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Objective To study the effects of main ingredients from microemulsion extract of Compound Longqincao in vitro on anti-influenza virus H1N1 activity; To analyze effects of main ingredients from microemulsion extract on influenza virus inhibition rate.Methods Uniform design was used to conduct the experiment. MTT method was used to detect the effect rate (ER) of anti-influenza virus H1N1 on A549 cells. Setting ER as the index, Minitab17 software was used to establish mathematical model to come up with regression equations of all factors. The effects of ingredients on ER were analyzed and the efficient composition ratio of the optimum anti-influenza virus H1N1 was chosen.ResultsIn the compound compatibility, baicalin showed the most obvious antivirus activity, and licorice glycosides had certain inhibition effects on pathological changes of cells. Five ingredients had coordinative or controlled relation with ER. When per milliliter liquids containing licorice glycosides, baicalin, leucine, aspartic acid, glutamic acid was 13.94μg, 49.44μg, 0.23 mg, 1.25 mg, and 2.50 mg, ER was the best. ER was 85.34%±4.72% after verification.ConclusionThe optimized combination of main ingredients from microemulsion extract of Compound Longqincaocan better play a role in anti-influenza virus H1N1.
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ObjectiveTo investigate the regulatory effects of traditional Chinese medicine (TCM) Shufengxuanfeijiedu formula on Janus kinase signal transducer and activators of transcription (JAK-STAT) of lung tissues in mice with influenza viral pneumonia.Methods According to random number table, 60 mice were randomly divided into six groups with 10 mice in each group: normal group (N), model group (M), Tamiflu control group (C) and low (SL), medium (SM), high dose (SH) Shufengxuanfeijiedu formula groups. The mouse model of influenza virus pneumonia was reproduced by dropping of 0.05 mL 4LD50 inflluenza virus FM1 strain which can be adapted to lung tissue into the nose; while the N received nose instillation of 0.05 mL normal saline. After successful modeling for 2 hours, distilled water was given orally (by lavage) to N and M; Duffy (oseltamivir) 2.5 g·mL-1·d-1 was administrated to C; the TCM SL, SM, SH were intragastrically administered with different doses of shufengxuanfeijiedu decoction into the corresponding groups respectively (the ingredients of prescription: chrysanthemum, mulberry leaf, almond, platycodon root, forsythia, bupleurum etc. forming granules), according to the suitable dose of granules used for human body surface, the dose used for mouse surface area was calculated, the high dose means the dose used in the medium dose group doubled, the low dose means 1/2 dose used in medium group, once a day, once 0.2 mL for consecutive 4 days. Afterwards, the lung tissues were collected, the mouse differential gene expressions related to JAK-STAT pathway were detected by gene chip technology, the standards for screening of differential gene expression were as follows: up-regulated gene was P 1; down-regulation gene wasP < 0.05, and log2ratio < -1. The levels in lung tissue kinase (JAK) andγinterferon (IFN-γ) mRNA expressions were determined by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR).Results Compared with those in N, the differential expression gene transcription activator, STAT5 [log2 (N/M) = 2.32], interleukin 4 receptor alpha subunit [IL4RA, log2 (N/M) = 4.77], interleukin 12 receptor [IL12R, log2 (N/M) = 1.58], JAK [log2 (N/M) = 2.41] were all obviously up-regulated, and IFN was significantly down-regulated [log2 (N/M) = -1.45] in M. Compared with those in M, C group IFN [log2 (C/M) = 1.51], various TCM dose groups [log2 (SL/M) = 1.46, log2 (SM/M) = 1.72, log2 (SH/M) = 1.40] differential expression gene IFN was significantly up-regulated, STAT5 [log2 (C/M) = -2.06, log2 (SL/M) = -1.41, log2 (SM/M) = -2.10, log2 (SH/M) = -1.89], IL4RA [log2 (C/M) = -2.52, log2 (SL/M) = -1.85, log2 (SM/M) = -2.74, log2 (SH/M) = -1.39), IL12R [log2 (C/M) = -1.48, log2 (SL/M) = -0.10, log2 (SM/M) = -1.58, log2 (SH/M) = -0.53], JAK [log2 (C/M) = -1.44, log2 (SL/M) = -0.88, log2 (SM/M) = -1.74, log2 (SH/M) = -0.53] were significantly down-regulated. In M, the JAK mRNA expression was obviously elevated (2-ΔΔCt: 3.17±0.94 vs. 1.01±0.13,P < 0.05), while the IFN-γ mRNA expression was decreased (2-ΔΔCt: 0.15±0.48 vs. 1.01±0.12,P < 0.05); compared with M, the JAK mRNA expressions in C, SM and SH groups were all obviously decreased (2-ΔΔCt: 2.02±0.63, 1.19±0.30, 1.59±0.67 vs. 3.17±0.94, allP < 0.05); while the IFN-γmRNA expressions in C, SL, SM and SH groups were elevated (2-ΔΔCt: 0.61±0.12, 0.41±0.13, 0.85±0.14, 0.78±0.20 vs. 0.15±0.48, allP < 0.05).Conclusions Shufengxuanfeijiedu formula can ameliorate the mice immune pathological injury of lung tissues induced by influenza virus by regulating JAK-STAT signal pathway and balancing Th1/2 via up-regulating the expression of IFN-γ.
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Objective To investigate the effects of Tangshen Formula on liver oxidative stress in diabetic rats, and their mechanisms thereof.Methods Rat model of type 2 diabetes mellitus and nonalcoholic fatty liver disease was estab?lished by intraperitoneally injecting small dose of chain urea with cephalosporins (STZ) and feeding high fat fodder . The model rats were randomly divided into control group, model group, Tangshen Formula group and metformin group.The se?rum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), cholesterol (CHO), low density lipoprotein-cholesterol(LDL-C), and superoxide dismutase (SOD), catalase(CAT), vitamin E(VE), nitric oxide (NO) and nitric oxide synthase (NOS) in liver were compared between four groups. Changes of pathological morphology were ob?served under light microscopy. Results There were significantly decrease in serum levels of ALT, AST, TG, CHO, LDL-C in Tangshen Formula group and metformin group compared with those of model group. There were significantly higher levels of SOD, CAT and VE, and lower levels of NO and NOS of liver homogenate in Tangshen Formula group than those of model group. There were higher levels of SOD and CAT, and lower levels of NO and NOS in liver homogenate in metformin group than those of model group. HE staining showed that liver fatty degeneration was significantly reduced in metformin group and Tangshen Formula group compared with that of model group. Conclusion The fatty liver in type 2 diabetic rats is signifi?cantly improved by Tangshen Formula treatment, which is probably associated with the regulation of the response level to oxi?dative stress in liver.
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Aim To investigate the regulatory effects of baicalin on apoptosis induced by virus H1 N1 in hu-man pulmonary carcinoma cell A549 . Methods The chips were used to screen the RNA samples in virus-in-fected A549 cells. Differentially expressed genes were selected in the pathway of apoptosis. The mRNA ex-pressions of caspase-3 and -8 were verified by Real-Time PCR. Results With the DNA microarray, the functions of differentially expressed genes involved in apoptosis biological pathways were analyzed by Kyoto Encyclopedia of Genes and Genomes ( KEGG ) Path-way databases. caspase-3, -7, -8, -10, TRAIL, MYD88 , IL1 A and IL1 B were up-regulated in virus-in-fected group. Oseltamivir could down-regulate gene ex-pressions of caspase-3 ,-4 and-8 . High-dose of baica-lin could down-regulate gene expressions of caspase-3 ,-4,-6 and -8. Except gene expressions of above, low-dose of baicalin could also down-regulate gene expres-sions of IL1RAP and Cn. Real-Time PCR experiments showed that baicalin could significantly decrease mR-NA expression of caspase-3 , -4 , -6 , -8 , IL1 RAP and Cn ( P < 0. 01 ) , compared with the virus-infected group. The results also figured that the interference ef-ficacy of low-dose baicalin was better than that of high-doses. As expected, real-time PCR data were in good agreement with the microarray assay. Conclusions Baicalin can be detected in their suppression effect of caspase-3,-4,-6, and -8 mRNA expression, so it re-sists against the apoptosis to fight against influenza vi-rus in vitro.
