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Objective To investigate the effects of modified Yupingfeng Powder medicated serum on the inflammatory response of rat lung epithelial type Ⅱ cells RLE-6TN;To explore the mechanism based on nuclear factor(NF)-κB signaling pathway.Methods The inflammatory reaction of RLE-6TN cells was induced by lipopolysaccharide,and the cells were divided into control group,model group,dexamethasone medicated serum group,and 5%,10%and 20%TCM serum group,and the cell model was intervened with different serum.After 24 hours,the content of lactate dehydrogenase(LDH)in cell supernatant was detected by kit,and the content of reactive oxygen species(ROS)in cells were detected by fluorescence staining,Hoechst/PI double staining was used to observe cell apoptosis,and ELISA was used to detect tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-1β contents in cell supernatant,RT-qPCR was used to detect Toll like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),and NF-κB inhibitory protein α(IκBα)mRNA expression,Western blot was used to detect protein expressions of TLR4,MyD88,NF-κBp65 and IκBα.Results Compared with the control group,the contents of LDH,TNF-α,IL-6,IL-1β in RLE-6TN cell supernatant and ROS content in cells of model group significantly increased(P<0.01),the cell apoptosis rate significantly increased(P<0.01),the expressions of TLR4 and MyD88 mRNA and protein significantly increased(P<0.01),the expression of IκBα mRNA significantly decreased(P<0.01),and the ratio of p-NF-κBp65/NF-κBp65 and p-IκBα/IκBα significantly increased(P<0.01).Compared with the model group,the content of LDH,TNF-α,IL-6,IL-1 in RLE-6TN cell supernatant and ROS content in cells significant decreased in the 20%TCM serum group and dexamethasone medicated serum group(P<0.01),the cell apoptosis rate significantly decreased(P<0.01),the expressions of TLR4 and MyD88 mRNA and protein significantly decreased(P<0.05,P<0.01),the expression of IκBα mRNA significantly increased(P<0.01),and the ratio of p-NF-κBp65/NF-κBp65 and p-IκBα/IκBα significantly decreased(P<0.05,P<0.01).Conclusion Modified Yupingfeng Powder medicated serum can reduce RLE-6TN cells inflammatory reaction,and its mechanism may be related to the regulation of NF-κB signaling pathway.
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Aim To research the neuroprotective effect of Haikun Shenxi (HKSX) medicated serum on N2a/ App695 cells and the underlying mechanism. Methods HKSX medicated serum was prepared and carbohydrate components in it was analyzed using high performance thin layer chromatography (HPTLC) . N2a/ App695 cells were intervened with HKSX medicated serum, the cytotoxicity of HKSX medicated serum was measured by MTT; AP[_
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Qualitative analysis of the ingredients absorbed into blood and their metabolites of Xihuang pill (XHP) were conducted using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) technology. Network pharmacology was used to explore the potential anticancer mechanisms of the ingredients against glioma, and their specific mechanisms were validated through molecular docking and experimental verification. SD rats were intragastrically administered with XHP, and rat serum samples were collected. Ingredients absorbed into blood and their metabolites were identified based on the retention time of chromatographic peaks, accurate molecular mass, characteristic fragment ions, and comparisons with reference substances and literature data. PharmMapper and SwissTarget Prediction databases were used to obtain the targets of the XHP-medicated serum, while GeneCards, OMIM, PharmGKB, TTD, and DrugBank databases were used to obtain glioma disease targets. The "component-target" network relationship diagram was constructed using Cytoscape 3.9.1 software. The protein-protein interaction (PPI) network diagram was constructed using the STRING database, and the targets were analyzed using GO and KEGG analyses. Molecular docking was used to verify the binding ability of core targets with their corresponding compounds in XHP-medicated serum. The potential mechanism of the anti-glioma effect of 11-keto-β-boswellic acid (KBA), a representative component of XHP-medicated serum, was verified using CCK-8 and Western blot assays. A total of 40 compounds were identified in the XHP-medicated serum, including 28 prototype components and 12 metabolites. The network pharmacology results showed that elemonic acid, 3-acetyl-β-boswellic acid, KBA, α-boswellic acid, and other 5 compounds might be the active ingredients of XHP-medicated serum in the treatment of glioma. Glutathione reductase (GSR), glucose-6-phosphate dehydrogenase (G6PD), ATP-citrate lyase (ACLY), aldo-keto reductase family 1 member B1 (AKR1B1) and glutaredoxin (GLRX) were identified as key targets, involving pathways such as glutathione metabolism and the pentose phosphate pathway. Further cell experiments showed that KBA significantly inhibited the proliferation of T98G cells with an IC50 of 30.96 μmol·L-1, and KBA (30 μmol·L-1) significantly downregulated the protein expression levels of GSR in T98G cells. In summary, XHP-medicated serum may exert its anti-glioma effect by regulating GSR and G6PD-targeted pathways involved in glutathione metabolism. These results provide valuable evidence for further investigating the mechanism of XHP in treating glioma. The animal welfare and experimental procedures were approved by the Ethical Committee of Laboratory Animals at Nanjing University of Chinese Medicine (approval No. ACU221001).
