Huang-Lian-Jie-Du decoction drug-containing serum inhibits IL-1ß secretion from D-glucose and PA induced BV2 cells via autophagy/NLRP3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du decoction (HLJDD), a famous traditional Chinese medicine prescription with heat-clearing and detoxifying effects, has been widely used to treat diabetes, dementia, stroke, and other diseases. However, the detailed mechanisms of HLJDD against type 2 diabetes associated cognitive dysfunction (DACD) through inhibiting interleukin-1ß (IL-1ß) mediated neuroinflammation remain to be further elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effect and potential mechanism of HLJDD on IL-1ß secretion in a DACD model of BV2 cells induced by D-glucose and palmitic acid (PA). MATERIALS AND METHOD: sUltra-performance liquid chromatography-quadrupole/electrostatic field orbital well high-resolution mass spectrometry technology was used to analyze the compounds in HLJDD drug-containing serum. The cytotoxicity was detected by cell counting kit-8. Enzyme-linked immunosorbent assay was used to measure the secretion of IL-1ß in BV2 cells. Reactive oxygen species, glutathione, superoxide dismutase, and malondialdehyde kits were used to detect the intracellular oxidative stress levels. The autophagy level was determined by autophagy staining kit and transmission electron microscope. The expression levels of autophagy-related 7 (Atg7), P62, LC3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3(NLRP3), Caspase1, and IL-1ß were detected by real-time PCR, immunofluorescence, and western blotting. The Atg7siRNA was transfected into BV2 cells to produce autophagy inhibitory effect. Then the effect of HLJDD drug-containing serum on IL-1ß secretion in D-glucose and PA induced BV2 cells and the potential mechanism of autophagy-NLRP3 inflammasome activation were further observed. RESULTS: Eighty-eight compounds were preliminarily identified in HLJDD drug-containing serum, among which geniposide, baicalin, palmatine, berberine, wogonoside, wogonin, and geniposidic acid were identified as the main prototype components of HLJDD into the blood. In this study, the DACD model of BV2 cells induced by high concentrations of glucose and PA was successfully constructed. HLJDD drug-containing serum significantly reduced the secretion of IL-1ß and the activity of NLRP3 inflammasome with improving the oxidative stress level. Interestingly, the enhanced autophagy level was also found. After transfection of Atg7siRNA into BV2 cells, the effect of HLJDD drug-containing serum on autophagy promotion was reversed, but the inhibitory effects on IL-1ß secretion, NLRP3 inflammasome activation, and oxidative stress were reduced. CONCLUSIONS: These results indicated that the inhibition of HLJDD drug-containing serum on the IL-1ß secretion in D-glucose and PA induced BV2 cells was related to autophagy promotion, the decreased NLRP3 inflammasome activation, and the improved oxidative stress. Moreover, the improvement of HLJDD drug-containing serum on IL-1ß secretion, NLRP3 inflammasome activation, and oxidative stress were all closely associated with Atg7 mediated autophagy promotion. Geniposide, baicalin, palmatine, berberine, wogonoside, wogonin, and geniposidic acid may be the potential active ingredients of HLJDD drug-containing serum.

