Material basis and mechanism of Curcuma longa tuberous roots with and without vinegar processing in treating primary dysmenorrhea / 中国中药杂志

Liquid chromatography-mass spectrometry was employed to analyze the chemical components in Curcuma longa tuberous roots(HSYJ), C. longa tuberous roots processed with vinegar(CHSYJ), and rat serum after the administration. The active components of HSYJ and CHSYJ absorbed in serum were identified based on the secondary spectrum of database and literature. The targets of primary dysmenorrhea was screened out from database. The protein-protein interaction network analysis, gene ontology(GO) functional annotation, and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed for the common targets shared by the drug active components in serum and primary dysmenorrhea, and the component-target-pathway network was constructed. AutoDock was used to conduct molecular docking between the core components and targets. A total of 44 chemical components were identified from HSYJ and CHSYJ, including 18 absorbed in serum. On the basis of network pharmacology, we identified 8 core components(including procurcumenol, isobutyl p-hydroxybenzoate, ferulic acid, and zedoarondiol) and 10 core targets \[including interleukin-6(IL-6), estrogen receptor 1(ESR1), and prostaglandin-endoperoxide synthase 2(PTGS2)\]. The core targets were mainly distributed in the heart, liver, uterus, and smooth muscle. The molecular docking results showed that the core components were well bound to the core targets, indicating that HSYJ and CHSYJ may exert therapeutic effect on primary dysmenorrhea via estrogen, ovarian steroidogenesis, tumor necrosis factor(TNF), hypoxia-inducible factor-1(HIF-1), IL-17 and other signaling pathways. This study clarifies the HSYJ and CHSYJ components absorbed in serum, as well as the corresponding mechanism, providing a reference for further elucidating the therapeutic material basis and clinical application of HSYJ and CHSYJ.

Manufacturing classification system for oral solid dosage forms of traditional Chinese medicines(Ⅰ): classification of processing routes / 中国中药杂志

Oral solid dosage(OSD) occupies a key position in the market of Chinese patent medicines and new traditional Chinese medicines. Processing route is the foundation for the research and development of traditional Chinese medicine OSDs. On the basis of prescriptions and preparation methods of 1 308 traditional Chinese medicine OSDs recorded in the Chinese Pharmacopoeia, we summarized the patterns of processing routes of both modern dosage forms(tablets, granules, and capsules) and traditional dosage forms(pills and powder) and constructed a manufacturing classification system(MCS) based on the processing routes. Based on the MCS, statistical analyses were conducted respectively on medicinal materials, pharmaceutical excipients, extraction solvents in the pretreatment process, crushed medicinal materials, methods of concentration and purification, and methods of drying and granulation, aiming to uncover the process features. The results showed that each dosage form can be prepared via different routes with different processing methods of decoction pieces and raw materials for dosage preparation. The raw materials for dosage form preparation of traditional Chinese medicine OSDs included total extract, semi-extract, and total crushed powder, which accounted for different proportions. The raw materials for traditional dosage forms are mainly decoction pieces powder. Semi-extracts are the main raw materials for tablets and capsules, which account for 64.8% and 56.3%, respectively. Total extracts are the main raw materials for granules, with a proportion of 77.8%. Compared with tablets and capsules, traditional Chinese medicine granules with dissolubility requirements had a larger proportion of water extraction process, a higher proportion of refining process(34.7%), and a lower proportion of crushed medicinal mate-rials in semi-extract granules. There are four ways to add volatile oil to the modern dosage forms of traditional Chinese medicine. In addition, some new technologies and processes have been used in concentration, filtration, and granulation processes of traditional Chinese medicine OSDs, and the application of pharmaceutical excipients is diversified. The results of this study are expected to provide reference for the processing route design and upgrading of OSDs for new traditional Chinese medicines.

