Mechanism Study On Leonurine Alleviating Colitis By Activating Nrf2 Signaling Pathway

Inflammatory bowel disease(IBD)is a common chronic inflammatory gastrointestinal disease,the main clinical forms being Ulcerative colitis(UC)and Crohn’s disease(CD),which is caused by a combination of intestinal epithelial barrier damage,genetic factors,intestinal intrinsic immune disorders,and intestinal microbial dysregulation.With the development of social economy,the prevalence of inflammatory bowel disease in our country is on the rise year by year.As a monomer of traditional Chinese medicine,leonurine,the active ingredient of motherwort,works in a multi-target manner and has a wide range of biological activities,including antioxidant,anti-inflammatory and anti-apoptotic.Studies have confirmed that leonurine regulates many signaling pathways.Leonurine suppresses the expression of Toll-like receptor 4(TLR4)and nuclear factor kappa-B(NF-κB).It inhibits several signaling pathways,such as the NF-κB,the mitogen activated protein kinase(MAPK),the protein kinase B(PI3K/Akt),and the nuclear factor erythroid 2-related factor 2(Nrf2),etc.However,there are fewer studies on the role of leonurine and its molecular mechanism in IBD.In this study,we preliminarily investigated the regulatory role of leonurine and the related mechanisms in a mouse model of UC induced by 4% dextran sulfate sodium(DSS)and a cellular model of UC induced by lipopolysaccharide(LPS).Objectives:1.To study the regulatory effects of leonurine in a mouse model of UC.2.To study the regulatory effects of leonurine on colonic epithelial cells.3.To study the effects of Nrf2 silencing by siRNA and its influence on the efficacy of leonurine on colonic epithelial cells.Methods:1.A mouse model of colitis was constructed by administering 4% DSS.The mice were randomly divided into five groups: control group,DSS group,DSS + leonurine(7.5mg/kg)group,DSS + leonurine(15 mg/kg)group and DSS + leonurine(30 mg/kg)group.The body weights,disease activity index(DAI),and colon lengths of each group were measured respectively.Serum and colon tissues were collected from mice of each group.Serum levels and m RNA levels of inflammatory factors including tumor necrosis factor(TNF)-α,interleukin(IL)-6 and IL-1β were measured by enzyme-linked immunosorbent assay(ELISA)and quantitative real-time polymerase chain reaction(qRT-PCR),respectively.The levels of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in the serum were measured by biochemical assays.Cellular apoptosis in the colon tissue of each mouse was observed by fluorescence microscope.Western blotting(WB)was performed to investigate the protein levels of apoptosis related molecules such as B-cell lymphoma-2(Bcl-2),Bcl-2 associated X(BAX),and cleaved caspase-3.Besides,protein levels of TLR4,p-NF-κB,NF-κB,Nrf2 and heme oxygenase 1(HO-1)were detected by WB in the mouse colon tissues from each group.2.Human colon cancer epithelial cells(HT-29)and human colon cancer cells(Caco-2)were cultured in vitro.For the establishment of UC cell model,HT-29 and Caco-2 cells were treated with LPS.The optimal concentration and duration of treatment of leonurine for the two cell lines were determined by detecting cell proliferation with cell counting kit-8(CCK-8).3.HT-29 and Caco-2 cells were cultured in vitro and divided into normal group,negative control group and transfection group(siRNA-Nrf2-1,siRNA-Nrf2-2,or siRNA-Nrf2-3),respectively.The si-RNA-Nrf2 sequences with high knockdown efficiency were screened out by qRT-PCR.4.The two cell lines were treated with leonurine in the presence of siRNA-Nrf2.Six groups were set up as follows: Group A = blank control =(HT-29 or Caco-2);Group B= blank control + LPS;Group C =(HT-29 or Caco-2)+LPS+ siRNA-Nrf2-NC;Group D=(HT-29 or Caco-2)+LPS+siRNA-Nrf2;Group E =(HT-29 or Caco-2)+LPS+ leonurine;Group F =(HT-29 or Caco-2)+LPS+ siRNA-Nrf2 +leonurine.The cell viability of each group was determined by CCK-8;the levels of cytokines(IL-6,IL-1β,and TNF-α)of each group were detected by ELISA;the levels of intracellular reactive oxygen species(ROS),MDA,SOD and GSH of each group were detected by flow cytometry;and the mitochondrial membrane potential was detected by the mitochondrial membrane potential assay kit with(JC-1)in cells of each group;the protein levels of apoptosis-related proteins BAX,cleaved caspase-3 and Bcl-2,as well as the protein levels of Nrf2 and HO-1,were detected by WB in each group.Results:1.Leonurine reversed DSS-induced weight loss,shortened colon length and decreased DAI scores in a dose-dependent manner and attenuated the disease phenotype in DSS mice.2.Leonurine reduced the levels of inflammatory factors in DSS mice.ELISA showed that serum levels of inflammatory factors TNF-α,IL-6 and L-1β in the DSS group were significantly elevated(p < 0.01),which