Cynanchum komarovii extract for the treatment of rheumatoid arthritis by acting on synovial cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynanchum komarovii (CK), the northwest Chinese region's common medicinal herb, was traditionally utilized to treat arthritis, toothache, bald sores and cholecystitis. Various forms of arthritis can be treated with CK, based on "Medicinal Plants of Chinese Desert Areas". However, the exact mechanism of action in rheumatoid arthritis (RA) is uncertain. AIM OF THE STUDY: To evaluate the in vitro and in vivo effects of CK extracts on RA and to preliminarily investigate its anti-RA mechanism of action. MATERIALS AND METHODS: The main components of CK extract were analyzed by HPLC method. The effects of CK on the proliferation and apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) cells and the expression of apoptosis-related proteins in HFLS-RA cells were evaluated by CCK8 assay, flow cytometry and WB assay. To verify the anti-RA effect of CK extracts in vivo, a collagen-induced arthritis (CIA) rat model was established. The rats were divided into six groups: normal group, model group, CK high-dose group (1000 mg/kg, CK-H), CK medium-dose group (500 mg/kg, CK-M), CK low-dose group (250 mg/kg, CK-L) and methotrexate-positive drug group (MTX); the drug was administered continuously for 28 days. Body weight changes, joint swelling, arthritis index, bone density, ankle lesions, immune organ index, splenic lesions and inflammatory factor expression were used to evaluate the in vivo anti-RA activity of the extract. RESULTS: The findings of in vitro experiments showed that 10% CK-containing serum decreased the expression level of Bcl-2, increased the expression levels of Bax and Cleaved Caspase-3 in synovial cells, and prevented TNF-α induced aberrant proliferation and apoptotic antagonism in HFLS-RA cells. According to in vivo studies, CK extract at doses above 250 mg/kg was effective in controlling the levels of inflammatory factors, lowering the arthritis index, and improving foot swelling in CIA rats. When administered at doses up to 1000 mg/kg, CK extract significantly improved synovial lesions, increased bone density, and decreased abnormally elevated immune organ index in CIA rats. CONCLUSIONS: CK has significant anti-RA activity, and its anti-RA mechanism of action may be related to the regulation of the expression levels of apoptosis related proteins and the promotion of apoptosis in synovial cells.

Shenweifang-containing serum inhibits transforming growth factor-ß1 induced myofibroblast differentiation in normal rat kidney interstitial fibroblast cell.

OBJECTIVE: To investigate the efficacy of Shenweifang (SWF)-containing serum on transforming growth factor (TGF)-ß1-induced fibroblast-myofibroblast transition in normal rat kidney interstitial fibroblast cells (NRK-49F). 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-49F cells were treated with TGF-ß1 and cultured using serum from the gavaged rats. RESULTS: TGF-ß1 treatment increased the expression of α-smooth muscle actin, proliferating cell nuclear antigen, collagen I, 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-ß1-induced abnormal changes in this in vitro system. SWF-containing serum reversed abnormalities in morphology, cell cycle progression, and proliferation in TGF-ß1-treated NRK49F cells, probably by blocking the TGF-ß1/Smads and TGF-ß1/MAPK/JNK pathways.

Drug-containing serum of rhubarb-astragalus capsule inhibits the epithelial-mesenchymal transformation of HK-2 by downregulating TGF-ß1/p38MAPK/Smad2/3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheum palmatum L; Astragalus membranaceus (Fisch.), is referred to as 'Dahuang, Huangqi' in China. As an important medicinal plant, the rhizome of rhubarb and astragalus is traditionally used in the treatment of kidney diseases associated with renal failure, inflammation and tumors. AIM OF THE STUDY: This study aimed to investigate the effect of a drug-containing serum of rhubarb-astragalus capsules (composed of rhubarb and astragalus) and to elucidate its mechanism in the epithelial-mesenchymal transformation of renal tubular epithelial cells. MATERIALS AND METHODS: Epithelial-mesenchymal transformation (EMT) of HK-2 cells was induced by TGF-ß1, and rhubarb-astragalus and losartan drug-containing serum from rats, as well as SB203580 (a specific inhibitor of p38 MAPK), were used. High-performance liquid chromatography analysis was performed to determine the main components of the drug-containing serum of rhubarb-astragalus from rats. Western blotting and immunofluorescence analysis were used to determine the levels of protein expression, and real-time quantitative PCR analysis was used to detect the levels of gene expression. RESULTS: The drug-containing serum of rhubarb-astragalus contained emodin (0.36 µg/ml) and danthraquinone (0.96 µg/ml). Rhubarb-astragalus significantly decreased the protein expression levels of α-SMA, FN, vimentin and N-cadherin in HK-2 cells that were increased by TGF-ß1, while it significantly increased the E-cadherin protein expression level that was decreased by TGF-ß1. Rhubarb-astragalus also significantly decreased the protein expression levels of TGF-ß1 and p38 MAPK and the mRNA expression levels of α-SMA, vimentin, TGF-ß1, p38 MAPK, Smad2 and Smad3 in HK-2 cells that were increased by TGF-ß1. It is worth noting that SB203580 (a p38 MAPK inhibitor) had similar effects as rhubarb-astragalus in this study. CONCLUSION: The drug-containing serum of rhubarb-astragalus can inhibit EMT in HK-2 cells by downregulating the TGF-ß1/p38 MAPK/Smad2/3 pathway.

