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ObjectiveTo investigate the effect of Yiqi Jiedu prescription-containing serum on the proliferation of medullary thymic epithelial cells （mTEC） and regulatory T （Treg） cells in myasthenia gravis （MG） patients with thymus hyperplasia. MethodAccording to serological methods，35 SD rats were adaptively fed for one week and randomized into the low-，medium-， and high-dose Yiqi Jiedu prescription groups，control group， and prednisone group，with seven rats in each group， which were then gavaged with the corresponding drugs for one week for preparing the drug-containing serum. The effect of Yiqi Jiedu prescription-containing serum at different concentrations on the proliferation of mTEC and Treg cells were determined by cell counting kit-8 （CCK-8） assay. Besides， the effect of mTEC and Yiqi Jiedu prescription-containing serum on Treg cell proliferation were observed through co-culture. ResultThymocytes were cultured for a period of time. Their mean positive rate revealed by flow cytometry using mTEC characteristic marker Ulex europaeus agglutinin Ⅰ （UEAI） was 92.54%. Treg cells were sorted by magnetic beads. The purity of Treg cells after repeated magnetic bead sorting was as high as 92%. mTEC and Treg cells showed high positive expression rates，and their cell purity met the requirements of subsequent experiments. When the concentration of Yiqi Jiedu prescription-containing serum was 2.5%-15%，it exhibited an inhibitory effect against mTEC and Treg cells. When the concentration was equal to or greater than 20%，it promoted cell proliferation，which was further enhanced with the extension of action time. The results after 48 h of culture showed that compared with the control group，prednisone and low-dose Yiqi Jiedu prescription had no significant effect on the proliferation of these two kinds of cells，but the medium- and high-dose Yiqi Jiedu prescription remarkably reduced their proliferation inhibition rate （P<0.01）. After co-culture with mTEC， the control group was not significantly different from the prednisone group and the low-dose Yiqi Jiedu prescription-containing serum group in the proliferation of Treg cells，while the medium- and high-dose Yiqi Jiedu prescription-containing serum groups significantly lowered the proliferation inhibition rate （P<0.01）. ConclusionYiqi Jiedu prescription-containing serum affects the proliferation of mTEC and Treg cells in MG patients with thymus hyperplasia. Compared with the solely cultured Treg cells isolated from MG patients，the Treg cells co-cultured with mTEC exhibit enhanced proliferation in MG patients，suggesting that mTEC can regulate the proliferation of Treg cells. This effect becomes more obvious after the intervention with Yiqi Jiedu prescription-containing serum，indicating that intervention effect of Yiqi Jiedu prescription on Treg cells can be produced during its treatment of mTEC， which may be one of the mechanisms of Yiqi Jiedu prescription-containing serum in alleviating MG.
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Objective To investigate the effect of compound Fufangteng mixture-containing serum on the proliferation of bone marrow mesenchymal stem cell (BMSC) and its mechanism. Methods Rat BMSC were isolated, cultured and purified in vitro by direct adherence method. Cell morphology was observed. Surface markers were identified by flow cytometry. The rats were treated with compound Fufangteng mixture at a dose of 3 mL/(kg·d) by gavage for 14 d, and then the drug-containing serum was collected. BMSC were divided into the blank control group, drug-containing serum group, Notch1 small interfering ribonucleic acid (siRNA) group and Notch1 siRNA+drug-containing serum group. The proliferation rate of BMSC was detected and the relative expression levels of Notch1 signaling pathway-associated messenger ribonucleic acid (mRNA) and proteins were measured in each group. Results Microscopic observation showed that the first generation BMSC were seen in the long spindle shape, and grown in the parallel or spiral pattern. The third generation BMSC positively expressed CD90 and CD44, whereas were negative for CD45. Compared with the blank control group, the proliferation rate of BMSC in the drug-containing serum group and Notch1 siRNA+ drug-containing serum group was significantly increased, whereas that of BMSC was significantly decreased in the Notch1 siRNA group (all P < 0.05). Compared with the Notch1 siRNA group, the proliferation rate of BMSC was significantly increased in the Notch1 siRNA+drug-containing serum group (P < 0.05). Compared with the blank control group, the relative expression levels of Hey1 and Delta-like ligand (DLL)1 mRNA and proteins were significantly up-regulated in the drug-containing serum group, whereas those were significantly down-regulated in the Notch1 siRNA group and Notch1 siRNA+drug-containing serum group (all P < 0.05). Compared with the Notch1 siRNA group, the relative expression levels of Hey1 and DLL1 mRNA and proteins were significantly up-regulated in the Notch1 siRNA+drug-containing serum group (all P < 0.05). Conclusions Compound Fufangteng mixture-containing serum may promote the proliferation of rat BMSC, and its mechanism is probably associated with the activation of Notch1 signaling pathway.