Effect of processing with vinegar on efficacy of Curcuma longa in treatment of dysmenorrhea in rats with syndrome of liver depression and Qi stagnation / 中国中药杂志

This study compared the effects of Curcuma longa before and after processing with vinegar on the rat model of dysmenorrhea with the syndrome of liver depression and Qi stagnation to reveal the mechanism of vinegar processing in improving the role of C. longa in soothing liver and relieving pain. The rat model of dysmenorrhea with the syndrome of liver depression and Qi stagnation was established according to the Preparation of the Animal Model of Dysmenorrhea(Draft) and the chronic unpredictable stress me-thod. The changes in the body weight, organ indexes, writhing latency, writhing score, and serum levels of six liver function indicators, sex hormones, pain factors, and blood rheological indicators were measured to evaluate the efficacy of C. longa processed with vinegar or not in treating dysmenorrhea in the rats with syndrome of liver depression and qi stagnation. Compared with the model group, the C. longa group(processed with vinegar or not) showed slow weight loss, increase in writhing latency, and decrease in writhing response(P<0.05). The inhibition rates on writhing in raw C. longa, vinegar-processed C. longa, and positive groups were 33.780%, 64.611%, and 62.466%, respectively. The significantly higher inhibition rate of the vinegar processing group indicated that vinegar-processed C. longa demonstrated more significant therapeutic effect. The vinegar-processed C. longa group showed lower levels of alanine aminotransferase(ALT), alkaline phosphatase(ALP), aspartate aminotransferase(AST), direct bilirubin(DBIL), and total bilirubin(TBIL) and higher level of albumin(ALB)(P<0.05), which indicated that vinegar processing enhanced the therapeutic effect of C. longa on liver injury. The serum levels of estradiol(E_2) and oxytocin(OT) were lower in the vinegar-processed C. longa group(P<0.05), indicating that the vinegar-processed C. longa could regulate the sex hormone levels, reduce the activity of uterine smooth muscle and contraction of uterus, and alleviate the symptoms of dysmenorrhea in rats. Moreover, the vinegar-processed C. longa group showed lower interleukin-6(IL-6) and arginine vasopressin(AVP) levels and higher beta-endorphin(β-EP) level(P<0.05), which indicated that vinegar-processed C. longa regulated the levels of pain factors to exert the pain-relieving effect. Drug intervention decreased the whole blood viscosity low-cut, medium-cut and high-cut values, plasma viscosity, whole blood reduction viscosity low-cut and high-cut values, erythrocyte cumulative pressure, and equation K value of erythrocyte sedimentation rate(P<0.05), and the vinegar-processed C. longa group outperformed other groups. This result indicated that vinegar processing enhanced the function of C. longa in improving the local blood rheology. C. longa processed with vinegar can enter the liver to relieve the da-mage to the heart, liver, kidney, and uterus, repair the liver function, and recover the sex hormone levels and immune function by regulating the levels of sex hormones and pain factors and improving the blood rheology. It activates the pain-relieving mechanism to relieve the pain, protect the liver, and fight inflammation, which is consistent with the theory that vinegar processing facilitates C. longa entering the liver to sooth liver and relieve pain.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer's disease by the high-content fluorescence imaging system / 中国天然药物

Ubiquitin-proteasome system (UPS) plays an important role in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important. In this study, we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD. At first, stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells, together with G418 screening. The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1. By employing the high-content fluorescence imaging system, together with stable YFP-CL1 HT22 cells, the UPS activators were successfully screened from our established TCM library. The representative images were captured and analyzed, and quantification of the YFP fluorescence intensity was performed by flow cytometry. Then, the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP (APP), pRK5-EGFP-Tau P301L (Tau P301L), or pRK5-EGFP-Tau (Tau) transiently transfected HT22 cells using fluorescence imaging, flow cytometry, and Western blot. In conclusion, our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the high-throughput screening of the UPS activators. Three compounds, namely salvianolic acid A (SAA), salvianolic acid B (SAB), and ellagic acid (EA), were identified to significantly decrease YFP fluorescence intensity, which suggested that these three compounds are UPS activators. The identified UPS activators were demonstrated to clear AD-related proteins, including APP, Tau, and Tau P301L. Therefore, these findings provide a novel insight into the discovery and development of anti-AD drugs.

