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??OBJECTIVE To study the chemical constituents of Microtropis triflora Merr. et Freem. METHODS The compounds were isolated and purified by various chromatographic technigues such as silica gel, Sephadex LH-20, and pre-HPLC. Their structures were identified on the basis of chemical properties and spectral analysis. RESULTS Ten triterpenes were isolated and elucidated as 3, 25-epoxy-1??, 3??, 11??, 12, 23, 25-hexahydroxy-urs-12-en(1), ??-amyrin(2), ??-amyrin palmitate(3), 3??-hydroxy-11-oxo-olean-12-enyl-3-palmitate(4), lupeol(5), friedelin(6), 3-oxo-28-friedelanoic acid(7), oleanolic acid(8), salaspermic acid(9), and orthosphenic acid(10). CONCLUSION Compound 1 is a new compound and has cytotoxic activity against Bcap37 and SMMC7721 cells with IC₅₀ values of 27.86 and 11.38 ??g??mL^-1, respectively.     

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??OBJECTIVE To establish a method for determining arsenic (As) and antimony (Sb) released from pharmaceutical glass packing materials to provide reference for improving the existing quality standard. METHODS The samples were filled with 4% acetic acid to leach As and Sb. The extracting temperature was 98 ??, and the extracting time was 2 h. Graphite furnace atomic absorption spectrometry (GFAAS) was used for the determination. RESULTS As and Sb showed good linear relationship in a certain concentration range with linear correlation coefficients of 0.999 9 and 0.999 5. The recovery rates were 94.7%-106.9% and 100.0%-106.3%. The relative standard deviations of precision were 2.3% and 2.9%. The relative standard deviations of repeatability were 6.5% and 6.9%. The limits of detection were 1.67 and 2.54 ng??mL^-1, respectively. The elements were nearly not detected in 16 batches of samples. CONCLUSION The method is efficient and accurate and can be used for determination of As and Sb released from pharmaceutical glass packing materials, which brings supplementary contents to the existing quality standard.     

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??OBJECTIVE To prepare capsaicin-solid lipid nanoparticles (CAP-SLNs) and study their physical and chemical properties. Then, the CAP-SLNs were modified with chitosan (CTS) and the pharmacokinetics across colon of rats was studied in vivo. METHODS CAP-SLNs were prepared by emulsion-solvent evaporation method. The mean size, encapsulation efficiency and drug loading of the nanoparticles were investigated. RESULTS The average diameter of CAP-SLNs was (118.89??25.0) nm, the encapsulation efficiency was (38.56??2.6)%, and the drug-loading was (6.17??0.21)%. After colon-specific delivery in rats, the AUC_{0-360 min}(243.63??61.46) mg??min??L^-1 and ??_max(1.23??0.18) mg??L^-1 of CTS-CAP-SLNs were 1.81-fold and 1.95-fold higher than CAP. CONCLUSION It is simple and feasible to prepare CAP-SLNs by emulsion-solvent evaporation method. The pharmacokinetic parameters in rats are improved remarkably compared with CAP.     

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??OBJECTIVE To validate the conventional gel filtration chromatography (GFC) method for determination of the polymer impurities in ??-lactam antibiotics and achieve combination of GFC and RP-HPLC system. METHODS The effectiveness of the conventional GFC method was identified by 2D-GFC??LC-TOFMS and the polymer impurities found by GFC were identified by RP-HPLC. RESULTS The polymer impurities found by GFC mainly were degradation impurities and open ??-lactam ring impurities, which could not effectively characterize the sensitizing polymer impurities in cefotiam hydrochloride. CONCLUSION An effective method to characterize the polymer impurities in ??-lactam antibiotics may be established on the basis of online column switching technique which effectively combines the advantages of GFC and the ability of RP-HPLC to identify the special impurities.     

