Label-free target discovery technology of small molecule drug and its application in traditional Chinese medicines / 药学学报

The discovery of drug targets plays a crucial role in drug research. Accurate information of small molecule drug-protein interaction can be provided by label-free target discovery technology without any structural modification at the small molecule. So, the label-free drug target discovery technology had become the powerful tool to discover the targets of drugs. Due to the “multi-component and multi-target” characteristics of traditional Chinese medicines (TCMs), the research on its targets and mechanism had been restricted. Based on potential of the label-free target discovery technology in the research of TCMs, this paper summarized the label-free target discovery technology and its application in TCMs research. It will provide a reference for the discovery of targets of TCMs and a new view for promoting the modernization of TCMs.

Effect of wheat-grain moxibustion on the expression of Beclin-1/GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy / 中国针灸

OBJECTIVE@#To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14) on the expressions of Beclin-1 and GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy (CSR), and to explore the possible analgesic mechanism of wheat-grain moxibustion for CSR.@*METHODS@#A total of 48 SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group and a wheat-grain moxibustion+3-MA group, 12 rats in each group. The CSR model was prepared by spinal cord insertion method. Three days after modeling, the rats in the model group were intraperitoneally injected with 1 mL of 0.9% sodium chloride solution; the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time) on the basis of the model group; the rats in the wheat-grain moxibustion+3-MA group were intraperitoneally injected with 3-MA solution and wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time). The three groups were intervened for 7 days, once a day. The gait score and mechanical pain threshold were observed before treatment and 7 days into treatment; after the treatment, the expressions of mRNA and protein of Beclin-1 in spinal dorsal horn were detected by real-time fluorescence quantitative PCR and immunohistochemistry; the expression of GRP78 protein in spinal dorsal horn was detected by Western blot method; the autophagosomes and ultrastructure in spinal dorsal horn neurons were observed by electron microscope.@*RESULTS@#After the treatment, compared with the sham operation group, in the model group, the gait score was increased and the mechanical pain threshold was decreased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was increased (P<0.01). Compared with the model group and the wheat-grain moxibustion+3-MA group, in the wheat-grain moxibustion group, the gait score was decreased and mechanical pain threshold was increased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was decreased, and the expressions of mRNA and protein of Beclin-1 were increased (P<0.01). Under electron microscope, the ultrastructure of spinal dorsal horn neurons in the wheat-grain moxibustion group was not significantly damaged, and its structure was basically close to normal, and the number of autophagosomes was more than the other three groups.@*CONCLUSION@#Wheat-grain moxibustion at "Dazhui" (GV 14) has analgesic effect on CSR rats. The mechanism may be related to moderately up-regulate the expression of Beclin-1, enhance autophagy and reduce endoplasmic reticulum stress.

Observation on clinical effect of acupuncture combined with wheat-grain moxibustion for mild to moderate postpartum depression / 中国针灸

OBJECTIVE@#To compare the clinical effect of acupuncture combined with wheat-grain moxibustion and oral sertraline hydrochloride dispersible tablets in the treatment of mild to moderate postpartum depression.@*METHODS@#Sixty patients with mild to moderate postpartum depression were randomly divided into an observation group and a control group, 30 cases in each group. Both groups were treated with psychotherapy. The control group was treated with oral sertraline hydrochloride dispersible tablets, 50 mg each time, once a day; the observation group was treated with acupuncture at Qihai (CV 6), Zusanli (ST 36), Xuehai (SP 10), Hegu (LI 4), Sanyinjiao (SP 6), Taixi (KI 3), etc. combined with wheat-grain moxibustion at Xinshu (BL 15), Pishu (BL 20), Ganshu (BL 18) and Shenshu (BL 23), once every other day, 3 times a week. Both groups were treated for 4 weeks as a course, with 2 consecutive courses of treatment. Before and after treatment and follow-up of 3 months after the end of treatment, the Hamilton depression scale (HAMD), Edinburgh postnatal depression scale (EPDS) and World Health Organization quality of life-BREF (WHOQOL-BREF) score of the two groups were compared, and the clinical effect was assessed.@*RESULTS@#After treatment and during follow-up, the HAMD and EPDS scores of the two groups were lower than before treatment (@*CONCLUSION@#Acupuncture combined with wheat-grain moxibustion can improve the depressive symptoms of patients with mild to moderate postpartum depression and improve their quality of life, and the clinical effect is more lasting and stable than oral sertraline hydrochloride dispersible tablets.