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BACKGROUND: The mitochondrial apoptotic pathway is an important pathway in cell apoptosis. Previous studies have found that Bushen Zhuangdu Fang can improve intervertebral disc degeneration by inhibiting the mitochondrial apoptotic pathway in animal experiments. However, its mechanism of action is to be clarified. OBJECTIVE: To observe the effect of serum containing Bushen Zhuangdu Fang on mitochondrial apoptotic pathway key proteins of human nucleus pulposus cells, and to explore the mechanism by which this drug-containing serum improves intervertebral disc degeneration. METHODS: Thirty-seven male Sprague-Dawley rats were randomly divided into blank group, low-dose Chinese medicine group (0.506 g/kg per day), medium-dose Chinese medicine group (1.012 g/kg per day) and high-dose Chinese medicine group (2.024 g/kg per day). After 2 weeks of continuous administration, drug-containing serum was prepared. Human nucleus pulposus cells were randomly divided into normal group, cell model group, low-dose drug-containing serum group, medium-dose drug-containing serum group, and high-dose drug-containing serum group. The cell model group was treated with 200 μmol/L H2O2 for 6 hours, and the normal group received no treatment. The three drug-containing serum groups were treated with corresponding treatments for 48 hours. The pathological changes of nucleus pulposus cells were observed by transmission electron microscopy. Apoptotic rate of nucleus pulposus cells was detected by flow cytometry and mitochondrial membrane potential was detected by flow cytometry. Apaf1, Bcl-2, Bax and Cytc expressions were detected by real-time quantitative PCR and western blot assay. RESULTS AND CONCLUSION: Compared with the normal group, the apoptotic rate of nucleus pulposus cells with obvious apoptotic morphology was significantly increased (P < 0.05), the expression of Apaf1, Cytc, and Bax were significantly increased at mRNA and protein levels (P < 0.05), and the mitochondrial membrane potential and expression of Bcl-2 mRNA and protein were significantly decreased in the cell model group (P < 0.05). After treatment with drug-containing serum, the apoptotic rate of nucleus pulposus cells decreased significantly (P < 0.05), the expression of Apaf1, Cytc, Bax and their proteins decreased significantly (P < 0.05), and the mitochondrial membrane potential, Bcl-2 and their proteins increased significantly (P < 0.05). Therefore, the serum containing Bushen Zhuangdu Fang can effectively inhibit apoptosis of nucleus pulposus cells in a dose-dependent manner. The drug-containing serum may alleviate intervertebral disc degeneration by reducing the expression of Apaf1, Cytc and Bax and increasing the expression of Bcl-2 at protein and gene levels, and inhibiting the mitochondrial apoptotic pathway.
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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-1β, 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-1β, 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.
Subject(s)
Animals , Humans , Rats , AMP-Activated Protein Kinases , Arthritis, Rheumatoid , Capsules , Forkhead Box Protein O3 , Leukocytes, Mononuclear , Oxidative Stress , Rats, Sprague-Dawley , Scutellaria baicalensisABSTRACT
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.