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ObjectiveTo observe the effects of Jianpi Bushen Huoxue prescription （JPBSHX） on rat brain microvascular endothelial cells （RBMECs） based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway， aiming to provide a theoretical basis for the treatment of ischemic stroke. MethodTwelve 8-week-old male SPF-grade SD rats were selected. Eight of them were randomly chosen and given 3.25 g·mL-1 JPBSHX solution by gavage at a dose of 10 mL·kg-1 for 5 consecutive days to prepare the medicated serum， which was then preserved for later use. The remaining four rats were given the same volume of normal saline. Follow-up operations were the same as those of the above eight rats. Normal rat serum was collected and stored for later use. RBMECs were revived， cultured， passaged， and randomly divided into five groups： normal group （20% normal rat serum+80% high glucose DMEM）， model group （hypoxia-reoxygenation injury） （20% normal rat serum+80% glucose-free DMEM）， medicated serum group （20% JPBSHX-medicated serum+80% glucose-free DMEM）， medicated serum+HIF-1α inhibitor group （20% JPBSHX-medicated serum+HIF-1α inhibitor 1 mg +80% glucose-free DMEM）， and medicated serum+VEGF inhibitor group （20% JPBSHX-medicated serum +VEGF inhibitor 1 mg+80% glucose-free DMEM）. The relative protein expression levels of Claudin-1 and Claudin-5 in RBMECs， the expression levels of HIF-1α and VEGF in RBMEC culture supernatants， the repair ability of RBMECs， and the number of nodes， microvessels， and their lengths after 72 h of culture were observed in each group. ResultAfter 24 h of reoxygenation， the scratch healing rate in the model group was significantly lower than in the normal group （P<0.01）. Compared with the result in the model group， the scratch healing rates significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group （P<0.05）. However， the healing rates in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group were significantly lower than that in the medicated serum group （P<0.05）. The number of nodes， microvessels， and total length of microvessels in the model group were significantly lower than those in the normal group （P<0.01）. These indicators significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group compared with those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group compared with those in medicated serum group （P<0.05）. The relative expression levels of Claudin-1 and Claudin-5 proteins were significantly lower in the model group than in the normal group （P<0.01）. These levels were significantly higher in medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. The expression levels of HIF-1α and VEGF in the RBMEC culture supernatants were significantly lower in the model group than those in the normal group （P<0.01）. These levels were significantly higher in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. ConclusionJPBSHX can promote the proliferation， migration， and angiogenesis， such as tubule formation， of RBMECs damaged by hypoxia-reoxygenation injury， and this effect may be achieved through the regulation of the HIF-1α/VEGF signaling pathway.
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Our studies were aimed to explore the effect and mechanism of the inhibition of the formation of vasculogenic mimicry (VM) in human glioblastoma cells by Xihuang pill (XHP) medicated serum through regulating the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. The medicated serum of XHP was prepared by gavage for 7 days to male SD rats (approval number of animal experiment ethics: 202105A051). The hypoxia model of U251 cells was established using 200 μmol·L-1 of CoCl2. After treatment with XHP-medicated serum, cell viability and proliferation of U251 cells were detected by CCK-8 and cell cloning experiment. Cell apoptosis and cell cycle of U251 cells were determined by flow cytometry. Cell migration and invasion were evaluated by wound healing and Transwell invasion assay. The formation of VM was assessed by three-dimensional cell culture of U251 cells. The protein expression levels of HIF-1α, VEGFA, VEGFR2, phosphorylated-VEGFR2 (p-VEGFR2), vascular endothelial-cadherin (VE-cadherin), Eph receptor tyrosine kinases A2 (EphA2), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 14 (MMP14) and laminin γ2 in U251 cells were detected by Western blot. The results showed that 10% XHP-medicated serum had little effect on the cell viability, proliferation, apoptosis and cell cycle of U251 cells under hypoxia. Compared with the model group, 10% XHP-medicated serum at 1.0, 1.5 and 2.0 h significantly decreased the migration rate (P < 0.01) and the number of invading U251 cells (P < 0.01). 10% XHP-medicated serum at 2.0 h significantly suppressed the formation of VM tubular structures in U251 cells under the condition of hypoxia (P < 0.01). Western blot experiment showed that 10% XHP-medicated serum significantly down-regulated the expression of HIF-1α, VEGFA, phospho-VEGFR2, VE-cadherin, EphA2 and MMP14 proteins (P < 0.05). In conclusion, XHP could inhibit the formation of VM in human glioblastoma U251 cells to suppress the angiogenesis by down-regulating the HIF-1α/VEGFA/VEGFR2 signaling pathway.