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??OBJECITVE To establish an HPLC-MS/MS method for simultaneous determination of six active components in Dengyinnaotong capsules, ie, scutellarin, bilobalide, ginkgolide A, ginkgolide B, ginsenoside Rg₁ and notoginsenoside R₁. METHODS The chromatographic separation was carried out at 30 ?? on a Phenomenex Luna C₁₈ (4.6 mm??150 mm, 5 ??m) column eluted by gradient program. The flow rate was 0.3 mL??min^-1. The six compounds were separated within 10.0 min. RESULTS The regression curves for the six compounds showed good linearity in wide ranges. The recoveries were around from 94.8% to 108.5%. CONCLUSION The established method is accurate, reliable, specific and reproducible, which can be used for the quality control of Dengyinnaotong Capsules.     

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??OBJECTIVE To report a clinical case and review the therapy of renal anemia in children with EPO-?? non-response to provide ideas on optimization of dosing regimens and new ways of clinical practice. METHODS One case of renal anemia was described and analyzed, and relevant literature was accessed and reviewed. RESULTS The level of hemoglobin increased to 100 g??L^-1 and the clinical symptoms were alleviated after transfering to EPO-?? from EPO-??. CONCLUSION For pediatric patients with chronic renal anemia, EPO-?? instead of EPO-?? is effective.     

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??OBJECTIVE To establish a new gas chromatography method coupled with ultrasound-assisted extraction and solid-phase microextraction for rapid analysis of organochlorine pesticide residues (OCPs) in 13 Miao medicines in Guizhou. METHODS Flos Lonicerae Japonicae was used as an optimization object. Before being analyzed by GC, the sample was extracted by organic solvent and ultrasound, then enriched by SPME. RESULTS Under the optimized conditions, the linearities of eight kinds of OCPs ranged from 12.5 to 500 ng??g^-1, all with good correlation coefficients. The spiked recoveries ranged from 70.4% to 119.8% and relative standard deviations (RSDs) ranged from 8.9% to 14.1%. The detection limits were 1.03-3.45 ng??g^-1. CONCLUSION UAE-SPME-GC method simplifies the preparation steps for traditional Chinese medicines by direct sample enrichment and analysis thus sparing further purification. It is a convenient, environmentally-friendly and rapid method.
     

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??OBJECTIVE To obtain the adequate QRAR models of the half-life (t_1/2), clearance(CL), volume of distribution (V_d) and area under concentration-time curve (AUC) of quinolones and elucidate the advantages and limitations of using mixed micellar liquid chromatography for describing and estimating the biological parameters. METHODS The BMC_Brij35/SDS-QRAR models using mixed micellar system of Brij35/SDS (85??15) as a mobile phase under adequate experimental conditions were developed for the biological parameter estimation of quinolones. The correlation between retention factors and biological activities was investigated using second order polynomial models. The predictive and interpretative ability of the chromatographic models was evaluated in terms of cross-validated data (RMSEC, RMSECV and RMSECVi). RESULTS The BMC_Brij35/SDS-QRAR models of t_1/2, CL, V_d and AUC were statistically significant and both interpolation and extrapolation of parameters were reasonably adequate. CONCLUSION The mixed micellar liquid chromatography can simulate the resting membrane potential and the conformation of the long hydrophilic polyoxyethylene chains, which may become a simple, economic, and highly reproducible option for establishing QRAR model.     

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??OBJECTIVE To prepare green fluorescent protein-hemagglutinin A-cervical cancer(GFP-HA-PTP) fusion protein trageting HPV transformed cervical cancer cells with endosome escape capability, and further investigate its penetrating ability for cervical cancer cell lines. METHODS pET15b-GFP-HA-PTP expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded GFP-HA-PTP fusion protein was purified by Ni-NTA His-Bind resin and conformed by Western blotting assay. Specific penetrating analysis of GFP-HA-PTP was performed under fluorescence microscopy. RESULTS GFP-HA-PTP fusion protein was highly expressed in BL21 cells. Western blotting analysis result showed that the fusion protein was expressed correctly. The fusion protein can selectively penetrate into cervical cancer cell lines Siha and Caski with endosome escape efficiently. CONCLUSION GFP-HA-PTP can specificly penetrate into cervical cancer cell lines after endosome escaping with high efficiency.
     