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Objective::To investigate the effect of serum containing Yanghetang (YHT) on the apoptosis of MCF-7 cells in breast cancer based on the mitogen-activated protein kinase (p38)/signal transduction and transcriptional activator 3 (STAT3) signal pathway. Method::YHT liquid with crude drug 1 g·mL-1 was prepared. Female SD rats were randomly divided into control group (distilled water), and high, medium and low-dose YHT groups (24, 12, 6 g·kg-1). YHT-medicated serum was prepared, and 10%medicated serum was used to intervene MCF-7 cells. Cell proliferation and cytotoxicity assay (CCK-8) was used to detect the effect of serum containing YHT on MCF-7 cell proliferation, apoptosis of MCF-7 cells was detected by flow cytometry protein expressions of p38 and STAT3 were detected by Western blot, Quantitative Real-time PCR (Real-time PCR) was used to detect the expressions of B-cell lymphoma/leukemia-xl(Bcl-xl) and Survivin mRNA. Result::CCK-8 assay showed that YHT serum inhibited the proliferation of MCF-7 cells in a time and dose-dependent manner compared with the blank group. The inhibitory effect was most obvious in the high-dose group, with the inhibition rates of 38%, 45%and 54%at different time points (P<0.01). Flow cytometry showed that, compared with the blank group, the apoptosis rate in the medium and high-dose groups increased significantly in a dose-dependent manner, with the apoptosis rates at 11.6%and 16.5%respectively (P<0.05, P<0.01). Western blot analysis showed that, compared with the blank group, the expressions of p38 and STAT3 protein was decreased in high, medium-dose YHT groups (P<0.01) in a dose-dependent manner. Compared with the blank group, the expressions of Bcl-xl and Survivin mRNA were decreased in high, medium-dose YHT groups (P<0.05, P<0.01) in a dose-dependent manner. Conclusion::YHT serum can promote the apoptosis of MCF-7 cells in breast cancer, which may be related to the p38/ STAT3 signaling pathway.
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OBJECTIVE: To study the effect of serum containing Jieduquyuziyin-prescription (JP) on signal pathway of interleukin-1 receptor-associated kinase 1 (IRAK1) in mononuclear macrophages of mice stimulated by lipopolysaccharide (LPS), and to explore the effect of Jieduquyuziyin-prescription on IRAK1 and NF-κB inflammatory signaling pathways, which providing a good theoretical support for its anti-inflammatory clinical medication. METHODS: In this study, mice mononuclear macrophages cultured in vitro were randomly divided into blank group, LPS group, JP serum group, blank serum group, LPS plus JP serum group, LPS plus blank serum group, IRAK1 inhibitor group, inhibitor plus LPS group, inhibitor plus JP serum group and inhibitor plus blank serum group. After intervention for 24 h, the activity of JP on macrophages was tested by CCK8 method. The IRAK1 expression in macrophages was tested by immunofluorescence chemical staining. The content of TNF-α in the supernatant of the cells was detected by ELISA. The mRNA expressions of IRAK1, NF-κB, TNF-α and IL-6 were detected by RT-PCR. The protein expressions of IRAK1, p-IRAK1 and NF-κB were detected by Western-blot. The LC-MS was used to detect the active ingredients in JP serum. RESULTS: The results show that 2.5% of JP serum is the optimal concentration. Jieduquyuziyin-prescription could down-regulate the expression of TNF-α and IL-6 and inhibit the expression of IRAK1 and activate NF-κB(P<0.05). Paeoniflorin and ferulic acid were detected in the JP serum. CONCLUSION: Jieduquyuziyin-prescription can inhibit the expression of IRAK1 and NF-κB in mouse monocyte-macrophage cells after LPS stimulation and provide a good theoretical support for its anti-inflammatory clinical medication.