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Aim To explore the protective effect of Zishen Huoxue Prescription on OGD/R-induced primary hippocampal neuron damage in rats and the possible mechanism. Methods After the isolated primary hippocampal neurons were identified by immunofluorescence, OGD/R induced neuronal damage, and the changes of autophagic flux at different re-oxygenation time were observed by confocal laser scanning microscopy. After OGD/R-induced primary hippocampal neurons were intervened with serum containing Zishen Huoxue Prescription, cell viability was detected by CCK-8, cell apoptosis was detected by flow cytometry, autophagosomes were detected by transmission electron microscopy, and autophagy-related protein expressions were detected by Western blot. Results 10% Zishen Huoxue Prescription-containing serum could significantly improve cell viability and reduce the proportion of cell apoptosis, increase the number of autophagosomes in neurons, and up-regulate the expression of autophagy-related protein PINK1, Parkin, and pATG16L1. Conclusions Zishen Huoxue Prescription can effectively resist OGD/R-induced apoptosis of primary hippocampal neurons in rats, and its effect may be related to the regulation of PINK1-Parkin pathway to promote mitophagy.
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ObjectiveTo observe the effects of Wumeiwan-medicated serum on the proliferation， invasion， migration， and apoptosis of human pancreatic cancer SW1990 cells and explore the underlying mechanism. MethodThe Wumeiwan-medicated serum was prepared and the pancreatic cancer SW1990 cell line was cultured in vitro. The optimal time of Wumeiwan-medicated serum was selected for subsequent experiments by cell counting kit-8（CCK-8）. SW1990 cells were divided into a control group and low- （2%）， medium- （4%）， and high-dose （8%） Wumeiwan-medicated serum groups. The colony-forming， migration， and invasion abilities were detected by clonogenic assay， wound healing assay， and transwell migration assay. Flow cytometry was used to detect the effect of Wumeiwan-medicated serum on the apoptosis of pancreatic cancer SW1900 cells. Western blot was used to detect the expression levels of apoptosis-related proteins， such as B-cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax）， cytochrome C （Cyt C）， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， cleaved cysteinyl aspartate-specific protease-9 （cleaved Caspase-9）， as well as phosphatidylinositol 3-kinase（PI3K）， phosphorylated PI3K（p-PI3K）， protein kinase B （Akt）， and phosphorylated Akt（p-Akt）in PI3K/Akt pathway in SW1990 cells. ResultCompared with blank group， Wumeiwan groups showed decreased absorbance （A） 72 h after drug intervention （P<0.01）. Compared with the low-dose group， the medium- and high-dose groups showed decreased A （P<0.01）. Compared with the medium-dose group， the high-dose group showed decreased A （P<0.01）. It indicates that Wumeiwan can inhibit SW1990 cell proliferation in a dose-dependent manner after 72 h， and the optimal action time is 72 h. Compared with the blank group， the Wumeiwan groups showed weakened invasion of SW1990 cells （P<0.01）， reduced colony-forming and migration abilities （P<0.05， P<0.01） in a dose-dependent manner， and increased total apoptosis rates （P<0.01）. The inducing effect of Wumeiwan on apoptosis increased with the increase in dosage. Compared with the blank group， the Wumeiwan groups showed decreased protein expression of Bcl-2 （P<0.01）， increased protein expression of cleaved Caspase-3， cleaved Caspase-9， Cyt C， and Bax （P<0.05， P<0.01） in a certain dose-effect relationship， reduced protein expression of p-PI3K and p-Akt （P<0.05， P<0.01） with the increase in dosage， and declining p-PI3K/PI3K and p-Akt/Akt （P<0.05， P<0.01） with the increase in dosage. ConclusionWumeiwan-medicated serum can significantly inhibit the malignant biological behaviors of pancreatic cancer SW1990 cells and induce apoptosis. The mechanism may be related to the inhibition of the PI3K/Akt signaling pathway and down-regulation of protein phosphorylation level in the PI3K/Akt signaling pathway.
