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Objective To observe the effect of sinomenine (SIN) on the expression of cyclooxygenase (COX2),alpha-7 nicotinic acetylcholine receptor(α7nAChR) and adenosine receptor(A2A) in A549 cells,and to explore the relative mechanism for cell proliferation.Methods The effect of SIN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the proliferation of A549 cells was determined by methyl thiazolyl tetrazolium (MTT) assay.The effect of SIN and NNK on the migration of A549 cells was detected by cell wound scratch assay.The effect of SIN and NNK on COX2 expression in A549 cells was determined by Western blotting method.The effect of SIN and NNK on the expression of α7nAChR and A2A mRNA and protein was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting method.Results NNK increased the proliferation and migration of A549 cells,while SIN inhibited the proliferation and migration of A549 cells.COX2 expression level was increased in NNK group but was decreased in SIN group.The expression levels of α7nAChR and A2A were up-regulated in NNK group but were down-regulated in SIN group.Conclusion SIN plays a role in inhibiting the proliferation and migration of A549 cells by suppressing COX2 expression.SIN has an inhibitory effect on the expression of α7nAChR and A2A.
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Objective Study on the absorption,distribution,transformation,excretion and toxicity of four forms of arsenic in rats.Methods Four thousand and fifty data were obtained from 28 days animal metabolism experiments of 81 rats exposed to the four As species.Distribution,metabolism,excretion,and subacute toxicity of 4 As species were compared by analyzing the arsenate [As(V)],arsenite [As(Ⅲ)],methyl arsenate (MMAV) and dimethyl arsenate (DMAV) content of rat feces (excrement and urine),blood,seven organs,and liver and kidney pathology.Results After oral administration of each As species,82.9％ of As(Ⅲ),85.1％ of As(V),95.0％ of MMAV and 96.2％ of DMAV were accumulatively secreted via feces and urine,while 16.2％ of As(Ⅲ),14.1％ of As(V),4.65％ of MMAV and 0.120％ of DMAV were detected in blood.The DMAV accumulated in blood and urine after dosing As(Ⅲ),As(V) and MMAV,and the content of DMAV in the blood was 8 times greater than urine.Four kinds of arsenic had effects on rat liver and kidney function,the influence on liver was acute phase effect while that on renal was cumulative effects.The effect of As(Ⅲ) was the most significant,followed by MMAV.Four kinds of arsenic could cause tissue deformation and inflammatory cells infiltration,but showed no difference between groups.Conclusion The distribution,metabolism,excretion,and toxicity of the four kinds of arsenic in rats were different in varying degrees.The research of health risk for arsenic should consider its different forms.
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Objective To investigate the effect of sinomenine on the purinergic receptors A2A and P2X7 in endotoxemia mouse model and RAW264.7 macrophage model stimulated by lipopolysaccharide(LPS). Methods BALB/c mice were randomly divided into blank control group, model group and sinomenine group. Thirty minutes after the rats of sinomenine group were pretreated with intraperitoneal injection of sinomenine (40, 80, 160 mg/kg), the mice were given intraperitoneal injection of 15 mg/kg LPS to induce endotoxemia model. The serum levels of tumor necrosis factor-alpha(TNF-α) and interleukin-6(IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of purinergic receptor A2A and P2X7 in the liver and spleen were detected by reverse transcription-polymerase chain reaction(RT-PCR). RAW264.7 macrophages were divided into blank control group, LPS group and sinomenine group. Sinomenine group was firstly treated with sinomenine(300μmol/L) for 2 h, and then LPS group and sinomenine group were treated with LPS (100 ng/mL) for another 8 hours. TNF-α in the cell supernatant was measured by ELISA, and the expression levels of A2A and P2X7 in RAW264.7 cells were detected by RT-PCR. Results Stimulation with LPS could induce the increase of the mouse serum levels of TNF-α and IL-6 as well as the expression of A2A and P2X7 in mouse liver and spleen, and sinomenine had a counteraction on the above indexes(P<0.05) . In-vitro stimulation with LPS could induce the increase of the content of TNF-α and the expression of A2A and P2X7 in RAW264.7 cells , and sinomenine decreased TNF-α content and P2X7 expression (P<0.05) , but had an effect on enhancing A2A expression. Conclusion Sinomenine suppresses the expression of purinergic receptor P2X7 in mouse spleen and liver as well as in RAW264.7 macrophages, but its effect on the expression of A2A in various tissues and cells varies, whose related mechanism is needed further study.