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Objective: To investigate the effect of Clerodendrum bungei-containing serum on liver cancer MHCC97-H cells and its possible mechanism from the perspective of phosphatidylinositol 3-kinase(PI3K)/protein kinase (Akt) signaling pathway. Method: The medicinal serum of 15% C. bungei was used to treat MHCC97-H cells. The effect of serum containing C. bungei on cell proliferation was observed by cell counting kit-8(CCK-8) method, in order to select the best time and concentration. The apoptosis was detected by Annexin V-FITC/PI double staining method. Western blot was used to detect the posphatase and tensin homologous gene deleted from chromosome 10 in key proteins (PTEN), phosphoprotein kinase B (p-Akt) and phosphatidylinositol 3-kinase (PI3K)-related protein expression of PI3K/Akt signaling pathway. Real-time PCR was used to detect C. bungei-containing serum on cells for 72 h after activation of nuclear factor-activated B cell kappa light chain(NF-κB) and tumor necrosis factor-α (TNF-α) mRNA expression. Result: The results of CCK-8 showed an inhibitory effect of the C. bungei-containing serum on the proliferation of tumor cells in a dose and time-dependent manner. Among them, the high-dose group had the most obvious inhibitory effect, and the maximum inhibition rates at 24, 48,72 h were 28%, 32%, and 43%, respectively. The results of flow cytometry showed that with the increase of drug-containing serum concentration, the cell growth was observed. The inhibition rate of cells was increased to different degrees, and the inhibition effect was significantly increased in the 72 h intervention group (PC. bungei-containing serum group was 19.48% and 19.72%, compared with the blank group. The difference was significant (PC. bungei-containing serum (PPC. bungei-containing serum could down-regulate the expression of NF-κB and up-regulate the expression of TNF-α mRNA (PConclusion: The medicinal serum of C. bungei can effectively inhibit the proliferation of MHCC97-H hepatoma cells and promote its apoptosis, which may be related to the PI3K/Akt signaling pathway and its key factors.
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OBJECTIVE： To study improvement effects of different proportions of total glucosides of ginseng （TGG）， total glucosides of moutan cortex （TGM） and paeonol containing serum on the injury of human umbilical vein endothelial cells （HUVEC） injury induced by hydrogen peroxide （H2O2）， screen the optimal proportion and investigate its mechanism. METHODS： The rats were randomly divided into blank group （distilled water）， TGG group （TGG， 2.025 g/kg）， TGM group （TGM， 4.05 g/kg） and paeonol group （paeonol， 1.08 g/kg）， with 12 rats in each group. They were given relevant medicine twice a day for consecutive 7 days. 1 h after last medication， the blood samples were collected via abdominal aorta to prepare drug containing serum. Using survival rate of HUVEC as evaluation indexes， different proportions of TGG， TGM and paeonol containing serum as factors， L9（34） orthogonal test was designed to optimize the optimal proportion of 3 kinds of drug containing serum. HUVEC were divided into blank group， model group， TGG group， TGM group， paeonol group and optimal proportion group. Except that blank group were treated with relevant medium， other group were treated with 1.2 mmol/L H2O2 to induce HUVEC injury， and then TGG group （volume fraction of drug containing serum was 0.000 5％）， TGM group （volume fraction of drug containing serum was 0.000 5％）， paeonol group （volume fraction of drug containing serum was 1％） and optimal proportion group were intervened with drug containing serum. The levels of LDH， NO and ET-1 in cells were detected by microplate method and ELISA. RESULTS： The optimal proportion of drug containing serum were TGG 0.000 5％， TGM 0.000 5％ and paeonol 1％. Compared with blank group， the levels of LDH and ET-1 were higher in model group （P＜0.01）， while NO level was lower （P＜0.05）. Compared with model group， the levels of NO were higher in TGG group， TGM group and optimal proportion group （P＜0.01）， while the levels of LDH and ET-1 were lower （P＜0.05 or P＜0.01）. Compared with TGG group， TGM group and paeonol group， the level of LDH was lower in optimal proportion group （P＜0.05 or P＜0.01）， while the level of NO was higher （P＜0.05 or P＜0.01）. CONCLUSIONS： TGG and TGM combined with paeonol can significantly improve HUVEC injury induced by H2O2， and the mechanism of which may be associated with the decrease of LDH and ET-1 and the increase of NO.
