Effect of Chinese Herbal Drug-Containing Serum for Activating-Blood and Dispelling-Toxin on ox-LDL-Induced Inflammatory Factors'Expression in Endothelial Cells

Objective： To investigate the effects of drug-containing serum of Chinese herbal compound, Xiongshao Capsule （芎芍胶囊, XS, for activating-blood） and Huanglian Capsule （黄连胶囊, HL, for dispellingtoxin） on the oxidized low-density lipoprotein （ox-LDL）-induced inflammatory factors in human umbilical vein endothelial cells （HUVECs）. Methods： Thirty-two rats were randomly divided into four groups： the blank control group treated with distilled water, the positive control group treated with simvastatin （1.8 mg/kg）, the test group I treated with Chinese herbal compound of XS （0.135 g/kg）, and the test group 1T treated with Chinese herbal compound of XS （0.135 g/kg） and HL （0.135 g/kg）. All the treatments were administered for 7 successive days by gastrogavage. Rats＇ blood serum was harvested 1 h after the last administration to prepare respective drug- containing serum. HUVECs were exposed to ox-LDL （100 μg/mL） to induce cell injury model and incubated with corresponding drug-containing serum for 24 h. Untreated HUVECs were set for blank control. Levels of interleukin-6 （IL-6）, tumor necrosis factor-α （TNF-α）, and soluble intercellular adhesion molecule-1 （slCAM-1） in supematant of cultured HUVECs were determined by enzyme-linked immunosorbent assay （ELISA）. HUVEC surface expressions of ICAM-1 and E-selectin were determined by flow cytometry. Results： Levels of IL-6, TNF-α, and sICAM-1 in the supernatant of HUVECs as well as the cell surface expressions of ICAM-1 and E-selectin significantly increased after 24-h ox-LDL stimulation （P〈0.01）, while the abnormal elevations, except slCAM-1 in the test group Ⅰ, were all reduced in the treated groups （the positive control and the two test groups） significantly （P〈0.01 or P〈0.05）. Besides, the effect in the test group Ⅱ seemed somewhat higher than that in the test group Ⅰ but with no statistical significance （P〉0.05）. Conclusion： Drug-containing serum of XS plus HL has a certain inhibitory effect on the vascular endothelial inflammation response induced by ox-LDL.

Drug-containing serum of Xinfeng capsules protect against H9C2 from death by enhancing miRNA-21 and inhibiting toll-like receptor 4/phosphorylated p-38(p-p38)/p-p65 signaling pathway and proinflammatory cytokines expression

OBJECTIVE: To investigate effect of drug-containing serum of Xinfeng capsules on myocardial cell growth.METHODS: Drug-containing serum of Xinfeng capsules rat models were established by intragastricly administrated Xinfeng capsules. MTT assay wasused to evaluated H9C2 cells viability. H9C2 cells were divided into normal control group, triptolide group, lipopolysaccharide(LPS) group, drug-containing serum group and mi RNA-21 inhibitor group. micro RNA-21(mi RNA-21) inhibitor was structured and transfected into H9C2 cells. Western blot and immunofluorescence assay were applied to examine toll-like receptor 4(TLR4), phosphorylated p-38(p-p38) and p-p65 expression. Quantitative real-time PCR(q RT-PCR) was used to evaluated m RNA levels of mi RNA-21. Enzyme linked immunosorbent(ELISA) was used to measure tumor necrosis factor α(TNF-α), IL-6 and IL-17 levels.RESULTS: Drug-containing serum treatment significantly increased cell viability compared to LPS treated group. q RT-PCR results indicated that mi RNA-21 levels were significantly decreased in drugcontaining serum group compared to LPS group.Early and late apoptosis in drug-containing serum group were significantly decreased compared to LPS group. Western blot and immunofluorescence assay results showed that TLR4, p-p38 and p-p65 levels in drug-containing serum group were significantly decreased compared to LPS group. ELISA findings indicated that drug-containing serum significantly decreased inflammatory cytokine levels of TNF-α, IL-6 and IL-17.CONCLUSION: Drug-containing serum of Xinfeng capsules protect against lipopolysaccharide instructed H9C2 cells from death by enhancing mi RNA-21 and inhibiting TLR4/p-p38/p-p65 signaling pathway and proinflammatory cytokines expression.

How do Chinese medicines that tonify the kidney inhibit dopaminergic neuron apoptosis?