[Effect of Huangqin Qingre Chubi Capsules containing serum on oxidative stress and protein expression of AMPK and FoxO3a in rheumatoid arthritis patients].

To study the effect of Huangqin Qingre Chubi Capsules containing serum on the protein expressions of AMPK and FoxO3 a in peripheral blood mononuclear cells of patients with rheumatoid arthritis(RA), in order to explore the mechanism of anti-oxidation. Peripheral anticoagulant was collected from patients and normal people. Monocytes(PBMC) were isolated through density gradient centrifugation, and the logarithmic phase cells were cultured. Drug containing serum was prepared through intragastric admini-stration to SD rats. The rats were divided into five groups, namely normal group, model group, AMPK blocker group(compound C 10 µmol·L~(-1)), medium-dose HQC+AMPK blocker group, and middle-dose HQC group. The cell inhibition rate was calculated by MTT method. The levels of IL-1ß, IL-4, LPO, MDA, SOD and TAOC were detected by ELISA. The expressions of AMPK, p-AMPK, p-FoxO3 a and FoxO3 a were detected by Western blot. The HQC containing serum had an inhibitory effect on human monocytes in peripheral blood. The best concentration was observed in middle-dose HQC, and the best time was 24 hours. Middle-dose HQC group was better than model group, AMPK blocker group and middle-dose HQC + AMPK blocker group in terms of increase of SOD, p-AMPK, p-FoxO3 a and decrease of LPO. It was better than model group and AMPK blocker group in terms of increase of IL-4, TAOC, AMPK, FoxO3 a and decrease of IL-1ß, MDA. The differences were statistically significant(P<0.05 or P<0.01). The HQC containing serum may increase the levels of TAOC and SOD, decrease the level of MDA and LPO, activate AMPK, directly phosphorylate FOXO3 a, enhance its transcriptional activity, and improve the state of oxidative stress in RA patients.

Chang'an II Decoction ( II )-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 ( II ))-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-α and 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-κ Bp65) 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-α were significantly inhibited in the Chang'an II-H group. Additionally, Chang'an II Decoction significantly attenuated translocation of NF-κ Bp65 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-κ Bp65.

Effect of Bugu granules in a drug-containing serum on chondrocyte apoptosis and the Trx2 signaling pathway.

Traditional Chinese medicine for invigorating the kidney and promoting blood circulation is commonly prescribed for the treatment of osteoarthritis associated with kidney deficiency and blood stasis. However, the specific mechanisms of these medicines are still unclear. The present study aimed to evaluate the protective effects of Bugu granules against sodium nitroprusside-induced chondrocyte apoptosis and elucidate the underlying molecular mechanisms. Drug-containing serum was prepared by administering rats with Bugu granules and harvesting the serum. Chondrocytes were exposed to different dilutions of serum, and apoptosis assessed by flow cytometry after staining with annexin V­FITC/PI. Flow cytometry showed that chondrocyte apoptosis increased significantly after incubation with 2 mol/L sodium nitroprusside for 24 h (t = -48.221, P = 0.000), and the apoptotic rate of chondrocytes decreased with increasing concentrations of drug-containing serum (F = 33.965, P = 0.000). Cellular levels of Trx2, ASK1, caspase­3, and reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay. The cellular content of Trx2 increased gradually with increasing concentrations of drug-containing serum (F = 2610.593, P = 0.000), while that of ASK1 (F = 2473.545, P = 0.000), caspase­3 (F = 209.921, P = 0.000), and ROS (F = 1666.435, P = 0.000) all decreased significantly. The mRNA expression levels were analyzed by RT-qPCR, which revealed that expression levels of Trx2 and caspase­3 mRNA increased and decreased significantly, respectively, following exposure to Bugu granules in the drug-containing serum (F = 6.974, P = 0.003 and F = 3.691, P = 0.191; respectively), but the expression of ASK1 mRNA was not significantly different between treatment groups (F = 1.784, P = 0.191). Taken together, these results support the hypothesis that the Trx2 signaling pathway is activated by Bugu granules, which in turn inhibits chondrocyte apoptosis. This may play a role in preventing the development of osteoarthritis.