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Objective:To investigate the effect of endothelin-1 (ET-1) on the expression of phosphorylated myosin light chain Ⅱ(p-MLCⅡ)and myosin light chain Ⅱ(MLCⅡ)protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San(DSS)drug-containing serum. Method:After HSC-T6 cells were seeded, DMEM and blank rat serum with final concentrations of 2.5%, 5%, 10%, 15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium(MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group (5%, 10%, 15%) and DSS drug-containing serum group (5%, 10%, 15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), DSS drug-containing serum low (5%), medium (10%) and high dose (15%) groups. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), model group (10%), DSS drug-containing serum low (5%), medium (10%), high dose (15%) groups and Y-27632 inhibitor group (100 μmol·L-1). Except the blank serum control group, the other groups all received 10 nmol·L-1 ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡ and MLCⅡ in HSC-T6 cells induced by ET-1. Result:Serum concentrations of 5%, 10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group, the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state (PPPPPConclusion:DSS drug-containing serum may down-regulate the expression of p-MLCⅡ and MLCⅡ by down-regulating the content of ET-1 and inhibiting the autocrine of ET-1.
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Objective To study the effects of Jiedu Quyu Ziyin Prescription (JQZP)-treated freeze dried powder and drug-containing serum on the inflammatory signal pathway of monocyte-macrophage induced by LPS (lipopolysaccharide) in mice. Methods Monocyte-macrophage cells were cultured in vitro and randomly divided into blank group, LPS stimulation group, drug-containing serum group, freeze dried powder group, LPS + drug-containing serum group, and LPS + freeze dried powder group. After 24 h intervention, the optimal concentrations of drug-containing serum and freeze dried powder were screened by CCK8 method and the cell viability was measured respectively. The content of tumor necrosis factor (TNF-α) in cell serum was measured by ELISA. Real-time PCR was employed to test the expression of TNF-α mRNA and nuclear transcription factor kappa-light-chain-enhancer of activated B cells (NF-κB). Western-blot was used to detect the expression of NF-κB protein. The LC-MS was used to detect the active ingredients in the drug-containing serum. Results Compared with the blank group, the expression of TNF-α level, NF-κB, TNF-α mRNA and NF-κB protein in LPS stimulation group were significantly increased (P < 0.05). Compared with the LPS stimulation group, the TNF-α level, NF-κB, TNF-α mRNA and the expression of NF-κB protein in the LPS plus serum group were significantly lower than those in the LPS plus freeze-dried powder group (P < 0.05). Paeoniflorin and ferulic acid were detected in the drug-containing serum. Conclusion JQZP freeze-dried powder and drug-containing serum all have the effect of inhibiting the inflammatory signaling pathway.
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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 (<0.05). The serum containing Xihuang pill increased TP53 and Bax in MDA-MB-435 cells (<0.05), and the ratio of Bcl-2/Bax was decreased (<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 (<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.
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This paper was aimed to study the protective effects and related mechanisms of Zhen-Gan Xi-Feng (ZGXF) decoction containing serum on 6-OHDA-induced oxidative stress in PC12 cells.The ZGXF decoction containing rat serum with low-,medium-,and high-dose (8,16,or 32 g.kg-1) or blank serum was used to preprocess PC12 cells for 1 h,and cultured together with 100 μM 6-OHDA for 24 h.And then,cells were collected.The fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) and fluorescence microplate reader were used to detect the level of reactive oxygen species (ROS).Real-time quantitative PCR was used to analyze the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2),Nfe2l2,heme oxygenase-1 (HO-1),glutamate-cysteine ligase catalytic subunit (GCLc),and GCL modulatory subunit (GCLm).The luciferase report gene system was used to detect the antioxidant response element (ARE) activation.The results showed that ZGXF decoction-containing serum inhibited the 6-OHDA-induced oxidative stress,upregulated the Nfe2l2,HO-1 and GCLc mRNA expressions in cells processed with 6-OHDA.However,it has no significant effect on GCLm mRNA expression.It was concluded that ZGXF decoction-containing serum had protective effects on 6-OHDA-induced oxidative stress in PC 12 cells.Its mechanism may be correlated with the upregulation on Nfe2l2 mRNA expression,which activated ARE and further induced its downstream gene of phase Ⅱ detoxifying enzyme,as well as the HO-1 and GCLc mRNA expressions of antioxidant enzyme gene.