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ObjectiveTo investigate the effect of Gegen Qinliantang （GGQLT）-medicated serum on free fatty acid （FFA）-induced nonalcoholic steatohepatitis （NASH） in vitro model of human hepatoma cells HepG2. MethodNASH model of HepG2 cells was established in vitro， and the cells were intervened with different volume fractions of GGQLT-medicated serum and resveratrol. Intracellular lipid deposition in each group was detected by oil red O staining， the level of reactive oxygen species （ROS） in each group were detected by flow cytometry， the levels of glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， triglyceride （TG） and malondialdehyde （MDA） in each group were detected by kits. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of nuclear transcription factor （NF）E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， Kelch-like epichlorohydrin-associated protein-1 （Keap1）， NF-κB， thioredoxin interacting protein （TXNIP）， interleukin-1β （IL-1β） in HepG2 cells of each group. The protein expression of Nrf2， TXNIP in cells of each group was detected by Western blot. ResultFFA induced large accumulation of intracellular lipids. Compared with the normal group， the activities of GSH-Px and SOD were significantly decreased （P<0.01） and the contents of TG， ROS and MDA were significantly increased （P<0.05， P<0.01） in the model group. Compared with the model group， all GGQLT groups and resveratrol group could elevate intracellular SOD activity to different degrees （P<0.05， P<0.01） and significantly reduce the levels of intracellular ROS and MDA （P<0.05， P<0.01）， GGQLD high- and medium-dose groups and resveratrol group significantly elevated GSH-Px activity （P<0.01）， GGQLD medium- and low-dose groups and resveratrol group significantly decreased TG content （P<0.05， P<0.01）. Compared with the model group， GGQLT high- and medium-dose groups and resveratrol group could significantly upregulate the mRNA expression levels of Nrf2， HO-1 and NQO1 （P<0.01）， all GGQLT groups and resveratrol group could significantly downregulate the TXNIP protein expression level， as well as significantly downregulate the mRNA expression levels of Keap1， NF-κB （P<0.05， P<0.01）. Nrf2-siRNA transfection of cells revealed that Nrf2 expression was significantly downregulated （P<0.01） in the Nrf2-siRNA group of cells by comparing with NC-siRNA group at the corresponding dose of drugs， and the inhibitory effects of GGQLT and resveratrol on TXNIP， IL-1β were attenuated. ConclusionFFA induces the production of ROS and inflammatory factors in HepG2 cells， and GGQLT can improve the anti-inflammatory and antioxidant capacities of cells， and its mechanism may be related to the regulation of Nrf2/TXNIP signaling pathway， so as to improve NASH.
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Objective:To investigate the protective effect and molecular mechanism of Angelicae Sinensis Radix-Chuanxiong Rhizoma medicated serum （ASRCRS） against oxidative damage of PC12 cells induced by H<sub>2</sub>O<sub>2</sub>. Method:Oxidative damage of PC12 cells was induced by H<sub>2</sub>O<sub>2</sub><italic> in vitro</italic>， and intervention was performed in the low-， medium-， and high-dose ASRCRS groups with a final volume fraction of 15%. The cell viability was determined by methyl thiazolyl tetrazolium （MTT） assay. Cell morphology was observed by an inverted fluorescence microscope. The content of lactate dehydrogenase （LDH） and malondialdehyde （MDA）， the activity of superoxide dismutase （SOD）， and the distribution of reactive oxygen species （ROS） in the cell supernatant were detected by the kits. Cell apoptosis was detected by Annexin V-FITC/PI double staining. The protein expression levels of nuclear factor E<sub>2</sub>-related factor 2 （Nrf2）， Kelch-like epichlorohydrin associated protein-1 （Keap1）， heme oxygenase-1 （HO-1）， and SOD1 were detected by Western blot. Result:Oxidative damage was induced by 300 μmol·L<sup>-1</sup> H<sub>2</sub>O<sub>2</sub> for 24 hours. Compared with the normal group， the model group showed abnormal cell morphology， reduced cell viability （<italic>P</italic><0.01）， increased LDH and MDA （<italic>P</italic><0.01）， blunted SOD activity， elevated intracellular distribution of ROS， down-regulated protein expression of Nrf2， HO-1， and SOD1 （<italic>P</italic><0.05， <italic>P</italic><0.05）， and up-regulated protein expression of Keap1 （<italic>P</italic><0.01）. Compared with the model group， ASRCRS groups displayed improved cell morphology， increased cell viability， inhibited cell apoptosis， potentiated SOD activity （<italic>P</italic><0.01）， suppressed release of LDH （<italic>P</italic><0.01） and generation of ROS， decreased content of MDA （<italic>P</italic><0.01）， up-regulated protein expression of Nrf2， HO-1 and SOD1 （<italic>P</italic><0.05， <italic>P</italic><0.01）， and down-regulated protein expression of Keap1 （<italic>P</italic><0.01）. Conclusion:ASRCRS could protect PC12 cells from oxidative damage induced by H<sub>2</sub>O<sub>2</sub> by up-regulating the expression of Nrf2 to activate the Nrf2/antioxidant response element （ARE） signaling pathway， enhancing the ability to resist oxidative damage， and inhibiting cell apoptosis.