Impact of Particle Size on Density of Binary Powder Mixture of Chinese Medicine / 中国实验方剂学杂志

Objective:To investigate the influence of particle size on density of binary powder mixture of traditional Chinese medicine （TCM）， and to provide reference for formulation design of TCM preparations. Method:Three groups of binary powder mixtures with different particle size ratio （<italic>α</italic>） were constructed， namely Oroxyli Semen-microcrystalline cellulose PH102 （MCC PH102） （<italic>α</italic>=0.071 7）， Stellariae Radix-MCC PH200 （<italic>α</italic>=0.158 7） and Angelicae Sinensis Radix-MCC KG802 （<italic>α</italic>=0.840 6）. Binary powder mixtures with nine mass ratios （90∶10， 80∶20， 70∶30， 60∶40， 50∶50， 40∶60， 30∶70， 20∶80 and 10∶90） were prepared for each group， and 27 binary powder mixtures containing TCM were obtained. The particle size distribution， density and other parameters of six single materials and 27 binary powder mixtures were characterized. Based on the packing theory and multivariate analysis， the effects of particle size related parameters on the filling structure and density of the binary powder mixtures were elucidated. Result:The <italic>α</italic> of Angelicae Sinensis Radix-MCC KG802 binary mixture system was larger than the replacement rate （<italic>α</italic>_r=0.741 0）， and its density had a good linear relationship with the mass ratio， which conformed to the replacement mechanism. The <italic>α</italic> of Oroxyli Semen-MCC PH102 binary mixture system was smaller than the critical ratio （<italic>α</italic>_c=0.154 0）， and its density was nonlinear with the mass ratio of components， which conformed to the filling mechanism. The <italic>α</italic> of Stellariae Radix-MCC PH200 binary mixture system was between <italic>α</italic>_c and <italic>α</italic>_r， its density was affected by both of replacement mechanism and filling mechanism. Based on the partial least squares （PLS） model， the variable importance in the projection （VIP） analysis further proved that the mixing mass ratio （VIP value=1.62）， <italic>α</italic> （VIP value=1.13） and <italic>D</italic>₁₀ （the corresponding particle size when the particle size distribution accumulated to 10%， VIP value=1.06） were the key factors affecting the density of binary powder mixtures of TCM. Conclusion:In the binary powder mixtures of TCM， the linearity relationship between density and mass ratio is largely depended on particle size difference of components.

Material manufacturability classification in high shear wet granulation process of Chinese medicine / 中国中药杂志

The high shear wet granulation(HSWG) process of Chinese medicine has a complicated mechanism. There are many influencing factors that contribute to this process. In order to summarize the manufacturability of different kinds of materials in HSWG, this paper constructed a material library composed of 11 materials, including 4 Chinese medicine extracts and 7 pharmaceutical excipients. Each material was described by 22 physical parameters. Several binders were employed, and their density, viscosity and surface tension were characterized. Combining empirical constraints and the principle of randomization, 21 designed experiments and 8 verification experiments were arranged. The partial least squares(PLS) algorithm was used to establish a process model in prediction of the median granule size based on properties of raw materials and binders, and process parameters. The surface tension and density of binders, as well as the maximum pore saturation were identified as key variables. In the latent variable space of the HSWG process model, all materials could be divided into three categories, namely the Chinese medicine extracts, the diluents and the disintegrants. The granulation of Chinese medicine extracts required low viscosity and low amount of binder, and the resulted granule sizes were small. The diluent powders occupied a large physical space, and could be made into granules with different granule sizes by adjusting the properties of binders. The disintegrants tended to be made into large granules under the condition of aqueous binder. The combination use of material database and multivariate modeling method is conducive to innovate the knowledge discovery of the wet granulation process of Chinese medicine, and provides a basis for the formulation and process design based on material attributes.