[Effect of serum containing Xihuang pill on proliferation of human breast cancer cell line MDA-MB-435 and MCF-7 Cells].

To study the effect of serum containing Xihuang pill on the proliferation of human breast cancer cell lines MDA-MB-435 and MCF-7 and the gene and protein expressions of Bcl-2, Bax, TP53, in order to explore the effect and mechanism of Xihuang pill in resisting breast cancer. The serum of the rats was prepared by the method of MTT assay. The expressions of Bcl-2 and Bax were detected by RT-PCR. The serum levels of Bcl-2 and Bax and the mRNA expression of TP53 were detected by immunofluorescence. The rats with serum containing Xihuang pill could inhibit the proliferation of MDA-MB-435 cells and MCF-7 cells (P<0.05). The serum containing Xihuang pill increased TP53 and Bax in MDA-MB-435 cells (P<0.05), and the ratio of Bcl-2/Bax was decreased (P<0.05). Meanwhile, the serum containing Xihuang pill could up-regulate the mRNA expression of Bax in MCF-7 cells and decrease the expression of Bcl (P<0.05), but there was no significant difference between the expression of TP53mRNA and Bax protein expressions after the treatment of MCF-7 cells with Xihuang pill serum. Serum containing Xihuang pill can induce the apoptosis of human breast cancer cells, and the mechanism of estrogen receptor-negative breast cancer cell apoptosis may be induced by up-regulating the mRNA expression of TP53, which can induce the expression of Bax and promote the metastasis of Bax to mitochondria, and ultimately play the role of inducing apoptosis.

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 was used to evaluated H9C2 cells viability. H9C2 cells were divided into normal control group, triptolide group, lipopolysaccharide (LPS) group, drug-containing serum group and miRNA-21 inhibitor group. microRNA-21 (miRNA-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 (qRT-PCR) was used to evaluated mRNA levels of miRNA-21. Enzyme linked immunosorbent (ELISA) was used to measure tumor necrosis factorα (TNF-α), IL-6 and IL-17 levels. RESULTS: Drug-containing serum treatment significantly increased cell viability compared to LPS treated group. qRT-PCR results indicated that miRNA-21 levels were significantly decreased in drug- containing 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 instr- ucted H9C2 cells from death by enhancing miRNA- 21 and inhibiting TLR4/p-p38/p-p65 signaling pathway and proinflammatory cytokines expression.

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

OBJECTIVE: To investigate the effect of Banxia Xiexin decoction (BXD) on Helicobacter pylori (Hp)-related peptic ulcers (PUs) and the possible mechanism underlying BXD actions via the transforming growth factor-¦Â/small mothers against decapentaplegic (TGF-ß/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 mRNA and protein expression levels of TGF-ß1, Smad3, and Smad7 were measured by reverse transcription quantitative polymerase chain reaction and western blotting, respectively. RESULTS: The mRNA expression levels of TGF-ß1 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-ß1 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-ß/Smad signaling pathway by inhibiting the expression of TGF-ß1 and Smad3, and increasing the expression of Smad7.

[Intervention effects of Zuoguiwan containing serum on osteoblast through ERK1/2 and Wnt/ß-catenin signaling pathway in models with kidney-Yang-deficiency, kidney-Yin-deficiency osteoporosis syndromes].