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Objective:To investigate the effect of Huayu Jiedu prescription medicated serum（HJRMS）on the proliferation， invasion and migration of human lung cancer cells （H1299 cells） and its mechanism. Method:Cell counting kit-8 （CCK-8） method was used to detect the inhibitory effect of HJRMS on the proliferation of lung cancer cells， the effect of HJRMS on the invasion and migration of H1299 cells were determined by Transwell assay and wound healing assay. The protein expressions of Janus kinase 2 （JAK2）， signal transduction and activation transcription factor 3 （STAT3）， phosphorylated JAK2（p-JAK2） and phosphorylated STAT3 （p-STAT3） were detected by Western blot， the mRNA expression levels of JAK2 and STAT3 were detected by Real-time quantitative polymerase chain reaction（Real-time PCR）. Result:① Compared with control group， the proliferation of H1299 cells was significantly inhibited after treatment with 1%~16%HJRMS serum for 24， 48 h， respectively（<italic>P</italic><0.01）， and showed a certain concentration dependence. ② After treatment with HJRMS for 24 h， the scratch healing ability of cells in the 4%，8%HJRMS serum groups was inhibited（<italic>P</italic><0.05，<italic>P</italic><0.01）. ③ Compared with control group， the membrane permeability of H1299 cells in invasion and migration experiments in 2%，4%，8%HJRMS serum groups was decreased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）. ④ Western blot showed that compared with control group， 4%，8%HJRMS serum groups inhibited the expression of JAK2/STAT3 signaling pathway related proteins （JAK2， p-JAK2， STAT3， and p-STAT3） in lung cancer H1299 cells（<italic>P</italic><0.05， <italic>P</italic><0.01）. ⑤ Compared with control group， the mRNA expression levels of JAK2 and STAT3 in lung cancer H1299 cells treated with 8%HJRMS for 24 h decreased significantly （<italic>P</italic><0.05）. Conclusion:The HJRMS can inhibit the proliferation， invasion and migration of lung cancer H1299 cells， and its mechanism may be related to the inhibition of JAK2/STAT3 signaling pathway.
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Objective:To observe the influence of Chang'an Ⅰ prescription drug-containing serum on IgE-mediated RBL-2H3 cell degranulation model， and explore the mechanism of Chang'an Ⅰ prescription in inhibiting RBL-2H3 activation degranulation and releasing inflammatory mediators with v-yes-1 Yanaguchi sarcoma viral related oncogene homolog （Lyn）/spleen tyrosine protein kinase （Syk）/mitogen-activated protein kinase （MAPK） signal pathway. Method:Preparation for Chang'an Ⅰ prescription serum. Animal group， SD male rats were randomly divided into Chang'an Ⅰ prescription serum high， medium， low dose， and blank control groups with 10 rats in each group. Dosage： 10 mL·kg-1 distilled water was given to blank control group， while Chang'an Ⅰ prescription serum high， medium and low dose groups were respectively given to the Chang'an Ⅰ prescription concentrated crude drug with concentration of 1.15，2.30，4.60 g·kg-1， respectively once a day for 7 days continuously and then blood was taken from aorta ventralis and centrifuged. Ketotifen as the positive control drug. Mast cells are counted with toluidine blue staining. Cellular release of β-aminohexose was detected by colorimetric method. Contents of MCT， TNF-α， MCP-1 and histamine were measured by enzyme-linked immunosorbent assay （ELISA） kits， Lyn/Syk/MAPK protein levels were detected by immunoblotting. Result:For cell activation and degranulation， compared with the blank control group， the model group had more cell degranulation （P<0.05）， compared with model group， the cell degranulation rate of each dose group of Chang'an Ⅰ prescription decreased （P<0.05）. The release rate of β-hexosamine in each dose group of Chang'an Ⅰ prescription decreased significantly （P<0.01）. For the release of active mediators， compared with the blank control group， the contents of histamine， MCT， TNF-α and MCP-1 all increased in the model group （P<0.01）， compared with the model group， the contents in each dose group of Chang'an Ⅰ prescription all decreased significantly （P<0.01）. Compared with the normal group， the phosphorylation levels of Lyn and Syk， extracellular regulatory protein kinase 1/2（ERK1/2）， c-Jun N-terminal kinase （JNK）， and mitogen-activated protein kinase p38 increased in the model group （P<0.05）. Compared with the model group， the Lyn， Syk and ERK1/2， JNK and p38 protein phosphorylation levels reduced in Chang'an Ⅰ prescription group （P<0.05）. Conclusion:Chang'an Ⅰ prescription drug-containing serum down-regulates the phosphorylation levels of proteins Lyn， Syk， and ERK1/2， JNK， and p38， inhibits RBL-2H3 cell activation and degranulation， reduces the release of cytokines and chemokines， such as histamine， MCT， TNF-α and MCP-1， it may be one of its mechanisms for treating IBS-D visceral hypersensitivity.