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Objective To observe the anti-inflammatory effects of three kinds of aconine from Radix Aconiti Lateralis Preparata ( Fuzi) on macrophages, as well as the effects of their combination with sinomenine. Methods The effect of three monoester alkaloids from Fuzi, benzoylaconine ( BAC) , benzoylmesaconine ( BMA) and benzoylhypaconine ( BHA) , on RAW264.7 cells proliferation was detected by cell counting kit 8 (CCK8) in vitro. RAW 264.7 macrophage cells were stimulated by lipopolysaccharides (LPS) and then treated with different concentrations of BAC, BMA, and BHA. Tumor necrosis factor alpha ( TNF-α) and interleukin 6 (IL-6) secreted by RAW264.7 cells were measured by enzyme-linked immunosorbent assay (ELISA) . The anti-inflammatory effects of three monoester alkaloids combined with sinomenine were evaluated by Zhengjun Jin Q method. Results The concentrations of TNF-α and IL-6 secreted by LPS-stimulated RAW264.7 cells were increased significantly ( P<0.01) , and then were inhibited by BAC ( 20, 30 μmol/L) , BMA ( 40, 80, 160μmol/L) and BHA ( 40, 80, 160 μmol/L) significantly ( P<0.01) . Antagonistic effects were present when 30 μmol/L of BAC or 160 μmol/L of BMA or 160 μmol/L of BHA was respectively used together with 100 or 300 μmol/L of sinomenine (Q<0.85). Conclusion The three kinds of monoester alkaloids from Fuzi exert anti-inflammatory effect on macrophages, and the effective dose of BAC is lower than that of BMA and BHA. The combination of BAC, BMA or BHA at the analyzed dosage with sinomenine has antagonistic effect.
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Objective:To investigate sinomenine (Sinomenine,SIN) effect on RAW264.7 cells polarization to M1 or M2 phenotype induced by lipopolysaccharide (LPS) or interleukin-4 (IL-4) .Methods:RAW264.7 cells were induced to polarize to M1 by LPS ,and to M2 by IL-4.Sinomenine effects on LPS or IL-4 induced macrophages:TNF-αand IL-10 secretion induced by different condition were detected by Enzyme linked immunosorbent assay (ELISA);The expression level of mRNA of Arginase1(Arg-1),Nitric oxide synthase(iNOS),suppressor of cytokine signaling protein-2(SOCS2) and suppressor of cytokine signaling protein-3(SOCS3) of M1/M2 phenotypes were detected by real time PCR respectively.Results:Sinomenine inhibited the increase of TNF-αsecretion,iNOS and SOCS3 mRNA expression level induced by LPS.Sinomenine inhibited the increase of IL-10 secretion and Arg-1 mRNA expression level induced by IL-4,but SOCS2 mRNA expression level was not affected by Sinomenine.Conclusion: Sinomenine can inhibite the macrophage polarization to M1 and M2 induced by LPS and IL-4.Sinomenine plays a regulatory role on imbalance of M1/M2,and is conducive to maintain the dynamic balance.