To clarify the effects of Zuoguiwan containing serum on osteoblast proliferation and alkaline phosphatase(ALP) expression and its effects on the expression of ß-catenin, ERK1, ERK2 mRNA and protein of osteoblast through ERK1/2, Wnt/ß-catenin signaling pathway in models with osteoporosis(OP) kidney-Yang-deficiency, osteoporosis(OP) kidney-Yin-deficiency syndrome. Rat osteoporosis models were established by ovariectomy surgery, and 10 weeks after surgery, hydrocortisone was injected and thyroxine was administered by intragastric administration to establish OP kidney-Yang-deficiency rat model, and OP kidney-Yin-deficiency rat model. Osteoblasts were obtained from 24 h newborn rat skull and were identified by alkaline phosphatase and alizarin red staining. Zuoguiwan containing serum of OP, OP kidney-Yang-deficiency, and OP kidney-Yin-deficiency, as well as the blank serum were used to intervene the osteoblast, and the cells proliferation was detected by MTS. ELISA assay was used to detect ALP expression. RT-PCR assay was used to detect the mRNA expression of ERK1, ERK2, ß-catenin and protein expression levels were detected by Western blot. The results showed that Zuoguiwan containing serum in OP kidney-Yin-deficiency model had stronger effect than OP kidney-Yang-deficiency in promoting osteoblast proliferation, ALP expression, osteoblast ERK1/2, Wnt/ß-catenin signaling pathway related factors ß-catenin, ERK1, ERK2 mRNA and protein expression levels. This was consistent with the TCM theory of "Zuoguiwan nourishes kidney Yin", providing a scientific basis for the clinical and dialectical treatment of osteoporosis. Zuoguiwan could regulate the proliferation and differentiation of bone cells by ERK1/2 and Wnt/ß-catenin signaling pathway, which may be one of the mechanisms of Zuoguiwan for the prevention of osteoporosis.

Intervention effects of Zuoguiwan containing serum on osteoblast through ERK1/2 and Wnt/β-catenin signaling pathway in models with kidney-Yang-deficiency, kidney-Yin-deficiency osteoporosis syndromes / 中国中药杂志

To clarify the effects of Zuoguiwan containing serum on osteoblast proliferation and alkaline phosphatase(ALP) expression and its effects on the expression of β-catenin, ERK1, ERK2 mRNA and protein of osteoblast through ERK1/2, Wnt/β-catenin signaling pathway in models with osteoporosis(OP) kidney-Yang-deficiency, osteoporosis(OP) kidney-Yin-deficiency syndrome. Rat osteoporosis models were established by ovariectomy surgery, and 10 weeks after surgery, hydrocortisone was injected and thyroxine was administered by intragastric administration to establish OP kidney-Yang-deficiency rat model, and OP kidney-Yin-deficiency rat model. Osteoblasts were obtained from 24 h newborn rat skull and were identified by alkaline phosphatase and alizarin red staining. Zuoguiwan containing serum of OP, OP kidney-Yang-deficiency, and OP kidney-Yin-deficiency, as well as the blank serum were used to intervene the osteoblast, and the cells proliferation was detected by MTS. ELISA assay was used to detect ALP expression. RT-PCR assay was used to detect the mRNA expression of ERK1, ERK2, β-catenin and protein expression levels were detected by Western blot. The results showed that Zuoguiwan containing serum in OP kidney-Yin-deficiency model had stronger effect than OP kidney-Yang-deficiency in promoting osteoblast proliferation, ALP expression, osteoblast ERK1/2, Wnt/β-catenin signaling pathway related factors β-catenin, ERK1, ERK2 mRNA and protein expression levels. This was consistent with the TCM theory of "Zuoguiwan nourishes kidney Yin", providing a scientific basis for the clinical and dialectical treatment of osteoporosis. Zuoguiwan could regulate the proliferation and differentiation of bone cells by ERK1/2 and Wnt/β-catenin signaling pathway, which may be one of the mechanisms of Zuoguiwan for the prevention of osteoporosis.

