling,intragastric administration was started for 1week: 1.Weight detection of mice: Weight detection and record were performed on mice in each group every day.2.DAI scoring index record: During the experiment,the diet,water and stool of each group of mice were recorded,and statistically scored according to the changes in body weight.After the experiment,serum and colon were collected.3.HE staining was used to detect the pathological changes of colon;4.ELISA was used to detect the levels of MPO,IL-18 and serum IL-1β,TNF-α and IL-10.5.NLRP3,ROS and IL-18 protein expression in colon were detected by immunofluorescence method.6.The expressions of IL-18,NF-κB p65,LC3Ⅱ/LC3Ⅰand Parkin were detected by immunohistochemical method.7.Protein western blots were used to detect the expressions of NLRP3 inflammasome signaling pathway protein,TLR4,My D88,NF-κB p65,I-κB,p-I-κB,LC3Ⅱ/LC3Ⅰ,p62,Beclin1 and Parkin proteins in colon.In vitro experiments: Rat cryptic intestinal epithelial cells(IEC-6)were induced by lipopolysaccharide(LPS)to establish a cell inflammatory injury model,and two experimental grouping schemes were established:1.To explore whether the medicated serum of Shenling Baizhu Powder can improve the injury of IEC-6 cells by regulating autophagy and apoptosis:IEC 6 cells were divided into normal group(ZC),model group(10μg/ml,LPS group),Shenling Baizhu SAN medicated serum high-dose,medium-dose and low-dose groups(12%,6%,3%,LPS+SLBZSH,M,L),autophagy inhibitor 3-MA group(5μg/ml,3-MA group),autophagy inducer Rapamycin group(5μg/ml,Rapamycin group);CCK-8 detected the activity of IEC-6 cells.Adenovirus m RFP-GFP-LC3 infected IEC-6 cells to detect autophagy flow.Apoptosis of IEC-6cells was detected by flow cytometry.The mrna expression levels of autophagy genes ATG5,ATG12,ATG16L1,ATG16L2,apoptosis genes Fas,Fasl,Caspase-3,P53 and Bcl-2were detected by q RT-PCR.The protein expressions of LC3Ⅱ/LC3Ⅰ,Beclin1 and p62 were detected by WB.2.To investigate whether the drug-containing serum of Shenling Baizhu Powder can improve the inflammatory damage of IEC 6 cells by regulating the protein related to NF-κB/NLRP3 inflammatory-body signaling pathway:IEC 6 cells were divided into normal group(ZC group),model group(10μg/ml,LPS group),Shenling Baizhushan medicated serum high-dose,medium-dose and low-dose groups(12%,6%,3%,LPS+SLBZSH,M,L group),NLRP3 inhibitor MCC950 group(50u M,LPS+MCC950 group).The secretion of IL-1β and 1L-18 in the supernatant of IEC-6 cells was detected by ELISA.Protein immunoblotting was used to detect the expression of NLRP3 inflammasome signaling pathway protein,NF-κB p65 and p-NF-κB p65 in colonic histopathin.In vitro experiment:1.Study on the mechanism of regulating autophagy and apoptosis of cells induced by LPS-induced IEC-6 cells by the drug-containing serum of Shenling Baizhu Powder1.1.IEC-6 cell viability value detected by CCK-8: The experimental concentration was selected(0,1,2,10,20,40,50,100,200)μg/ml,and the experimental results showed that when C(LPS)=10μg/ml,the cell viability value(proportional to OD value)was significantly decreased compared with the blank group and each concentration group(P<0.05).Serum with different volume fractions had obvious effect on the proliferation of IEC-6 cells,and could significantly enhance cell viability.1.2.Detection of IEC-6 autophagy flow: Compared with the normal group,the formation of autophagosome spots in the model group was significantly inhibited.Compared with the model group,the autophagy spots in the Shenling Baizhu SAN group and rapamycin group were significantly increased,while the autophagy spots in the 3-MA group were not significantly different from the model group.Both red and green fluorescent proteins were expressed in the IEC 6 cells.The results indicated that the IEC-6 cell line with stable expression of m RFP-GFP-LC3 was successfully constructed.1.3.The apoptosis of IEC-6 cells was detected by flow cytometry: compared with normal group,the total apoptosis rate of IEC-6 cells in model group was significantly increased(P<0.05);Compared with model group,Shenling Baizhu Powder drug-containing serum group and 3-MA group significantly decreased the total apoptosis rate of IEC 6 cells(P<0.05),but