Health Care-seeking Behaviors in Mosuo and Pumi People in Yunnan Ninglang / 中国医学科学院学报

Objective To investigate the health care-seeking behaviors of Mosuo and Pumi people.Methods The subjects were enrolled by using the multi-stage stratified random sampling method and surveyed by the self-designed questionnaire.Results To tally 1669 subjects including 1121 Mosuo people and 548 Pumi people completed the survey.When Mosuo and Pumi people suffer from ailments,they preferred to buy drugs in drugstores(47.3% for Mosuo and 46.9% for Pumi),followed by visiting a local township hospital(27.0% for Mosuo and 24.3% for Pumi).When they suffered from severe diseases,they preferred to visit the county/city/state hospital(93.4% for Mosuo and 91.1% for Pumi).The mental disease were mainly treated in the county/city/state hospitals(49.3% for Mosuo and 52.7% for Pumi);notably,39.3% of the Mosuo respondents and 31.5% of the Pumi respondents skipped this question.Conclusion Health education,including awareness-raising activities on mental health,should be enhanced in Mosuo and Pumi people to further improve their health care-seeking behaviors.

Cardioprotection by hypoxia-induced rat adipose-derived stem cells through paracrine mechanisms / 中国组织工程研究

BACKGROUND: Adipose-derived stem cells (ADSCs) represent one of the promising cell sources for myocardial regeneration due to their easy accessibility and efficacy in the improvement of cardiac function following myocardial infarction. However, previously reported studies on the underlying mechanism of ADSCs-mediated cardioprotective effect mainly focused on the ADSCs cultured at room air. OBJECTIVE: To test the paracrine actions and anti-apoptotic effect of ADSCs under hypoxic conditions. METHODS: After isolation and culture, neonatal rat myocardial cells were injured by hydrogen peroxide and co-cultured with rat ADSCs under normoxia and hypoxia (10% O2) conditions. Ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in the cell pellet and levels of vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and basic fibroblast growth factor (bFGF) were tested by ELISA. Expression of apoptotic proteins Bax and Bcl-2 were determined by western blot. RESULTS AND CONCLUSION: GSH/GSSG, VEGF, IGF-1, and bFGF were decreased in neonatal rat myocardial cells injured by hydrogen peroxide. ADSCs significantly attenuated hydrogen peroxide-induced myocardial apoptosis by increasing the ratio of GSH/GSSG and the secretion of VEGF, IGF-1 and bFGF. ADSCs also down-regulated Bax expression and up-regulated Bcl-2 expression. To conclude, hypoxic conditions can enhance the anti-apoptosis and cardioprotective effects of ADSCs through the paracrine mechanism.

Changes of choroidal thickness after anterior - posterior joint surgery in patients with proliferative diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM:To determine the effect of anterior-posterior joint surgery on choroidal thickness in patients with proliferative diabetic retinopathy (PDR). · METHODS: A retrospective, case - control study enrolled 60 eyes of 60 patients with PDR diagnosed at Qingdao Municipal Hospital. The patients, who had conditions that warranted anterior - posterior joint surgery,were divided into a clinically significant macular edema group (PDR/CSME+;31 patients,31 eyes) and a non-CSME group (PDR/CSME-;29 patients,29 eyes). Twenty-seven eyes of 27 normal patients were included in the control group. All affected eyes underwent anterior - posterior joint surgery. After surgery, the subfoveal choroidal thickness (SFCT), and the nasal choroidal thickness (NCT) and temporal choroidal thickness (TCT), which were obtained at a distance of 1500μ m from the fovea in the nasal and temporal directions, respectively, were measured in the control and PDR groups by enhanced depth imaging spectral domain optical coherence tomography (EDI-SDOCT) at 1wk,1,3, and 6mo after surgery. Changes in choroidal thickness after anterior - posterior joint surgery were compared between the groups. ·RESULTS:The SFCT,NCT,and TCT were significantly thicker at 1mo than at 1wk, 3, and 6mo after surgery in the PDR/CSME+ and PDR/CSME- groups(P<0.05). The SFCT, NCT, and TCT were significantly thinner at 6mo than at 1wk,1,and 3mo after surgery in the PDR/CSME+and PDR/CSME- groups(P<0.05). The SFCT,NCT,and TCT in the PDR/CSME+ and PDR/CSME- groups at 1wk, 1, and 3mo after surgery were significantly thicker than those in the control group (all P<0.05), but the SFCT, NCT, and TCT at 6mo after surgery showed no significant difference compared with the control group (all P>0.05). There was no significant difference in the SFCT,NCT, or TCT at 1wk, 1, 3, or 6mo between the PDR/CSME+ and PDR/CSME- groups (P>0.05). ·CONCLUSION:The choroidal thickness of PDR patients increases within 1mo after surgery, and decreased after 1mo,but is not significantly different between the control group and the PDR groups at 6mo after surgery. Whether PDR is associated with CSME has no effect on the choroidal thickness after surgery.