Wistar rats were intragastrical y perfused with Chinese medicines used for tonifying the kidney. These included 0.180 g/mL of Herba Epimedi （Epimedium）, Semen Cuscutae （Dodder Seed）, or Herba Cistanches （Desertliving Cistanche）, 0.04 mg/mL monoamine oxidase-B inhibitor selegiline, or distil ed water for 14 consecutive days to prepare drug-containing serum or blank serum. MES23.5 cells in the logarithmic phase were cultured in media supplemented with 15%drug-containing serum for 24 hours, fol owed by incubation in culture solution containing 100μmol/L H2O2 for 3 hours. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） and flow tometry results showed that al drug-containing serums improved the survival rate of H 2 O 2-injured MES23.5 cells, inhibited pro-apoptotic FasL and caspase-3 expression, promoted anti-apoptotic Bcl-2 expression. However, drug-containing serums had little influence on Fas expression in H 2 O 2-injured MES23.5 cells. Enzyme-linked immunosorbent assay results showed that serum containing Herba Cistanches or Herba Epimedi increased the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cellline-derived neurotrophic factor in injured MES23.5 cells;serum containing Semen Cuscutae only increased brain-derived neurotrophic factor expres-sion; while expression of the above neurotrophic factors remained the same in cells treated with serum containing selegiline. These findings indicate that Chinese medicines used to tonify the kid-ney can protect nerve cells by regulating the expression of apoptosis-related factors and neuro-trophic factors in MES23.5 cells.

VEGFR2 mediated RAS/MAPK signaling pathway to explore the mechanism of Bushen Antai Granule in the treatment of recurrent abortion

Objective:To investigate the effect of Bushen Antai Granule on the mRNA and protein expression of Ras protein/mitogen activated protein kinase mediated by vascular endothelial growth factor receptor 2 with small interference RNA interference.Methods:Method to construct the placenta microvascular endothelial cells,and the preparation of kidney fetus granule drug-containing serum,select the best drug-containing serum concentration,it can be divided into normal group,the serum siRNA-NC normal serum group,drug serum,siRNA normal serum group,siRNA drug serum group,using real-time fluorescent quantitative PCR,Western blotting,immunofluorescence test respectively the RAS/MAPK mRNA and protein expression.Results:Results there was no significant difference in Ras and MAPK mRNA and protein expression between the normal group and the negative control group(P>0.05).The mRNA and protein expressions of Ras and MAPK in the drug serum group were significantly higher than those in the normal serum group(P<0.01).Ras and MAPK mRNA and protein expression were significantly decreased in siRNA1 normal serum group compared with normal serum group(P<0.01).Ras,MAPK mRNA and protein expression in siRNA1 drug serum group were significantly different from that in siRNA1 normal serum group(P<0.01).Conclusion:Conclusion The therapeutic effect of Bushen Antai Granule on recurrent abortion may be realized by upregulation of RAS/MAPK mRNA and protein expression.

Experimental Study on the Suppression of Sodium Nitroprussiate-lnduced Chondrocyte Apoptosis by Tougu Xiaotong Capsule(透骨消痛胶囊)-Containing Serum

Objective：To study the mechanism of action of Tougu Xiaotong Capsule（透骨消痛胶囊,TGXTC） ex vivo in suppressing chondrocyte（CD） apoptosis induced by sodium nitroprussiate（SNP）.Methods：Thirty New Zealand rabbits,2 months old,were randomized by lottery into five groups,six in each：the blank group treated with saline,the positive control group treated with Zhuanggu Guanjie Pill（壮骨关节丸,70 mg/kg）,and the three experimental groups,EGA,EGB,and EGC,treated with low dose（35 mg/kg）,moderate dose（70 mg/kg）,and high dose（140 mg/kg） of TGXTC,respectively.All treatments were administered via gastrogavage twice a day for 3 days.Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared.CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining.SNP of various final concentrations（0,0.5,1.0,and 2.0 mmol/L） was used to induce CD apoptosis,and the dosage-effect relationship of SNP in inducing CD apoptosis was determined.Serum samples from the blank,control,and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP.Cell apoptosis was determined by Hoechst 33342 staining,viability of CDs was quantified by MTT,CD apoptosis rate was determined by annexin V-FITC/PI staining,levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR,and contents of caspase-3 and caspase-9 proteins were determined by colorimetry.Results：CD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner.The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study.Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis,decrease in p53 mRNA expression,inhibition of catalytic activities of caspase-3 and caspase-9,and increase in Bcl-2 mRNA expression when compared with the serum from the blank group（P0.05）.Conclusion：TGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression,and inhibition of caspase-3 and caspase-9 catalytic activities.