there was no significant difference in the total apoptosis rate of rapamycin group.1.4.q RT-PCR results: Compared with the normal group,the m RNA levels of ATG5,ATG12,ATG16L1,ATG16L2 and Bcl-2 in the model group were significantly decreased(P<0.05);Fas,Fasl,Caspase-3 and P53 m RNA were significantly increased(P<0.05);Compared with model group,ATG5,ATG12,ATG16L1 and ATG16L2 m RNA in Shenlingbaizhu powder drug-containing serum group and rapamycin group were significantly increased(P<0.05);ATG12 in 3-MA group was significantly increased(P<0.05),Fas,Fasl and Caspase-3m RNA levels in Shenling Baizhu SAN drug-containing serum group were significantly decreased(P<0.05),and Bcl-2 mrna levels were significantly increased(P<0.05).The expression of P53 m RNA in different groups was different from that of other genes.Compared with the model group,the expression of P53 mrna in 3% and 12%Shenlingbaizhu powder drug-containing serum groups was significantly decreased(P<0.05).Compared with model group,the m RNA levels of Fas,Caspase-3 and P53 in rapamycin group were significantly decreased(P<0.05),while the mrna levels of Bcl-2 were significantly increased(P<0.05).The m RNA expressions of Fas,Fasl,Caspase-3 and P53 in 3-MA group were significantly decreased(P<0.05),while the mrna levels of Bcl-2 were significantly increased(P<0.05).1.5.Protein expression results detected by WB: Compared with normal group,the protein expression levels of LC3Ⅱ/LC3Ⅰ and Beclin1 in model group were significantly decreased(P<0.05);The expression of p62 protein was significantly increased(P<0.05).Compared with model group,LC3Ⅱ/LC3Ⅰ and Beclin1 in 6%Shenlingbaizhu powder drug-containing serum group were significantly increased(P<0.05),while p62 protein expression was significantly decreased(P<0.05).Beclin1 was significantly increased in 3% Shenlingbaizhu powder serum group(P<0.05),and p62 protein expression was significantly decreased(P<0.05);The expression of p62 protein in 12% Shenlingbaizhu powder serum group was significantly decreased(P<0.05);In rapamycin group,LC3Ⅱ/LC3Ⅰ and Beclin1 were significantly increased(P<0.05),while p62 protein was significantly decreased(P<0.05).2.To investigate the mechanism of anti-LPS-induced injury of IEC 6 cells in the drug-containing serum of Shenlingbaizhu Powder based on NF-κB p65/NLRP3 inflammasome signaling pathway2.1.ELISA was used to detect the secretion of inflammatory cytokines IL-1β and IL-18,and the experimental results showed that compared with the normal group,the contents of IL-1β and IL-18 in the model group were significantly increased(P<0.05).Compared with the model group,the contents of IL-1β and IL-18 in the serum containing Shenlingbaizhu Powder were significantly decreased.IL-1β content in MCC950 was significantly decreased(P<0.05).2.2.Protein expression levels detected by WB: compared with normal group,the protein expressions of P-NF-κB p65,NF-κB p65 NLRP3,Caspase-1,Pro-IL-18,Pro-IL-1β,cleaved IL-1β were significantly increased in the model group(P<0.05);Compared with the model group,protein expression levels of NLRP3,Pro-IL-1β,cleaved IL-1β in 3%,6%,12% Shenling Baizhu powder drug-containing serum groups and MCC950 group,Caspase-1 protein expression levels in 6%,12% Shenlingbaizhu powder drug-containing serum groups and MCC950 group,respectively.The expression of Pro-IL-18 protein in 12% Shenlingbaizhu powder drug-containing serum group and MCC950 group was significantly decreased(P<0.05),and the expression levels of P-NF-κB p65 and NF-κB p65 in 3%,6% and 12%Shenlingbaizhu powder drug-containing serum group and MCC950 group were significantly decreased(P<0.05).Conclusion:1.Shenlingbaizhu powder can treat ulcerative colitis in mice by regulating autophagy pathway proteins and NLRP3 inflammasome signaling pathway related proteins.2.The drug-containing serum of Shenling Baizhu Powder can activate the expression of autophagy related pathway proteins and genes,promote autophagy of IEC-6 cells,and reduce the apoptosis rate of IEC-6 cells.3.The drug-containing serum of Shenling Baizhu Powder can significantly inhibit the expression of NLRP3 inflammasome signaling pathway related proteins in IEC-6 cells and alleviate the inflammation of IEC-6 cells.