Dose-toxicity-effect relationship between licorice combined with rhubarb in purgation / 中国中药杂志

The dose-toxicity-effect relationship between licorice combined with rhubarb in purgation was studied. A total of 108 ICR mice were divided into control group,model group,positive group,low,medium and high-dose rhubarb groups,and low,medium and high-dose rhubarb-liquorice decoction group. After 6 days of continuous administration of loperamide hydrochloride,the constipation model of mice was replicated,and each group was given lactulose,different doses of rhubarb and rhubarb-liquorice decoction for 14 days. After administration,the defecation characteristics,blood biochemistry,liver,kidney and colon pathological changes in each group were compared. Based on the objective weight given by factor analysis,the dose-toxicity-effect relationship was comprehensively analyzed by multi-index scoring method. Two common factors were extracted by factor analysis,representing effect and toxicity respectively. The results showed that rhubarb could exert a diarrhea effect at the dosage of 1/2,2 and 8 times of the high limit set forth in the Chinese Pharmacopoeia,increase the defecation volume and the intestinal tract propulsion rate,reduce the time of anal and the oral transmission,and increase the water content of feces. The combination with licorice could alleviate its diarrhea effect,especially at the dosage of 1/2 times of the high limit set forth in the Chinese Pharmacopoeia. However,rhubarb showed obvious hepatic and colon toxicities at the dosage of 2 and 8 times of the high limit set forth in the Chinese Pharmacopoeia,and the combination with licorice could significantly reduce its toxicity. It shows that licorice has a " mediating" effect on rhubarb by alleviating the purgation property and reducing the toxicity.

Screening of serum susceptibility biomarkers in patients with mild liver function abnormalities based on non-targeted metabolomics / 中国中药杂志

In the present study,non-targeted metabolomics technique was used to screen potentially susceptibility biomarkers in patients with mild liver function abnormalities during long-term use of Chinese herbal compound. According to the inclusion and exclusion criteria,we collected 7 cases of patients with abnormal liver function during the period of complete taking Chinese herbal medicine( 60 days),and 18 cases of patients with normal liver function in re-examination from the reproductive medicine center in our hospital. Ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOF/MS~E) technique combined with Progenesis QI software was used to analyze the differential biomarkers in serum of patients with wild liver function abnormalities and normal liver function. 11 potential biomarkers such as bilirubin,pantothenic acid,hippuric acid,sphingomyelin,palmitic acid,and oleic acid were tentatively identified. Metabolic disorders in patients with herbal-induced mild liver abnormality were mainly related to two pathways: pantothenic acid and coenzyme A biosynthesis and linoleic acid metabolism. It could provide a reference for the early warning of mild liver function abnormalities of patients that may be caused by long-term use of Chinese medicine compound in clinical application,and will lay a foundation for further understanding the endogenous substance changes in different levels of liver injury.

Chemistry and biology research on bitter-taste Chinese materia medica with function of regulating glycolipid metabolism / 中国中药杂志

The bitter taste is one of the important properties among five flavors of Chinese materia medica (CMM), characterized by downbearing and discharging, drying dampness, and consolidating Yin. In common CMM, bitter-taste CMM accounts for a large proportion, indicating the importance of it. Through the efficacy of clearing away heat and dampness, reducing fire and removing toxin, bitter-taste CMM has achieved good results in treating diabetes in clinical application, proving their definite therapeutic effect on regulating glucose and lipid metabolism (main features of diabetes). At present, there are many reports about the chemical constituents and pharmacological effects of CMM on diabetes, but there are few reviews on the chemistry and biology of bitter-taste CMM. This study summarized the properties and compatibility characteristics of bitter-taste CMM for treating diabetes, and mainly analyzed the chemistry and biology basis of bitter-taste CMM with function of regulating glycolipid metabolism, laying foundation for further researches on properties theory of CMM.