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Objective To investigate the effect of Shufeng Xuanfei and Jiebiao Qingli concoctions on Toll-like receptor (TLR) signal pathway of pneumonia infected with influenza virus in mice.Methods The pneumonia model was reproduced by nasal dropping of influenza virus A in mice.The mice were randomly divided into nine groups:normal group (C),model group (M),tamiflu group (D),Shufeng Xuanfei low-dose (SL),medium-dose (SM) and high-dose (SH) groups,Jiebiao Qingli low-dose (JL),medium-dose (JM) and high-dose (JH) groups,each n =12.Two hours after model-reproduction,the mice in C group and M group received distilled water by gavage.The mice in D group received 2.5 g· mL-1· d-1 oseltamivirphosphate.Shufeng Xuanfei formula in doses of 3.76,1.88,0.94 g· kg1 · d-1 were respectively administered to SH,SM and SL groups by gavage,Jiebiao Qingli formula in doses of 4.37,2.18,1.09 g ·kg-1 ·d-1 was given to JH,JM and JL groups by gavage,respectively.Each group was in equal dose of 0.2 mL daily over a 4-day period.Total RNA was extracted in each group.Then gene chips were used to screen these RNA samples.Some genes that were involved in TLR signal pathways were selected.These candidate genes were verified by real-time reverse transcription-polymerase chain reaction (RT-PCR).Results TLR7,MYD88,CCLS,IFNB1,IL6,IL12a,NFKBIA and IKBKB were up-regulated in model group compared with control group.Compared with model group,down-regulated genes in medium-dose,low-dose Shufeng Xuanfei formula and medium-dose Jiebiao Qingli formula included TLR3,TLR7,MYD88,CCL5,IFNB1,IL6,IL12a,NFKBIA and IKBKB (log2 signal intensity of SM,/M in medium-dose Shufeng Xuanfei formula group were-1.24,-2.02,-1.36,-1.95,-0.63,-1.33,-3.50,-1.33,-1.33,log2 signal intensity of SI/M in low-dose Shufeng Xuanfei group were-1.07,-2.43,-2.63,-2.30,-5.09,-3.19,-3.53,-1.95,-1.95,log2 signal intensity of JM/M in medium-dose Jiebiao Qingli formula group were -1.78,-0.55,-1.35,-1.47,-1.65,-2.03,-3.02,-1.57,-1.57,respectively).The results suggested that the effect of Shufeng Xuanfei formula was better than that of Jiebiao Qingli formula.By RT-PCR,compared with model group,low-dose,medium-dose and high-dose groups of Shufeng Xuanfei formula,medium-dose and high-dose groups of Jiebiao Qingli formula,and tamiflu group,significant decrease in TLR7,nuclear factor-κB (NF-κB),myeloid differential protein-88 (MyD88) mRNA expression were found.Medium-dose and low-dose Shufeng Xuanfei formula group (TLR7 mRNA:3.6 ±0.3,3.5 ± 1.2 vs.7.4 ± 1.6,NF-κB mRNA:1.1 ±0.2,1.0 ±0.2 vs.2.2 ±0.4; MyD88mRNA:1.4 ± 0.4,1.0 ± 0.3 vs.3.4 ± 0.9,all P＜0.01) and medium-dose Jiebiao Qingli formula group (TLR7 mRNA:4.9 ± 0.3 vs.7.4 ± 1.6,NF-κB aRNA:1.3 ± 0.7 vs.2.2 ± 0.4,MyD88 mRNA:1.6 ± 0.8 vs.3.4 ± 0.9,P＜0.05 or P＜ 0.01) were shown statistically significant decreases compared with the model group.Conclusions Medium-dose and low-dose Shufeng Xuanfei formula and medium-dose Jiebiao Qingli formula can inhibit the inflammatory reaction induced by influenza virus by down-regulating the NF-κB through TLR signal pathways dependent on MyD88.The regulation of Shufeng Xuanfci formula in TLR signal pathways was superior to that of Jiebiao Qingh formula.