小钻木脂素类化合物戈米辛R的体外抗炎机制研究

目的 通过脂多糖(LPS)诱导活化小鼠单核巨噬细胞系RAW264.7建立炎症模型,探讨小钻木脂素类化合物戈米辛R对炎症细胞增殖及炎症因子分泌的影响,评价其抗炎活性及机制。方法 采用MTT比色法检测经LPS诱导的RAW264.7细胞存活率并计算半数抑制浓度(IC₅₀);采用ELISA法检测细胞上清液TNF-α、IL-1β、IL-6等炎症因子含量;利用RT-PCR法检测细胞TNF-α、IL-1β、IL-6、IκB-α、NF-κB p65的mRNA表达量;Western blot法检测细胞IκB-α及NF-κB p65蛋白表达量。结果 与LPS模型组比较,戈米辛R可显著抑制LPS诱导的RAW264.7细胞增殖;降低细胞分泌TNF-α、IL-1β及IL-6的含量;升高LPS所致降低的IκB-α mRNA的表达量同时降低LPS引起升高的TNF-α、IL-1β、IL-6、NF-κB p65的mRNA表达量;升高IκB-α并降低NF-κB p65的蛋白表达量,显示出良好的剂量依赖性,具有统计学差异(P<0.05)。结论 戈米辛R可通过抑制TNF-α、IL-1β、IL-6、NF-κB p65的mRNA和NF-κB p65蛋白的表达,增加IκB-α mRNA和IκB-α蛋白的表达,减少致炎因子TNF-α、IL-1β、IL-6的释放,从而发挥显著抗炎活性。     

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??OBJECTIVE To investigate the protective effect and possible mechanism of Rhizoma Coptis(RC) on lipopolysaccharide(LPS)-induced inflammatory injury in rat hepatocytes(BRL). METHODS LPS-induced BRL cells injury model was established in vitro, then the damaged cells were given different interventions and treatment with 0.175, 0.1 mg?? mL^-1 RC aqueous extract as the test drug, and dexamethasone(Dex) as positive control drug. The optimal test doses of LPS and RC aqueous extract were selected and determined by cell counting kit-8(CCK-8), the cellular apoptosis rate was determined by flow cytometry, TLR4/NF-??B and TLR4/IRF3 signaling pathways and the mRNA level of related inflammatory mediators(TNF-??, IL-1??, IL-6) were detected by RT-PCR, the NF-??B p65 protein expression was analysed by Western blot and immunofluorescence techniques. RESULTS ??Compared with normal control group, 0.1 mg??mL^-1 LPS affected on BRL cells for 24 h, the cell survival rate was decreased significantly(P<0.01), the apoptotic rate increased significantly(P<0.01), the mRNA level of TLR4, NF-??B, IRF3, TNF-??, IL-1??, IL-6 were significantly increased(P<0.01), and the NF-??B p65 protein expression was increased. ??Compared with the model group, 0.1 and 0.175 mg??mL^-1 RC affected on LPS-induced BRL cells for 24 h, the survival rate of BRL cells was increased significantly(P<0.05), the apoptotic rate decreased significantly(P<0.01), the mRNA level of TLR4, NF-??B, IRF3, TNF-??, IL-1??, IL-6 and the NF-??B p65 protein expression were decreased significantly(P<0.01). CONCLUSION Rhizoma Coptis has obviously protective effect on LPS-induced inflammatory injury in rat hepatocytes(BRL), the mechanism of which may be related with inhibiting apoptosis, reducing the release of inflammatory factors such as TNF-????IL-1?? and IL-6, blocking NF-??B p65 protein nuclear translocation, interfering the R4/NF-??B and TLR4/IRF3 signaling pathway.     