Analysis on compatibility and application of Glycyrrhizae Radix et Rhizoma and its herb pairs based on data mining method / 中草药

To study the compatibility and application rules of Glycyrrhizae Radix et Rhizoma (GRR) in prescription by data mining. Using computer programming method to transform the information of 26 185 formulas containing GRR in traditional Chinese medicine recipe database to facilitate data analysis. Firstly, the herbs frequently compatible with GRR are found. Then the proportion of GRR and another medicine, family and genus of the diseases treated are counted, the efficacy of formulas as well as their relationship. Through data mining, it is found that GRR often used with Panax ginseng, Angelica sinensis, Poria cocos, Paeonia lactiflora, Zingiber officinale, Atractylodes macrocephala, and other drugs. The most commonly used ratio is 1:1. These herb-pairs are usually used for internal medical diseases and exerting clearing heat, relieving pain, resolving phlegm and relieving cough and asthma, interior-warming, reinforcing deficiency, and checking retching effect, etc. The properties and functions of GRR are the basis of its compatibility. The compatibility of GMM can increase efficiency and reduce toxicity. This paper will provide a basis for the research of the efficacy of GMM and provide reference for its clinical application.

The effects of lifestyle factors on the incidence of central obesity in Chinese adults aged 35-74 years / 中华预防医学杂志

<p><b>OBJECTIVE</b>To understand the incidence of central obesity and its characteristics, and explore the effects of lifestyle factors on incidence of central obesity in Chinese adults aged 35-74 years.</p><p><b>METHODS</b>A total of 27 020 Chinese adults aged 35 to 74 years were enrolled in a prospective follow-up study (the study cohort was built from 1998 to 2000, respectively) during 2007 and 2008. Central obesity was defined as waist circumference ≥ 90 cm in men, ≥ 85 cm and ≥ 80 cm in women, respectively. Multivariate logistic regression was used to estimate relative risks (RR) of central obesity for lifestyle factors after adjusting factors including genders, age, southern and geographic region, urbanization, lifestyles, and so on.</p><p><b>RESULTS</b>Among Chinese adults aged 35-74 years, the standardized annual incidence of central obesity (waist ≥ 90 cm) was 2.19% for men and this rate decreased gradually with age among people younger than 65 years old. The incidence of central obesity was 2.64% (waist ≥ 85 cm) and 4.06% (waist ≥ 80 cm) for women, respectively, and this rate increased obviously among people aged 55 to 74 years. Participants with ≥ 12 years' education (RR = 0.84, 95%CI:0.74-0.96) had a lower risk of central obesity(waist ≥ 90 cm for men, waist ≥ 85 cm for women). And this risk increased as the monthly household per capita income increased. Compared with the reference group, people involved in housework or retirees (RR = 1.17; 95% CI: 1.01-1.36), drinking alcohol (RR = 1.15, 95% CI: 1.01-1.32) or scented tea (RR = 1.49, 95%CI:1.28-1.72) had a higher risk of developing central obesity, while drinking milk (RR = 0.85, 95% CI: 0.74-0.97) or black tea (RR = 0.74, 95% CI: 0.58-0.95), had a lower risk of developing central obesity.</p><p><b>CONCLUSION</b>A healthy lifestyle plays a key role in the prevention and control of central obesity in Chinese adults, and a healthy way of lifestyle should be promoted in the whole society to decelerate the epidemic of the central obesity.</p>