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<p><b>OBJECTIVE</b>To investigate the influence of Dureping injection to the murinal celiac macrophage Ana-1 on TIR signal pathway.</p><p><b>METHOD</b>Ana-1 cell line was infected by influenza virus FM1 strain and treated with the Dureping injection in different concentrations (10.1 mg x L(-1) group) for 12 h and 24 h. Then we collected the cells, extracted mRNA and measured the expressions of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 respectively by RT-PCR.</p><p><b>RESULT</b>Dureping injection down-regulated the expression of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 mRNA in Ana-1 cell line infected by influenza virus, in a dose-dependent manner significantly.</p><p><b>CONCLUSION</b>Dureping injection has an obvious effect against influenza virus FM1 strain by regulating the TIR signal pathway.</p>
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Animals , Mice , Adaptor Proteins, Signal Transducing , Cells , Cells, Cultured , Epithelial Cells , Metabolism , Interleukin-1 Receptor-Associated Kinases , Genetics , Macrophages , Metabolism , Myeloid Differentiation Factor 88 , Genetics , Metabolism , NF-kappa B , Metabolism , RNA, Messenger , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , TNF Receptor-Associated Factor 6 , Genetics , Metabolism , Transcription Factor RelA , MetabolismABSTRACT
Objective:To observe the influence of ginsenoside Rg1 on transcriptional activation of NF-κB induced by hydrogen peroxide (H_2O_2) in 293T cell,and probe into the antioxidant mechanism of ginsenoside Rg1.Methods:In the experiment,cells was exposed to H_2O_2 after pretreatment with Rg1,cell proliferation and cytotoxicity studies were detected by MTT and Trypan blue.The quantities of generation of intracellular reactive oxygen species (iROS) was analyzed by flow cytometric analysis measured with fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA).NF-κB-responsive element-luciferase reporter gene was transfected and dual-luciferase cis-reporting systems were used to assay the transcriptional activity of NF-κB under the stimulated circumstance of oxidative stress induced by H_2O_2.Results:The results of MTT showed that ginsenoside Rg1 apparently protected the proliferation of 293T cell,which were repressed by H_2O_2 (P＜0.05).The results by trypan blue showed that H_2O_2 stimulated substantial cytotoxicity.This effect was markedly attenuated by treatment with ginsenoside Rg1.Oxidant production,measured as the fluorescence of dichlorofluorescein,was significant increased by 40%-50% through H_2O_2 stimulation.The decrease in iROS generation was significant blocked by 35%-40% through Rg1 and antioxidant.The relative luciferase reporter assay of NF-κB was apparently improved by H_2O_2-induced(P＜0.05),but Ginsenoside Rg1 significantly repressed the relative value of luciferase (P＜0.05).Conclusion:Ginsenoside Rg1 has the obvious protective function from the damage of oxidative stress damage,whose possible mechanism is to eliminate excessive free radicals of the cells effectively,to reduce transcriptional activation of nuclear factor kappa B(NF-κB),and subsequently to suppress the NF-κB circuit activation.
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Objective To study the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on transcription factor STAT1, STAT3 and NF-?B in human cervix cancer Hela cell. Methods The cell proliferation was assessed by MTT assay. The luciferase activity of transcription factor STAT1, STAT3 and NF-?B was determined by the Dual-Luciferase Reporter Assay System. Results ISA can not inhibited the growth of Hela cell. The luciferase activity of transcription factor NF-?B in Hela cells treated with ISA was inhibited, while the luciferase activities of transcription factor STAT1 and STAT3 were not inhibited. Conclusion ISA can inhibit inflammation, which may be related with suppression of NF-?B transcriptional activity.