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Objective::This study aims to explore the effect and mechanism of Yuehua capsule serum for autophagy of macrophages infected with multi-drug resistant mycobacterium tuberculosis. Method::The rats were undertaken intragastric gavage with Yuehua capsule by 3.02 g·kg-1 once a day which was produced through low temperature condensation drying method. After 7 days, blood of abdominal aorta of rats was collected to prepare Yuehua capsule serum. RAW264.7 andmultidrug resistant tuberculosis were cultured in vitro.According to cell counting kit-8(CCK-8), 10% drug-containing serum was considered as the effective concentration. The cultured cells were divided into four groups: model groups(10% fetal bovine serum). Yuehua capsule serum(10% Yuehua capsule serum). Autophagy inhibitor group+ 3-MA+ Yuehua capsule medicated serum(3-MA+ 10% Yuehua capsule serum). Rapamycin (Rap) positive control group(200 mg·L-1 Rap+ 10% Yuehua capsule serum). Except for the normal group, the cells of each group were cultured for 24 h and infected for 4 h according to cell-bacteria 1∶10.Testing index: observation of autophagosomes under transmission electron microscope, the test of expression of microtubule-associated protein light chain-3Ⅱ(LC-3Ⅱ), microtubule-associated protein LC 3-Ⅱ/microtubule-associated protein light chain 3-Ⅰ(LC3-Ⅰ) and Beclin-1 with Western blot, indirect immunofluorescence staining for LC3B, and mRNA of Beclin-1 as well as LC3 with real-time fluorescent quantitative polymerase chain reaction(Real-time PCR). Result::Compared with normal group, model group did not see autophagy body cells, cells in the LC-3 Ⅱ, LC-3 Ⅱ/LC-3 Ⅰ, Beclin-1 protein and LC3, Beclin-1 mRNA gene expression level had no significant change, the cells without fluorescent particles, spots, no fluorescence intensity.Compared with model group, Yuehua capsules serum group and Rap positive control group can be observed the formation of phage, mRNA andprotein expression levelof LC-3 Ⅱ, LC-3 Ⅱ/LC-3 Ⅰ, Beclin-1 and LC3, Beclin-1 were significantly increased (P<0.05). Autophagy inhibitor group+ 3-MA+ Yuehua capsule medicated serum did not see autophagy, the mRNA and protein expression level of LC-3 Ⅱ, LC-3Ⅱ/LC-3Ⅰ, Beclin-1 and LC3, Beclin-1 were no significantly increased. Conclusion::Yuehua capsule medicated serum could induce autophagy of macrophages of RAW264.7.The mechanism was probably accomplished through regulating the expression level of autophagy key protein LC3, autophagosome mature protein Beclin-1 and relevant gene, meanwhile the conversion of LC3-I to LC3-Ⅱ was accelerated.
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Objective The mechanism of luteal phase defect remains unclear. To investigate the mechanism of BuShen ZhuYun Decoction on the gonadotropin secretion in the pituitary gland, we observed the effects of medicated serum of BuShen ZhuYun Decotion on the secretion of gonadotropin-follicle-stimulating hormone (FSH) and luteotropic hormone (LH) in rat pituitary cells.Methods The BuShen ZhuYun Decotion was administered to the female SD rats by gavage to prepare the serum containing BuShen ZhuYun Decoction. The CCK-8 method was used to detect the effect of cetrorelix acetate powder for injection, medicated serum and gonadotropin releasing hormone (GnRH) on cell activity. In the maximum non-toxic concentration, we used cetrorelix acetate powder for injection to block the GnRH receptor (GnRHR) in pituitary cells and established the GnRHR antagonistic model. Then we treat the model group with medicated serum (model group). Moreover, we established the blank group (normal pituitary cells), the cetrorelix group (intervented with cetrorelix for 6 hours), and medicated serum group (intervented with medicated serum for 24 hours). 20nmol/L GnRH was used to stimulate cells for 6h. The contents of FSH and LH in the supernatant of each group and the mRNA expression of FSHβ, LHβ and GnRHR were detected.Results Compared with that of the blank group, the supernatant levels of FSH and LH in the Cetrorelix group decreased significantly \[(3.91±0.36) mIU/mL vs (2.26±0.22) mIU/mL, (8.94±0.57) mIU/mL vs (3.35±0.59) mIU/mL, P<0.05)\]. In contrast, the levels of LH significantly increased \[(8.94±0.57) mIU/mL vs (10.79±0.60) mIU/mL, P<0.05)\]; Compared with the cetrorelix group, the levels of FSH and LH in both medicated serum group and model group increased significantly (P<0.05). Compared with the blank group, the mRNA level of FSH and LH in the cetrorelix group decreased significantly \[(0.95±0.23) mIU/mL vs (0.58±0.12) mIU/mL, (0.98±0.14) mIU/mL vs (0.27±0.21) mIU/mL, P<0.01) \], and the mRNA expression of GnRHR increased in the cetrorelix group \[(0.97±0.13) mIU/mL vs (1.77±0.26) mIU/mL, P<0.01) \]; The mRNA levels of FSH and LH in the medicated serum group were increased (P<0.05). Compared with the cetrorelix group, the mRNA expression of FSHβ mRNA and LHβ were both increased in the medicated serum group and model group (P<0.05), the mRNA expression of GnRHR decreased (P<0.01).Conclusion It is suggested that the therapeutic mechanism of BuShen ZhuYun Decotion may be related to the improvement of GnRH receptor expression.