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Objective To optimize the effective methods of isolation,purification,culture in vitro and identification of SD rat ovarian granulosa cells,to research the effects of Oviductus Ranae on the proliferation of rat ovarian granulosa cells by CCK-8,and to contrastively analyze the best optimal action concentration and time of serum contsining Oviductus Ranae on granulosa cells to lay the foundation for further in vitro experiment.Methods Nonage SD rats aged 25 d were selected and intraperitoneally injected by pregnant mare serum,then killed after 48 h.Ovarian granulosa cells were collected and cultured in the DMEM-F12 culture solution.The hematoxylin & eosin(HE) staining and immunofluorescence technique were used to identify the ovarian granulosa cells.Twen ty-five SD rats were randomly divided into the normal control group,positive medicine control group,and low,middle and high do ses Oviductus Ranae groups.Blood was collected and serum was separated after 7 d mediaction gavage.The volume percent of 10 ％,20％,40％,80％ serum in each group was added into the in vitro medium system of ovarian granulosa cells culture.Then the cell proliferation situation at 24,48,72 h in each group was measured by CCK-8.Results Oviductus Ranae significantly increased the proliferation ability of granulosa cells in a certain dose-dependent relation.With the increase of Oviductus Ranae concentration con centration,its.proliferation ability was gradually increased,after 48 h action,which in the Oviducthus Ranae-Comtaining serum group with the volume fraction of 20％ was most significant (P＜0.05).Conclusion Establishing in vitro cultural method of rat o varian granulosa cells is conductive to further research the action and mechanism of Oviductus Ranae on ovary.
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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.
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Objective To study the effects of drug-containing serum of Ficus Hirta on oxidative damage of spleen lymphocyte due to aging in aged mice; To discuss its mechanism of action.Methods Forty aged mice were randomly divided into control group and high-, medium- and low-dose Ficus Hirta groups. Control group was given 0.9% sodium chloride solution for gavage, while high-, medium- and low-dose Ficus Hirta groups were given 6.6, 4.4, and 2.2 g/kg aqueous extract of Ficus Hirta for gavage. The spleen index was observed for optimum dose in aged mice. The optimum time and dilution of drug-containing serum of Ficus Hirta were confirmed by MTT method in lymphocyte proliferation test. The positive rate of senescent cells, the activity of T-SOD and the contents of MDA and ROS were determined in cellular antioxidant experiment after treated by optimal drug-containing serum for 48 h. Results Compared with the control group, the spleen index was significantly improved in high-, medium- and low-dose Ficus Hirta groups (P<0.05,P<0.01). 20% drug-containing serum of Ficus Hirta cultivated for 48 h had the best effects on lymphocyte proliferation in aged mice. 20% drug-containing serum of Ficus Hirta could significantly decrease the positive rate of senescent cells (P<0.01), improve T-SOD activity and decrease the contents of MDA and ROS (P<0.05,P<0.01).Conclusion The drug-containing serum of Ficus Hirta can improve the proliferative activity of spleen lymphocyte in aged mice and the mechanism of action may be involved in decreasing the positive rate of senescent cells and increasing antioxidant ability of lymphocyte.