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Objective To study the effect of elemene on transcription factor ELK1 and its target gene in human cervix cancer Hela cell. Methods The cell proliferation was assessed by MTT assay. The luciferase activity of transcription factor ELK1 was determined by the Dual-Luciferase Reporter Assay System. The protein expression of phosphorated ELK1 and its target gene c-fos were determined by Western Blot. Results Elemene can remarkably inhibited the growth of Hela cell and its IC50 was 80.6 ?g/mL. The luciferase activity of transcription factor ELK1 in Hela cells treated with elemene was inhibited. The protein expression of phosphorated ELK1 and its target gene c-fos in Hela cells treated with elemene were down-regulated. Conclusion Elemene can inhibit human cervix cancer Hela cell proliferation,which may be related with suppression of c-fos gene through inhibiting expression of phosphorated ELK1.
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Objective To investigate the protective effect of Tanreqing injection on the mice infected with influenza virus FM 1 . Methods The experiment includes protection of Tanreqing injection to the mice infected with influenza virus FM1, and effect of Tanreqing injection to the viral titers, pathology and lung index of mice with influenza virus FM 1 . Results Tanreqing injection can reduce the mortality of the mice with influenza virus FM 1 infected. And Tanreqing injection can improve viral titers, pathology, and lung index of the mice infected with influenza virus FM1. Conclusions Tanreqing injection can protect the mice infected with influenza virus FM 1 by resisting the influenza virus and meliorating the lung pathology of the mice infected with influenza virus FM 1 .
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Objective To investigate the effect of Dureping injection (DRP) on IL-4, IFN-? mRNA and CD40L protein expression of FM1 infected mice with virus pneumonia, and explore the mechanism of antiviral effect. Methods ICR mice were divided into 7 groups:DRP1, DRP2, DRP3, SHL, Ribavirin, sham and model group. The mice were sacrificed on the 7th after infected, and the lung index was calculated. Then, IFN-?mRNA and IL-10 mRNA in the lung tissue was measured with RT-PCR, CD40L protein in the lung tissue was measured with Western-blot. Results The lung index of mice in model group was significantly higher than sham group (P0.05). Conclusion DRP in vivo could down-regulate the lung index, inhibit the transcription IL-4 mRNA, enhance the transcription of IFN-? and inhibit the expression of CD40L protein in lung of FM1 infected mice.
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0.05). Compared with control cells, the relative luciferase activity of NF-?B in virus-infected cells was apparently up-regulated (P
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Objective:To research the anti-virus effect of Tan Re Qing injection on the mice with influenza virus FM1 infected.Method:Mice models of lung infection were set up induced by influenza virus FM1.The effect of Tan Re Qing injection on the pathology,viral titers,the level of T lymphocyte and IFN-?of mice with influenza virus FM1 infected were observed.Results:Tan Re Qing injection can reduce the viral titers and pathological damages,improve the T lymphocyte multiplication ability and the level of serous cytokine IFN-?of the mice with influenza virus FM1 infected.Conclusion:Tan Re Qing injection has anti-virus effect on mouse with influenza virus FM1 infected by reducing viral titers and improving the immune function.
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Objective: To observe the effect of different caesalpinia sappan extract on the Mice thymus tissue, peripheral T-cell proliferation and the production of INF-?. Methods: To make preparation of different caesalpinia sappan extract and give them to the mice, weighing the mice thymus and calculating the thymus index 7 days later. The MTT method was used to measure the proliferation ability of T-cells of the mouse spleen. A double antibody sandwich ELISA was used to detect INF-? content in T-cells culture supernatant. Results: The different caesalpinia sappan extract not only have clear inhibitory effect on the weight of thymus but also have inhibitory effect on the proliferation of T-cells and the production of INF-?. The inhibitory effect of caesalpinia sappan ethanol extract and n-butanol extract on the T-cells is much stronger.