Effect of Trichinella spiralis infection on expression and distribution of colonic epithelial E-cadherin in mice and its mechanism / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the effect of Trichinella spiralis (T.spiralis) infection on the expression and distribution of colonic epithelial E-cadherin in mice and its mechanism.</p><p><b>METHODS</b>BALB/c mice and STAT6-/- mice were infected with T.spiralis, and mice without infection were used as control. Seven days later, the horseradish peroxidase (HRP) was infused by rectal enema. Serum HRP was detected in the subsequent 0, 60 and 120 minutes. Then the mice were sacrificed and colon was taken out. The distribution of E-cadherin in colon was detected by immunofluorescence staining, and the expression of E-cadherin was detected by Western blot. The expression of interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA.</p><p><b>RESULTS</b>Serum HRP level in infected BALB/c mice was significantly higher than that in control mice (P<0.05), while it was not significantly different between infected STAT6-/- mice and controls (P>0.05). In infected BALB/c mice, E-cadherin located in cytoplasm of colonic epithelial cells, while in controls, it located in cellular membrane. E-cadherin expression down-regulated significantly in infected BALB/c mice as compared to controls. E-cadherin expression and distribution did not change obviously in infected STAT6-/- and control mice. IL-4 level in mesenteric lymph nodes of infected BALB/c mice [(193.0±12.5) μg/L] was significantly higher as compared to control BALB/c and infected STAT6-/- mice [(21.0±2.3) μg/L and (15.0±3.1) μg/L, all P<0.05].</p><p><b>CONCLUSION</b>T.spiralis infection can increase colonic epithelial permeability of mice, which may be associated with induction of Th2 cytokine secretion.</p>

Reduning injection for community-acquired pneumonia: meta-analysis / 中国中药杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of Reduning injection for treating community-acquired pneumonia.</p><p><b>METHOD</b>Literatures about randomized controlled trials of Reduning injection for treating community-acquired pneumonia were reviewed. The methodological quality of the trials was assessed by the Jadad scale, and evaluation was performed.</p><p><b>RESULT</b>Seven RCTS were included. Reduning combined group (Reduning injection plus antibiotics and basic therapy) was compared with antibiotics group (antibiotics plus basic therapy). Meta-analysis showed that the relative risk (RR) for the total cure rate was 1.34, and 95% confidence interval (CI) was [1.19, 1.51]; RR for the total effective rate was 1.10, and 95% CI was [1.06, 1.15]. The weighted mean difference (WMD) in disappearance time of fever between the two groups was - 1.42, and 95% CI was [- 2.58, -0.26]; The WMD between the two groups for the total obvious effect rate of cough and expectoration were - 2.36, and 95% CI was [- 3.41, - 1.31]; Improve the time of pulmonary rales MD -2.30, 95% CI [- 2.61, - 2.00]; The WMD between the two groups in absorption of chest x-ray shadow was -2.36 and 95% CI [-2.52, -2.20]. Serious systematic adverse reactions had not been reported in the trials.</p><p><b>CONCLUSION</b>The effect of combined therapy with Reduning injection plus antibiotics and basic therapy is better than that of antibiotics plus basic therapy. Reduning injection can improve the symptoms of cough and expectoration, shorten the fever time and facilitate the absorption of chest x-ray shadow without any significant adverse reactions. However, further high quality trials are needed.</p>

Protective effects of omega-3 fish oil on lead-induced impairment of long-term potentiation in rat dentate gyrus in vivo / 生理学报

In order to evaluate the effect of omega-3 fish oil supplement by gavage (0.4 mL/100 g body weight) on the chronic lead-induced (0.2% lead acetate) impairments of long-term potentiation (LTP) in rat dentate gyrus (DG) in vivo, we designed the experiments which were carried out in four groups of newborn Wistar rats (the control, the lead-exposed, the control with fish oil treatment and the lead-exposed with fish oil treatment, respectively). The excitatory postsynaptic potential (EPSP) and population spike (PS) amplitude were measured in the DG of rats with above different treatments at the age of 80-90 d in response to stimulation applied to the lateral perforant path. The results showed (1) postnatal chronic lead-exposure impaired LTP measured on both EPSP slope and PS amplitude in DG area of the hippocampus; (2) in the control rats, omega-3 fish oil had no effect on LTP while in the lead-exposed rats, omega-3 fish oil had a protective effect on LTP. These results suggest that omega-3 fish oil supplement could protect rats from the lead-induced impairment of LTP. Omega-3 fish oil might be a preventive substance in reducing LTP deficits induced by lead.