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Objective To observe the anti-cancer effect in vitro of Aitongxiao Granules containing serum on SMMC-7721 and Bel-7404 liver cancer cells, thus revealing the anti-cancer mechanism of the treatment in adjusting proliferation and apoptosis. Methods Male Sprague-Dawley rats were administered by gastrogavage Aitongxiao decoction of 86.4, 43.2, 21.6 g/kg twice a day for 1 week. Using serum pharmacologic method, the proliferation of SMMC-7721 and Bel-7404 liver cancer cells were determined by MTT chromatometry after co-cultured with medicated serum containing different concentration of Aitongxiao decoction. The apoptosis of SMMC-7721 and Bel-7404 liver cancer cells were detected by flow cytometry. Results The research in vitro showed that Aitongxiao Granules containing serum low, medium and high dose groups had varying degrees of inhibition on hepatoma cells, and this inhibition showed a concentration dependence within a certain range. Aitongxiao Granules containing serum could induce apoptosis of human hepatoma SMMC-7721 cells and Bel-7404 cells. Conclusion Aitongxiao Granules showed effect of inhibiting proliferation and inducing apoptosis on SMMC-7721 and Bel-7404 cells in vitro.
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Objective To investigate the effect of the serums containing Xuefuzhuyu capsules and Xuesetong capsules on the proliferation of vascular smooth muscle cells (VSMC) induced by 1ysophosphatidie acid (LPA). Methods Rat VSMCs were obtainded by tissue-piece inoculation and divided into seven groups:blank serum group, Xuefuzhuyu (Xuesetong) capsules-containing serum group, LPA+ Xuefuzhuyu (Xuesetong, captopril) containing serum group and LPA+blank serum group. Effects of different groups on the proliferation of VSMCs was evaluated by MTT colorimetry. Analysis of cell cycle and DNA content were performed by flow cytometry. Results The optical density (OD) and the S phase fraction (SPF) of LPA+blank serum group and Xuefuzhuyu capsules-containing serum group were more increased than the blank serum group (P 0.05, P 0.05). Conclusion Xuefuzhuyu capsules and LPA promoted the proliferation of VSMCs in vitro. Xuefuzhuyu and Xuesetong capsules can inhibit the proliferation induced by LPA, and effect of Xuesetong capsules was greater. Part of the mechanism may be associated with the inhibition of DNA synthesis and preventing the transition from G0/G1 to S phase of VSMCs.
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Objective To establish the determination method of Tubeimoside Ⅰ in Jiazhongxiao preparation and serum of rat. Methods RP-HPLC/UV method was used as follows:Diamonsil C18 column (250 mm?4.6 mm, 5 ?m) and Easyguard column, methanol-water (65∶35) as mobile phase with flow rate of 1.0 mL/min, UV detection at 214 nm. Results The mean recovery rates of Tubeimoside Ⅰ in preparation and serum of rat were (103.49?3.49)% and (104.70?4.69)% respectively. The linearity of Tubeimoside Ⅰ was shown in the range of 1.18～88.5 ?g/mL. Tubeimoside Ⅰ in preparation and serum of rat were 995.3 ?g/g and 3.19 ?g/mL. Conclusion The method was simple, reliable and rapid. It’s quite suitable to be used in the analysis of Tubeimoside Ⅰ in Jiazhongxiao preparation and serum of rat.
ABSTRACT
Objective To investigate the anticancer mechanism of Herba Hedyotis Diffusae on PG cell by observing the action of Herba Hedyotis Diffusae in inducing apoptosis of PG cells. Method After treating PG cells with different doses of drug containing serum of Herba Hedyotis Diffusae, FCM was used to detect the apoptosis of PG cell. Result After treating PG cell with Herba Hedyotis Diffusae, typical apoptosis characteristics could be seen and apoptosis rates were increased with the relation of dosage-effect, which were 6.86%, 9.77% and 11.69%, respectively. Conclusion Inducing the apoptosis of tumor cells maybe part of the anti-tumor mechanism of Herba Hedyotis Diffusae.