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Objective To study the relationship between the inflammatory cytokine levels and the expression of hepatic alpha-7 nicotinic acetylcholine receptor (α7 nAChR) in hyperlipidemia rats and to investigate the intervention effect of Yirui Capsules ( mainly composed of Radix Salviae Miltiorrhizae, Fructus Crataegi, Rhizoma Alismatis, etc). Methods Fifty male SD rats were randomly divided into blank control group ( fed with conventional diet, N=10) and high-fat diet group (fed with high-fat diet, N=40). After feeding for 14 d, the forty rats in high-fat diet group were further randomly divided into model control group, and low-, middle-, and high-dose Yirui Capsules groups (140, 280, 560 mg/kg, respectively), 10 rats in each group and the treatment lasting 30 days. And then, the serum levels of lipids were detected, C-reactive protein (CRP) and tumor necrosis factor (TNF) -α in the serum were detected by enzyme-linked immunosorbent assay ( ELISA) , and the expression of hepatic α7 nAChR was analyzed by immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) . Results Yirui Capsules in different dosages could improve serum lipid levels in hyperlipidemia rats to various degrees. The contents of serum inflammatory cytokines CRP and TNF-α and the mRNA expression of hepatic α7nAChR were increased in the model control group as compared with those in the blank control group ( P<0.01). The contents of inflammatory cytokines CRP, TNF-αand the mRNA expression of hepatic α7nAChR were decreased in the three Yirui Capsules groups as compared with those in the model control group ( P<0.01). The results of immunohistochemical staining for α7nAChR expression were consistent with RT-PCR results. Conclusion The expression of hepatic α7nAChR is increased in hyperlipidemia rats. Yirui capsules are effective on decreasing inflammatory cytokine levels, improving lipid metabolism, and down-regulating α7nAChR expression.
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Objective To investigate the effect of berberine on the polarization of mice RAW264.7 macrophages induced separately by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Methods Mice RAW 264.7 macrophages cultured in vitro were divided into model group, medication group, and blank control group. Both model group and medication group were given either LPS (in final dose of 100 ng/mL) or IL-4 (in final dose of 10 ng/mL). Additionally, the medication group was treated with berberine in final dose of 20 μmol/L. The blank control group was given the same volume of phosphate buffered saline ( PBS). Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of arginase-1 (Arg-1), inducible nitric oxide synthase ( iNOS) , suppressor of cytokine signaling2 ( SOCS2) and SOCS3. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of tumor necrosis factor alpha (TNF-α) and IL-10. Results The content of TNF-αand the mRNA expression levels of iNOS and SOCS3 in macrophages induced by LPS were increased, and then were down-regulated by berberine (P0.05). Conclusion Berberine has an effect on inhibiting the M1 and M2 polarization of macrophages in vitro, suggesting that berberine may play a regulatory role in the dynamic balance of M1/M2.
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Insertion of foreign genes into the genome map of Chinese herb always produces the intended beneficial effects, but also the unintended effects. Analysis of genomic function and metabolic end products based on the principle of substantial can reveal the influence on the quality of herbs, which is able to provide valuable refer-ence for the following evaluation on pharmacology and toxicology.
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<p><b>OBJECTIVE</b>To transform the antimicrobial peptide fusion gene of cecropin B and rabbit NP-1(CN) into Houttuynia cordata to improve its antimicrobic capability.</p><p><b>METHOD</b>The fusion gene of CN designed and synthesized artificially was recombined with expression vector pBI121. The recombined vector was transformed to Agrobacterium tumefaciens LBA4404, by which CN gene was transformed to the explants of H. cordata. The transgenic regeneration plantlets were selected by kanamycin and rapid screening PCR. The transgenic plants were identified by PCR-Southern of genomic DNA and RT-PCR. The disease resistances were detected by antibacterial zone trail of leaf extracts to E. coli K12 and infection by Rhizoctonia solani.</p><p><b>RESULT</b>Gene of interesting CN was inserted into genomic DNA and expressed in transformed H, cordata, whose resistance to E. coli K12 and Rh. solani was stronger than that of the non-transformed control.</p><p><b>CONCLUSION</b>The fusion gene CN can improve antimicrobic capability of transformed H. cordata.</p>