基于TLR4/MAPKs/NF-κB炎症信号通路探讨三痹颗粒对Ⅱ型胶原性关节炎大鼠的治疗作用及机制研究

目的探讨三痹颗粒对Ⅱ型胶原性关节炎大鼠的治疗作用以及对TLR4/MAPKs/NF-κB信号通路的影响。方法将60只Wistar大鼠随机分为空白对照组(CTL)、模型组、阳性对照组(地塞米松片,DXM)和受试药物低、中、高剂量组,每组各10只。建立胶原性关节炎(CIA)大鼠模型,各组于初次免疫2周后开始灌胃。连续灌服20 d,至免疫后35 d处死大鼠,观察大鼠状态、足肿胀度、关节炎指数(AI)、踝关节病变观察。采用ELISA法检测白介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)水平。采用qRT-PCR法和Western blot法检测Toll样受体4(TLR4)、核转录因子-κB(NF-κB)(p65)、p-NF-κB(p65)、p38、p-p38、细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、c-Jun氨基末端激酶(JNK)、p-JNK mRNA和蛋白的表达水平。结果实验结束时,DXM组、受试药物高、中、低剂量组大鼠足踝关节肿胀程度和AI值均低于模型组(P<0.05)。经H&E染色和肉眼观察,受试药物可明显减轻足水肿以及滑膜组织病理学变化。DXM组和受试药物低、中、高剂量组大鼠外周血IL-1β、IL-6、TNF-α水平均明显低于模型组大鼠(P<0.05);而受试药物高剂量组大鼠外周血IL-1β、IL-6、TNF-α水平与DXM组大鼠比较未见统计学差异(P>0.05)。另外,模型组大鼠足踝关节滑膜组织TLR4、p-NF-κB(p65)、p-p38、p-ERK1/2、p-JNK mRNA和蛋白表达量明显高于CTL组(P<0.05);而且DXM组以及受试药物低、中、高剂量组较模型组明显降低(P<0.05);尤其以DXM组和受试药物高剂量组降低最为明显,与CTL组比较,无统计学差异(P>0.05)。结论三痹颗粒可能通过负性调控TLR4/MAPKs/NF-κB信号通路保护胶原性关节炎大鼠模型足踝关节炎症损伤。     

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??OBJECTIVE To study the effects of Kadsura coccinea alcohol extract(KCAE) on rats with immunologic hepatic fibrosis and research the possible mechanisms in it. METHODS Totally 60 SD male rats were randomly divided into 6 groups: a normal control group,a model group, a compound Biejia-ruangan tablets group(0.7 g??kg^-1), KCAE high, middle and low dose groups(1.68, 0.84, 0.42 g??kg^-1) at ten in each groups. Except for the normal control group,other groups were duplicated intraperitoneal injection of porcine serum twice a week at dose of 0.5 mL??time^-1. The rats in treatment groups were intragastric administration respectively, meanwhile, the rats in normal control and model groups were treated with the same volume of distilled water, once a day for 15 weeks. The liver was weighed to calculate the liver index. Alanine aminotransferase(ALT), aspartate aminotransferase(AST), total protein(TP), albumin(ALB) and total bilirubin(TB) were evaluated by the Mind-Ray automatic biochaemical analyzer. The expression level of procollagen ??(PC??), collagen type ??(??-C), laminin(LN), hyaluronic acid(HA), transforming growth factor-??1(TGF-??1), interkeukin-10(IL-10), interferon-??(IFN-??) and tumor necrosis factor-??(TNF-??) in serum were detected by ELISA. The degrees of fibrosis were evaluated by HE and Masson straining, and the expression levels of TGF-??₁ in liver tissue were assessed by Western blot. RESULTS Compared with model group, the liver index of KCAE high-dose group was decreased significantly(P??0.01). The expression levels of ALT, AST, TP, ALB, TB were within normal range, the differences were not statistically significant(P>0.05). KCAE could decrease the level of PC??, IV-C, LN, HA, TGF-??₁, TNF-?? and increase the level of IFN-?? in serum. KCAE could alleviate the hepatic fibrosis in rats(P??0.01) and inhibit the expression of TGF-??₁ in the liver tissues significantly(P??0.01). CONCLUSION KCAE has an anti-immunologic hepatic fibrosis effect in rats and the mechanisms possibly involve effectively regulating inflammatory cytokines, reducing extracellular matrix expression and inhibiting the expression of TGF-??₁.     