Effect of Banxia Xiexin decoction on Helicobacter pylori-related peptic ulcers and its possible mechanism via the TGF-β/Smad signaling pathway

OBJECTIVE: To investigate the effect of Banxia Xiexin decoction(BXD) on Helicobacter pylori(Hp)-related peptic ulcers(PUs) and the possible mecha-nism underlying BXD actions via the transforming growth factor-β/small mothers against decapentaplegic(TGF-β/Smad) signaling pathway.METHODS: PU patients with cold-heat complex syndrome were randomly assigned to groups that received Chinese or Western medicines with 20 patients in each group. Serum was collected after 7 d of treatment. The healthy group included 20 individuals. Gastric mucosal epithelial cell line GES-1 was cultured in vitro and randomly divided into the following seven groups: control, model, healthy,Western Medicine, prior treatment, low dosage,and high dosage. After 72 h of treatment with the corresponding serum, the m RNA and protein expression levels of TGF-β1, Smad3, and Smad7 were measured by reverse transcription quantitative polymerase chain reaction and western blotting, respectively.RESULTS: The m RNA expression levels of TGF-β1 and Smad3 in GES-1 cells were increased after Hp introduction, and these increased levels were reduced by the BXD-containing serum. The protein levels of p-Smad3, but not TGF-β1 or Smad3, were significantly increased in Hp-treated GES-1 cells,and treatment with the BXD-containing serum markedly decreased the protein levels. Smad7 expression was significantly enhanced following treatment with the BXD-containing serum at transcriptional and protein levels in a dose-dependent manner.CONCLUSION: BXD regulates the TGF-β/Smad signaling pathway by inhibiting the expression of TGF-β1 and Smad3, and increasing the expression of Smad7.

Chang'an Ⅱ Decoction(肠安Ⅱ号方)-Containing Serum Ameliorates Tumor Necrosis Factor-α-Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats

Objective To investigate the effect of Chang’an Ⅱ Decoction(肠安Ⅱ号方))-containing serum on intestinal epithelial barrier dysfunction in rats.Methods Tumor necrosis factor(TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium.Caco-2 monolayers were treated with blank serum and Chang’an Ⅱ Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang’an Ⅱ Decoction intragastrically at doses of 0.49,0.98,1.96 g/(kg·d)for 1 week,respectively.After preparation of containing serum,cells were divided into the normal group,the model group,the Chang’an Ⅱ-H,M,and L groups(treated with 30 ng/mL TNF-αand medium plus 10%high,middle-,and low-doses Chang’an Ⅱ serum,respectively).Epithelial barrier function was assessed by transepithelial electrical resistance(TER)and permeability of fluorescein isothiocyanate(FITC)-labeled dextran.Transmission electron microscopy was used to observe the ultrastructure of tight junctions(TJs).Immunofluorescence of zonula occludens-1(ZO-1),claudin-1 and nuclear transcription factor-kappa p65(NF-κBp65)were measured to determine the protein distribution.The mRNA expression of myosin light chain kinase(MLCK)was measured by real-time polymerase chain reaction.The expression levels of MLCK,myosin light chain(MLC)and p-MLC were determined by Western blot.Results Chang’an Ⅱ Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α.It alleviated TNF-α-induced morphological alterations in TJ proteins.The increases in MLCK mRNA and MLCK,MLC and p-MLC protein expressions induced by TNF-αwere significantly inhibited in the Chang’an Ⅱ-H group.Additionally,Chang’an Ⅱ Decoction significantly attenuated translocation of NF-κBp65 into the nucleus.Conclusion High-dose Chang’an Ⅱ-containing serum attenuates TNF-α-induced intestinal barrier dysfunction.The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κBp65.

Shenweifang-containing serum inhibits transforming growth factor-β1–induced myofibroblast differentiation in normal rat kidney interstitial fibroblast cells

OBJECTIVE:To investigate the efficacy of Shenweifang(SWF)-containing serum on transforming growth factor(TGF)-β1–induced fibroblast-myofibroblast transition in normal rat kidney interstitial fibroblast cells(NRK-49 F).METHODS:Sprague-Dawley rats were gavaged with one of five solutions:(a)saline;(b)saline plus low-dose SWF;(c)saline plus medium-dose SWF;(d)saline plus highdose SWF;and(e)saline plus valsartan.NRK-49 F cells were treated with TGF-β1 and cultured using serum from the gavaged rats.RESULTS:TGF-β1 treatment increased the expression ofα-smooth muscle actin,proliferating cell nuclear antigen,collagenⅠ,Smad3,mitogen-activated protein kinase(MAPK)10,and c-Jun N-terminal kinase(JNK)3 and induced abnormalities in cell morphology,cell cycle progression,and cell proliferation.CONCLUSIONS:SWF-or valsartan-containing serum corrected(or partially corrected)TGF-β1–induced abnormal changes in this in vitro system.SWF-containing serum reversed abnormalities in morphology,cell cycle progression,and proliferation in TGF-β1–treated NRK-49F cells,probably by blocking the TGF-β1/Smads and TGF-β1/MAPK/JNK pathways.