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BACKGROUND: Restriction is a reliable means in the study of psychological stress, and restriction stress can suppress cellular immunity of T lymphocytes through hypothalamus-pituitary-adrenal axis.OBJECTIVE: To investigate the changes of Th1/Th2 balance due to restriction stress in cancer-loaded rats and the growth of cancer so as to explore the effect of psychological stress on cancer cells.DESIGN:Case control study based on experimental animal as subjects.SETTING: Teaching and Research Department of Microbiology and Immunology of Beijing University of Traditional Chinese Medical.MATERIALS: This study was carried out in the Immunology Laboratory of Beijing Traditional Chinese Medical University. Kunming rats of 6-8weeks old were selected. They were raised for 3 days before experiment to adapt to the environment and numbered according to their weight. Rats with the highest and lowest body weight were excluded, and the rest were randomly divided into 4 groups of 16 rats (8 male rats and 8 female rats)weighing 18 to 20 g.METHODS: S180 cancer cells were collected at 7 days of celiac subculture and rinsed with normal saline before made into cell suspensions of 1×1010 L-1 with RPMI 1640 medium. Rats were given subcutaneous injection of 0.2 mL cell suspension at the right axilla in cancer group and cancer-combined restriction group. Meanwhile, the same dosage of normal saline was used instead in normal control group and pure restriction group in the same way. After injection, movements of rats in pure restriction group and cancer-combined restriction group were restricted in specially-made tubes for 8 hours a day. Ten days later rats were killed to remove the tumor and thymus which were then weighed for calculating the thymus index. MTT colorimetry and mitogen-activated immunoblast method were used to examine the proliferation of spleen T lymphocytes and the production of Thl-type cytokines, such as interleukin-2(IL-2), interferon-γ (IFN-γ); meanwhile,ELISA technique was used to detect the level of serum Th2-type cytokines,such as interleukin-4(IL-4) and interleukin-10(IL-10).MAIN OUTCOME MEASURES: Primary outcomes: effect of restriction stress on T lymphocyte' production of IL-2 and interferon-γ, and on IL-4and IL-10 as well as the effect in promoting the cancer growth in cancer-loaded rats. Secondary outcomes: effect of restriction stress on T-lymphocyte proliferation and thymus index.RESULTS: Restriction stress significantly increased the cancer weight and decreased thymus index and the proliferation of spleen T lymphocytes. Meanwhile, IL-2 and IFN-γ produced by spleen cells also decreased, with serum IL-4 and IL-10 level obviously increased in cancer-loaded rats.CONCLUSION:The cellular immunity of cancer-loaded rats was obviously suppressed due to restriction stress, which was presented by decreased Thl-type cytokine production and increased Th2-type cytokine production,resulting in Thl/Th2 cytokine imbalance towards Th2. It may be the important mechanism of its promoting effect on the growth of cancer.
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Objective:To survey the effect of the Dureping Injection on the kinetic change of nitrous oxide(NO) in macrophage infected by the influenza virus FM1 strain.Methods:The murinal celiac macrophage(Ana-1) was infected by the virus,add the different concentration of the drug in the supernatant of the macrophage for 6 h,12 h,24 h and 36 h.The level of the NO in the supernatant was measured by the method of traditional Griess.Ana-1 cell line was infected by influenza virus FM1 strain,then treated with Dureping Injection 1 ?g/ml for 24 h.The cells were then collected,mRNA was extracted and the expression of NF-?B p65 was measured by RT-PCR;The nuclear protein was extracted and the expression of NF-?B p65 measured by Western blot.Results:60 ?g/ml,30 ?g/ml,10 ?g/ml and 1 ?g/ml group of Dureping Injection down-regulated amount of NO secreted by the Ana-1 cells infected by virus,and it was showed in dose relationship significantly;1 ?g/ml group of Dureping Injection down-regulated the expression of the NF-?B p65 mRNA and protein.Conclusion:Dureping Injection has an obvious effect against influenza virus FM1 strain by depressing the activity of NF-?B,which prevents the production of NO secreted by Ana-1 cells infected by the virus.