On-line comprehensive two-dimensional HepG2 cell membrane chromatographic analysis system for charactering anti-hepatoma components from rat serum after oral administration of Radix scutellariae: A strategy for rapid screening active compounds in vivo.

Cell membrane chromatography (CMC) is a bioaffinity chromatography technique for characterizing interactions between drugs and membrane receptors and has been widely used to screen active components from complex samples such as herbal medicines (HMs). However, it has never been applied in vivo due to its relatively high limit of detection (LOD) and the matrix interferences. In this study, a novel on-line comprehensive two-dimensional HepG2/CMC/enrich columns/high performance liquid chromatography/time-of-flight mass spectrometry system was developed to rapidly screen potential anti-hepatoma components from drug-containing serum of rats after oral administration of Radix scutellariae. A matrix interference deduction method with a home-written program in MATLAB was developed, which could successfully eliminate the interference of endogenous substances in serum. Baicalein, wogonin, chrysin, oroxylin A, neobaicalein and rivularin from Radix scutellariae extraction were significantly retained in the HepG2/CMC column. Three potential active components, wogonin, oroxylin A and neobaicalein were firstly screened from the drug-containing serum as well. The cell counting kit-8 assay demonstrated that wogonin, oroxylin A and chrysin showed high inhibitory activities in a dose-dependent manner on HepG2 cells at the concentration of 12.5-200 µM (p<0.05) and the IC50 values were 69.83, 16.66 and 51.6 µM, respectively. Wogonin and oroxylin A, which were screened both from Radix scutellariae extraction and the drug-containing serum, could be selected as lead compounds to obtain good anti-hepatoma effects. The proposed comprehensive 2D CMC system and matrix interference elimination strategy have significant advantages for in vivo screening of active components from complex biological samples and could be applied to other biochromatography models.

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

Wistar rats were intragastrically perfused with Chinese medicines used for tonifying the kidney. These included 0.180 g/mL of Herba Epimedii (Epimedium), Semen Cuscutae (Dodder Seed), or Herba Cistanches (Desertliving Cistanche), 0.04 mg/mL monoamine oxidase-B inhibitor selegiline, or distilled 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, followed by incubation in culture solution containing 100 µmol/L H2O2 for 3 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow tometry results showed that all drug-containing serums improved the survival rate of H2O2-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 H2O2-injured MES23.5 cells. Enzyme-linked immunosorbent assay results showed that serum containing Herba Cistanches or Herba Epimedii increased the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in injured MES23.5 cells; serum containing Semen Cuscutae only increased brain-derived neurotrophic factor expression; 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 kidney can protect nerve cells by regulating the expression of apoptosis-related factors and neuro-trophic factors in MES23.5 cells.

Rescue role of Bu-Shen-Yi-Qi Formula for trophoblast apoptosis induced by TNF-?/IFN-? / 中国免疫学杂志

Objective:To investigate the rescue role of Bu-Shen-Yi-Qi Formula on the apoptosis of first trimester trophoblasts induced by TNF-?/IFN-?.Methods:Cell viability was measured by MTT assay,and cell numbers in sub-G1 phase were determined by FCM.The amount of apoptotic cells was evaluated by TUNEL,caspase-3 enzyme activity was examined by caspase-3 active assay kit.Bcl-2 and XIAP protein expression were detected by Western blot.Results:(1)Cell viability decreased,but apoptotic cells increased and the cells in sub-G1 phase accumulated when cultured with TNF-? and IFN-? for a 24 h and the reaction became more serious following a 48 h culture.However,cells cocultured with drug-containg serum showed enhanced cell viability,less apoptotic cells and low enzyme activity.Also,cells in sub-G1 phase decreased and Bcl-2,XIAP protein expression were enhanced when the drug-containg serum were added.This change showed time and dosage dependent.Cell viability cultured with blank serum had no significant changes.Conclusion:Bu-Shen-Yi-Qi Formula rescue trophoblasts apoptosis induced by TNF-? and IFN-?.