was reduced by leonurine treatment(p < 0.01);qRT-PCR demonstrated that the m RNA levels of inflammatory factors TNF-α,IL-6 and L-1β in the DSS group were significantly elevated(p < 0.01),which was lowered by leonurine treatment.3.Leonurine had some alleviating effects on oxidative stress in DSS mice;MDA levels were increased in the DSS group and decreased in the leonurine group in a dose-dependent manner;SOD and GSH levels were decreased in the DSS group and increased in the leonurine group in a dose-dependent manner.4.Leonurine inhibited intestinal cell apoptosis in DSS mice.In the DSS group,crypt cells were almost destroyed and TUNEL-positive cells(red color)were significantly increased.Leonurine inhibited DSS-induced apoptosis.The apoptosis-related proteins BAX and cleaved caspase-3 showed a significant elevation in the DSS group(P < 0.01),and displayed a dose-dependent decrease in the leonurine group.Moreover,Bcl-2showed an opposite trend in the two groups.5.Leonurine inhibited the TLR4/NF-κB and activated the Nrf2/HO-1 signaling pathway.The protein levels of TLR4 and p-NF-κB were up-regulated in the DSS group and down-regulated in the leonurine group.The protein levels of Nrf2 and HO-1 was down-regulated in the DSS group and gradually up-regulated in a dose-dependent manner in the leonurine group.6.Leonurine was able to increase LPS cell viability through activation of Nrf2.Cell viability was decreased in the two LPS cell groups.Cell viability was decreased when Nrf2 was knocked down by siRNA-Nrf2,and increased in the presence of leonurine.Compared with adding leonurine alone,cell viability was decreased with the simultaneous silencing of Nrf2 by siRNA-Nrf2.7.The level of ROS in LPS cells was decreased by leonurine through the activation of Nrf2,and the level of cellular ROS was increased in the two LPS groups,which was further increased when Nrf2 was silenced by siRNA-Nrf2 transfection,but decreased by the addition of leonurine.Compared with adding leonurine alone,the level of cellular ROS was increased by the simultaneous knockdown of Nrf2.8.Leonurine inhibited apoptosis in LPS cells by activating Nrf2.JC-1 assay showed that: mitochondrial membrane potential was increased in the two LPS cell groups,which was increased upon Nrf2 silencing by siRNA-Nrf2 and decreased after treatment with leonurine.Compared with adding leonurine alone,simultaneously knockdown of Nrf2 contributed to an increase in the mitochondrial membrane potential.Western blotting demonstrated that the protein levels of Bax and cleaved caspase-3 were elevated,and Bcl-2 and HO-1 were reduced in cells.By contrast,when Nrf2 was knocked down,increased protein levels of Bax and cleaved caspase-3,and decreased levels of BLC-2 and HO-1 were observed.Notably,opposite trends were seen in cells treated with leonurine.Compared with adding leonurine alone,si-RNA-Nrf2 transfection at the same time lead to increased levels Bax and cleaved caspase-3 but decreased levels of Bcl-2 and HO-1.9.Leonurine alleviated the oxidative stress of LPS cells by activating Nrf2,and the two LPS cell groups showed an increase in MDA levels and a decrease in SOD and GSH levels.siRNA-Nrf2 transfection led to an increase in the levels of MDA and a decrease in SOD and GSH levels,while in the presence of leonurine decreased levels of MDA and elevated levels of SOD and GSH were seen.Compared with adding leonurine alone,additional knockdown of Nrf2 by siRNA-Nrf2 resulted in augmented levels MDA and reduced levels of SOD and GSH.10.Leonurine decreased the levels of inflammatory factors in LPS cells by activating Nrf2.The levels of inflammatory factors IL-6,IL-1β and TNF-α were increased in the two LPS cells.The levels of IL-6,IL-1β and TNF-α were significantly increased when Nrf2 was silenced by siRNA-Nrf2 transfection,but significantly decreased upon leonurine treatment.Compared with adding leonurine alone,when cells were transfected with siRNA-Nrf2 simultaneously,elevated levels of IL-6,IL-1β and TNF-αwere observed.11.Leonurine was able to increase LPS cell the level of Fe through activation of Nrf2.The level of Fe was decreased in the two LPS cells.The level of Fewere significantly decreased when Nrf2 was silenced by siRNA-Nrf2 transfection,but significantly increased upon leonurine treatment.Compared with adding leonurine alone,when cells were transfected with siRNA-Nrf2 simultaneously,decreased the level of Fe were observed.Conclusion:1 Leonurine ameliorates DSS-induced colitis mouse model and LPS-induced colitis cell model by inhibiting apoptosis,inflammatory response and oxidative stress;2.Nrf2 is involved in LPS-induced inflammatory response,oxidative stress and apoptosis in intestinal epithelial cells;3.Leonurine inhibits oxidative stress,inflammatory response and apoptosis in colitis intestinal epithelium by activating Nrf2.