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??OBJECTIVE To investigate the protection of lyophilized powder of catalpol and puerarin (C-P) on oxygen-glucose deprivation/reperfusion(OGD/R)-injured astrocytes and the possible mechanism in vitro.METHODS Primary astrocytes were isolated from the cerebral cortex of neonatal rats. The purity of astrocytes was identified by GFAP immunofluorescence. Astrocytes were divided into 6 groups:normal group, model group, excipients group, and three groups with gradient doses of C-P (12.25, 24.50, 49.00 ??g??mL^-1). After astrocytes suffered from OGD/R (OGD 6 h/R 12 h) with or without C-P, cell survival, LDH leakage, and apoptosis were analyzed by MTT, colorimetry, and TUNEL respectively. The apoptotic protein, caspase-3, was evaluated by Western blot. Meanwhile, oxidative indexes including SOD, GSH, ROS, MDA, and NO were detected by assay kits. In terms of inflammation, TNF-??, IL-1??, and PGE₂ in medium were evaluated via ELISA. iNOS, COX-2, NF-??B p65, p-NF-??B p65, I??B-??, and p-I??B-?? were examined by Western blot. RESULTS The purity of primary astrocytes was more than 97%. Compared with normal group, the cell survival rate of model group decreased significantly, while the LDH leakage rate and cell apoptosis rate markedly increased after OGD/R injury in model group. Meanwhile, SOD activity and GSH level in astrocytes markedly decreased. The level of MDA and ROS significantly increased. The content of NO, TNF-??, IL-1??, and PGE₂ released in medium also sharply increased. However, those changes of related biochemical indexes above could be reversed by different gradient doses of C-P. There was no significant difference between excipients group and model group. Further study found that C-P could inhibit the protein expression of caspase-3, iNOS, and COX-2, as well as the increase of phosphorylation level of NF-??B p65 and I??B-?? significantly. CONCLUSION C-P shows a protective effect on the OGD/R injured astrocytes in vitro, and its protection mechanism involves in anti-inflammation, antioxidant, inhibiting the cell apoptosis and activation of NF-??B signaling pathway.     

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??OBJECTIVE To investigate the anti-inflammatory effects of extract of Scutellaria baicalensis Georgi (SGE) and underlying mechanism by using LPS-induced microglial BV2 cells. METHODS MTT assay was used to observe the cell viability. The content of NO in cell supernatant was measured by Griess reagent. The levels of IL-1??, IL-6 and TNF-?? were detected by ELISA kits. The intracellular TLR4 expression was assayed by Western blotting. RESULTS The levels of NO, IL-1??, IL-6 and TNF-?? were significantly increased induced by LPS in the supernatant of BV2 cells (all P<0.01). However, co-treatment with SGE 100 ??g??mL^-1 significantly decreased the production of related inflammatory factors including NO (P<0.01), IL-1??(P<0.01), IL-6 (P<0.01) and TNF-?? (P<0.05). Furthermore, SGE significantly inhibited the TLR4 expression induced by LPS in BV2 cells. CONCLUSION SGE is able to alleviate LPS-induced inflammatory responses in BV2 cells through down-regulation of TLR4 protein expression suggesting that SGE has therapeutic potential for the treatment of neuroinflammatory diseases.     