Clinical observation on three-combination needling method for treatment of primary trigeminal neuralgia / 中国针灸

<p><b>OBJECTIVE</b>To probe into a better therapy for primary trigeminal neuralgia.</p><p><b>METHODS</b>Eighty-six cases were randomly divided into an observation group (n = 46) and a control group (n = 40). The observation group were treated with the three-combination needling method, i. e. acupuncture, acupoint-injection and fire-needle therapy, and the control group with acupuncture and acupoint-injection. After treatment of 2 courses, their therapeutic effects were assessed.</p><p><b>RESULTS</b>The total effective rate of 93.5% and the cured rate of 60.9% in the observation group were better than 65.0% and 22.5% in the control group, with significant differences (both P < 0.01).</p><p><b>CONCLUSION</b>The three-combination needling method has obvious clinical therapeutic effect on primary trigeminal neuralgia.</p>

Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand / 生物工程学报

Hepatitis C virus (HCV) is an important human pathogen that causes chronic liver disease worldwide. It is desirable to develop vaccines to prevent HCV infection, or at least to prevent progression to chronicity. We once constructed an optimized hepatitis C virus core and envelope 2 fusion antigen DNA vaccine, which could induce humoral and cellular immune responses against HCV core and E2 protein in BALB/c mice efficiently. Flt3 (Fms-like tyrosine kinase 3) -ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells, particularly dendritic cells. We reasoned that a DNA vaccine coexpressing the antigen and FL may activate immune responses more effectually. In this study, The influence of FL on this HCV DNA vaccine was evaluated. The cDNA encoding signal peptide and extracellular domain of murine FL was inserted into the plasmid pST-CE2t, and the resulting plasmid pST-CE2t/FL was transfected into COS7 cells. The HCV core and E2 protein were detected by Western blotting, and the soluble murine FL was detected by ELISA. Eight-week-old female BALB/c mice were inoculated intramuscularly with 100 microg pST-CE2t, pST-CE2t/FL or mock vector, respectively, and boosted at the same dosage 3 weeks later. Anti-HCV core and E2 total IgG and isotypes were measured at weeks 1,3,5,7. Splenocyte proliferative response to recombinant HCV core and E2 protrein were detected at week 7. SP2/0 cells expressing HCV core protein were used as target cells for the detection of cytotoxic T lymphocyte (CTL) response. Western blot analysis showed that a protein band with molecular weight about 70 kD from lysate of COS7 cells transfected with plasmid pST-CE2t/FL could be detected by anti-HCV core or E2 monoclonal antibodies, which indicated that pST-CE2t could express glucosylated HCV core and E2 fusion protein. Murine FL could be detected in the culture supernatant of COS7 cells transfected with pST-CE2t/FL. Plasmid pST-CE2t immunized mice developed higher anti-HCV core and E2 IgG seroconversion rates and titers than pST-CE2t/FL group did at different various times, but the IgG2a/IgG1 ratio of anti-HCV E2 protein in pST-CE2t/FL group is much higher than pST-CE2t group. Splenocytes from pST-CE2t or pST-CE2t/FL immunized mice could proliferate with stimulation of HCV core or E2 protein in vitro, although pST-CE2t/FL group showed much stronger response. Splenocytes from mice immunized with pST-CE2t/FL induced 79.03% +/- 9.95% of target cell lysis at the effector/target ratio of 100:1, which was significantly greater than the lysis (62.2% +/- 8.62%) observed in mice immunized with pST-CE2t. Our data demonstrated that the incorporation of FL can preferentially enhance the cellular response to this HCV fusion antigen DNA vaccine. In contrast, HCV specific antibodies were inhibited by FL in vaccinated mice. More and more data supports that recovery from acute HCV infection may depend upon the generation of broad-based cellular immune responses to viral proteins. So, FL may be of potential value as an adjuvant in the development of DNA-based immunization for prophylactic and therapeutic vaccine against HCV infection.