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This study was aimed to investigate the effect and mechanism of Zhenwu Tang on AVP-V2R-AQP2 pathway in NRK-52E cells . Forty eight male SD rats were randomly divided into eight groups with 6 animals in each group. Distilled water or 22.68 g·kg⁻¹·d⁻¹ Zhenwu Tang(calculated by raw drug dosage meter) was given by gavage. Blood samples were collected by cardiac puncture， and the medicated serum was centrifuged from the blood by 3 000 r·min⁻¹. NRK-52E cells were treated with different medicated serum or dDAVP. The condition of cell proliferation was detected by RTCA. The distribution of V2R and AQP2 in cells were detected by immunofluorescence. The expression of V2R， PKA and AQP2 were detected by Western blot and AQP2 mRNA level was detected by real-time PCR. Results showed that the level of AQP2 mRNA(<0.01) and protein expression of V2R， PKA and AQP2(<0.05， <0.01， <0.05) of Z7d group which was treated with Zhenwu Tang medicated serum for 24 h were significantly higher than that of normal rat serum group. And the expression level of V2R， p-AQP2 and AQP2(<0.01， <0.05， <0.01) of Z7d+dDAVP group were significantly increased comparing to normal rat serum group. The results indicate that the applying of Zhenwu Tang medicated serum could increase the expression level of V2R， PKA and AQP2 which exist in AVP-V2R-AQP2 pathway in NRK-52E， and there is synergistic effect between Zhenwu Tang medicated serum and dDAVP. So the pathway of AVP-V2R-AQP2 may be one of the mechanism for which Zhenwu Tang regulate balance of water transportation.
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Animais , Masculino , Ratos , Aquaporina 2 , Metabolismo , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico , Metabolismo , Medicamentos de Ervas Chinesas , Farmacologia , Rim , Biologia Celular , RNA Mensageiro , Ratos Sprague-Dawley , Receptores de Vasopressinas , Metabolismo , Transdução de SinaisRESUMO
To investigate the ATP-sensitive potassium channel (KATP channel) protein expressions during different periods under hypoxia condition and explore the effect of Qibai Pingfei capsule medicated serum (hereinafter referred to as QBPF) on the correlation between the protein expressions of KATP channel and nitric oxide in rat pulmonary arterial smooth muscle cells(PASMCs). Qibai Pingfei capsules were given to SD rats via continuous gavage for 10 days to obtain QBPF. Primary rats PASMCs were cultured by the direct adherent culture method. Western blot was applied to detect the protein expression levels of KATP channel (Kir6.1 and SUR2B) in PASMCs. Then the noncompetitive inhibitor of NO synthase--Nω-nitro-L-arginine methyl ester(L-NAME) and KATP channel inhibitor--glyburide(GLYB) were applied respectively to evaluate the effect of QBPF on the protein expressions of KATP channel. The protein expressions of Kir6.1 and SUR2B were increased after 6-hour hypoxia treament, peaked at the 24-hour hypoxia treament, and decreased in both 48-hour and 72-hour hypoxia groups. Especially, QBPF could further up-regulate the Kir6.1 and SUR2B protein expressions under 24-hour hypoxia condition; however, such up-regulation effect could be blocked by KATP channel inhibitor GLYB and NO specific inhibitor L-NAME, indicating that QBPF played the role of opening KATP channel. The regulatory mechanism was probably associated with up-regulating KATP channel protein expression via NO relative pathway, involving pulmonary vasodilation, and thus relieving the occurence and development of COPD.