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??OBJECTIVE To observe the effect of Dracocephalum heterophyllum flavonoids(DHBF) of Uygur Medicine on cardiomyocyte hypertrophy, which were induced by angiotensin ??(Ang??), and it could provide a basis for further study to the mechanism. METHODS SD rats,0-3 d of age, neonatal rat myocardial cells cultured in vitro, the experiment was divided into control group, Ang??(1 ??mol??L^-1)group, different concentrations of DHBF(10, 25, 50 ??mol??L^-1)+Ang??(1 ??mol??L^-1) groups, cardiomyocyte hypertrophy were induced by Ang?? 1 ??mol??L^-1 and was intervened using DHBF respectively, CCK-8 method was used to observe the activity of myocardial cells,RT-PCR technique was used to detect the expression of m RNA of cardiac hypertrophy gene atrial natriuretic peptide(ANP) and brain natriuretic peptide(BNP),the internal factor was glyceraldehyde-3-phosphate dehydrogenase(GAPDH) .Confocal laser scanning was used to detect the surface area of myocardial cell was [Ca²⁺]_i;the activity of Ca²⁺-ATP was measured by the enzymatic reaction of fragmentation cell; the concentration of NO and the activity of NOS were determined by colorimetry. RESULTS Compared with the control group, the activity of myocardial cell was(85%??5%) in the Ang??group,which was increased significantly after it was dealed with DHBF and Ang??(P<0.05);RT-PCR results showed the expression of mRNA of ANP and BNP were increased by using Ang??, which were lower by using DHBF and Ang??. The surface area of myocardial cell were increased by using Ang??, which could be reversed by using DHBF and Ang??. Confocal laser scanning showed the concentration of[Ca²⁺]_i was increased by using Ang??,but which was lower significantly by using DHBF and Ang??(P<0.05). The enzymatic reaction of fragmentation cell results showed the activity of Ca²⁺-ATP was decreased by using Ang??, but which was increased significantly by using DHBF and Ang??(P<0.05). Colorimetry results showed the concentration of NO and the activity of NOS were decreased by using Ang??, but which was increased significantly by using DHBF and Ang??(P<0.05). CONCLUSION DHBF can improve the activity of hypertrophy cardiomyocytes which were induced by ang??, downregulate expression of mRNA of ANP and BNP, reduce surface area of cardiomyocytes induced by Ang??,the mechanism of action may be related to promoting the release of NO, regulating the concentration of[Ca²⁺]_i and the activity of Ca²⁺-ATP.     

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??OBJECTIVE To study the effects of fibroblast growth factor-21(FGF-21) on acute inflammation induced by lipopolysaccharide (LPS)and its mechanism. METHODS Mouse model of acute inflammation was established by injection of LPS and treated with FGF-21 at high, medium and low doses, the pathological changes were detected with HE staining, the expression level of TNF-??, IL-1??, IL-6 and IL-17 in serum and peritoneal macrophage were determined by ELISA and Real-time PCR. NF-??B p65 in macrophage cells was analyzed by confocal laser scanning microscope and Western-blotting. RESULTS FGF-21 treatment reduced lung damage and inflammatory cell infiltration and decreased the expression level of TNF-??, IL-1??, IL-6 and IL-17 in both serum and peritoneal macrophage. The results of confocal laser scanning microscope and Western-blotting both showed that FGF-21 could inhibit NF-??B transferring to the nucleus. CONCLUSION FGF-21 could regulate the immune system by acting on macrophage. Relieving the inflammatory response in mice through NF-??B signal pathway may be one of the mechanisms FGF-21 regulating immune system.     

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??OBJECTIVE To observe the effect of chlorogenic acid on the secretion of inflammatory cytokines and regulation mechanism of P38MAPK, NF-??B signaling pathway in human hepatic stellate cells induced by TGF-??1. METHODS Different concentrations of CGA worked in normal and activated hepatic stellate cells to make sure the appropriate drug concentration.The exponential growth phase cells were randomly divided into normal HSC group, normal HSC+CGA group, after cultured 48 h, the cells were cultured with 0??50??100 mg??L^-1 CGA for 24 h; HSC(TGF-??1) group?? HSC(TGF-??1 + CGA) group: after 24 h, the cells were induced by 10 ??g??L^-1 TGF-??1 for 24 h, and then cultured with 0, 50, 100 mg??L^-1 CGA for 24 h. The expression of ??-SMA protein was detected by immunocytochemistry, the expression of p-P38, P65 protein was detected by Western-blot, the expression of TNF-??, IL-6 mRNA was detected by real time quantitative PCR, and the content of TNF-??, IL-6 in the supernatant was detected by ELISA method. RESULTS The appropriate concentrations of CGA were 50 and 100 mg??L^-1, these concentration has no effect on normal HSC(P>0.05); after stimulation by TGF-??1, the expression of ??-SMA, p-P38, P65, TNF-??, IL-6 was increased(P<0.01), when activated HSC cells were treated with 50 and 100 mg??L^-1 CGA, the expression of ??-SMA, p-P38, P65, TNF-??, IL-6 was decreased(P<0.05, P<0.01). CONCLUSION CGA can inhibit the proliferation of activated HSC, regulate the secretion of inflammatory factors such as TNF-??, IL-6 by P38MAPK and NF-??B signaling pathway, inhibit the occurrence of liver fibrosis.