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<p><b>OBJECTIVE</b>To explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts.</p><p><b>METHODS</b>Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR).</p><p><b>RESULTS</b>The mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5.</p><p><b>CONCLUSIONS</b>BSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.</p>
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Adulto , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Proteínas Morfogenéticas Ósseas , Metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Genética , Metabolismo , Medicamentos de Ervas Chinesas , Farmacologia , Fibroblastos , Metabolismo , Patologia , Osteogênese , Genética , Fosforilação , RNA Mensageiro , Genética , Metabolismo , Soro , Metabolismo , Transdução de Sinais , Proteínas Smad , Metabolismo , Espondilite Anquilosante , Genética , PatologiaRESUMO
Objective To investigate the immunological reaction mechanism of medicated serum in Zibai Gelatin for SiHa cells of the cervical cancer infected by high risk human papilloma virus (HPV). Methods Through immunohistochemical comparison and the cell culture, and after medicated serum was administrated to SiHa cells of cervical cancer for 24 h, 48 h, and 72 h, optical densities of IL-6, IL-10, CD83 and TNF-αin the same time but different concentrations and different times but the same concentration were observed. Relationships of dose-effect and time-effect between expressions of IL-6, IL-10, CD83 and TNF-α and medicine action were analyzed by calculating average optical density. Results With the increase of medicine concentration and administration time of Zibai Gelatin, the expressions of IL-6 and TNF-αgradually decreased, while the expressions of CD83 and IL-10 gradually increased (P<0.05). Conclusion Zibai Gelatin with medicated serum can inhibit local inflammatory reaction by improving local immunologic function of cervix, which is beneficial to reduce cervix high-risk HPV.
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[Abstract ] Objective Modern pharmacological studies confirmed Zhigancao Decoction total extract, single active ingredients and their combinations could obviously inhibit arrhythmia.This study was to investigate the effects of Zhigancao Decoction medicated serum combined with myocardial tissue/silicon substrate microelectrode arrays (MEA) on rapid atrial pacing(RAP). Methods New Zealand white rabbits were randomly divided into normal control group, normal serum control group, Zhigancao Decoction medica-ted serum group and water decoction group, 8 in each group.After the establishment of an atrial fibrillation rabbit model, the field ac-tion potential duration ( fAPD) of the right atrial appendage ( RAA ) tissue was measured and the effections of Zhigancao Decoction medicated serum and water decoction on the fAPD of RAA were observed. Results The successful modeling of rapid atrial pacing induced atrial fibrillation in rabbits contributed to the significant shortening of fAPD 12 h after pacing compared to that before pacing ([174.30 ±1.36]ms vs[162.48 ±0.88]ms, P<0.05).After giving 10%~25% Zhigancao Decoction medicated serum and water decoction, the fAPDs of RAA tissue in rabbits with atrial fibrillation were prolonged, which represented positive dose-response relation-ship.The fAPDs of the rabbits given serum containing 10%, 15%, 20%and 25%Zhigancao Decoction were respectively (170.81 ± 0.61)ms, (171.00 ±0.46)ms, (179.08 ±0.67)ms, (179.76 ±2.26)ms, which were longer than those of water decoction group ([163.82 ±0.780]ms, [163.66 ±0.95]ms, [174.06 ±1.32]ms, [176.84 ±1.19]ms), and the difference was statistically sig-nificant (P<0.05). Conclusion The fAPD can be taken as one index of cardiac electrophysiological change, and 10%~25%Zh-igancao Decoction medicated serum can lead to fAPD extension in rabbit model of atrial fibrillation, which might be the electrophysio-logical mechanism of anti-atrial fibrillation.
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Objective To study the effect of Bushengyijing Fang medicated serum on human sperm quality in vitro. Methods Human sperm was co-cultured with Bushengyijing Fang medicated serum in vitro. Human sperm quality was evaluated by computer assisted semen analysis (CASA) and the sperm move high test. Results The co-cultured Bushengyijing Fang medicated serum significantly increased the sperm motion velocity (VAP, VCL, VSL) (P
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Objective: To study the effect of Jinxin Oral Liqiud medicated serum on RS virus virus (RSV) infected cell apoptosis and the regulation gene as Bcl-2, Bax on the different point in early time. Methods: Cell culture, serum pharmacology and Annexin V/PI technique combining with flow cytometry were used to detect cell apoptosis, Bcl-2 and Bax expression. Results: The total apoptosis rates of human embryonic lung fibroblast in JOL group in early time of 12h and 24h were significantly higher than that in RSV infected group (P