Association between Polymorphisms of rs3924999,rs35753505 in NRG1 Gene and the Efficacy of Risperidone in Patients with Schizophrenia / 昆明医科大学学报

Objective To investigate the association between polymorphisms of rs35753505, rs3924999in neuregulin－1 (NRG1) gene and the efficacy of risperidone after 12 weeks treatment in Han patients with schizophrenia from Yunnan of China.Methods A case－control study was conducted: 114 schizophrenic Han inpatients with 12 weeks single therapy of risperidone were randomly selected from July 2012 to March 2013 and 187 normal Han persons whose age and years of education matched the controls.TaqMan allelic genotyping technology was used to analyze NRG1 genotyping.Treatment effect of risperidone was evaluated by the positive and negative symptoms scale (PANSS), PSP, Raven’s Standard Progressive Matrices, Wechsler Intelligence Scale and Number Cancellation Test.Results (1) There was no statistically difference in genotypes, allele frequencies of rs35753505, rs3924999 polymorphic locibetween schizophrenic inpatients and normal persons. (2) The baseline clinical data of patients with schizophrenia in different NRG1 gene polymorphism was not significant. (3) The value difference of Number Cancellation Test One between pre and post treatment of risperidone was related with different genotypes of two polymorphismsin NRG1 gene, there was statistically difference in two genotypes of rs35753505 loci: G/A group was higher than G/G group (P=0.010) and in three genotypes of rs3924999 loci: A/A group was higher than A/G group (P=0.032) . (4) The value difference of Reven's Standard Progressive Matrices between pre and post treatment of risperidone was related with rs35753505 polymorphic loci: G/A group was higher than G/G group (P=0.004) (5) The result of correlation regression analysis between pre and post treatment of risperidone showed that the value difference of Number Cancellation Test Two was related with the baseline clinical data of course,dose,immediate memory,net score of Number Cancellation Test Two,Number Cancellation Test Three and the genotypes of rs35753505 loci.Conclusion After 12 weeks treatment of risperidone: the improvement degree of patients'attention directivity and concentrated force in different genotypes of rs35753505loci was different:G/A group was higher than G/G group.

Optimization for prescription of Baoxieling Hydrogel Patch using Box-Behnken test design and research on its transdermal permeability in vitro / 中草药

Objective: To optimize the prescription of Baoxieling Hydrogel Patch (BHP) using Box-Behnken test design and to investigate its transdermal absorption properties in vitro. Methods: Taking the comprehensive scores of the early adhesion, uniformity, ductility, consistence, skin adhesive ability, repeated exposing paste, and residue as response values, Box-Behnken test design was used to optimize the amounts of sodium polyacrylate NP 800, aluminum glycinate, and fillers, and to validate the optimal formulation. The percutaneous permeation of cinnamaldehyde, eugenol, evodiamine, and rutaecarpine in the optimal formulation was studied by in vitro transdermal delivery experiment with Franz diffusion cells and their contents were determined by HPLC. Results: The optimal ratio of the prescription was as follows: sodium polyacrylate NP 800-aluminum glycinate-fillers (0.82:0.02:1.56). The foremost factors were fillers and aluminum glycinate. Its transdermal absorption met zero order dynamic process. Conclusion: The optimal prescription has uniform paste, suitable consistence, easy ductility, moderate adhesion, and perfect transdermal effect. It could provide the foundation for the development of new prescription of Baoxieling.

Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes / 生理学报

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).

Insertion of TC motif into hepatitis B virus core protein c/e1 site does not affect the expression of S and e antigen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen.</p><p><b>METHODS</b>Different oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay.</p><p><b>RESULTS</b>Western blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different.</p><p><b>CONCLUSION</b>Insertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.</p>

Activation of δ-opioid receptors inhibits L-type Ca(2+) current and transient outward K(+) current in rat ventricular myocytes / 生理学报

In the present study, whole-cell patch-clamp technique was used to observe the effects of SNC162, a selective agonist of δ-opioid receptors, on L-type Ca(2+) current (I(Ca-L)) and transient outward K(+) current (I(to)) in rat ventricular myocytes. The results showed that SNC162 significantly inhibited I(Ca-L) and I(to) in rat ventricular myocytes. The maximal inhibition rate of I(Ca-L) and I(to) reached (46.13±4.12)% and (36.53±10.57)%, respectively. SNC162 at 1×10(-4) mol/L inhibited the current density of I(Ca-L) from (8.98±0.40) pA/pF to (4.84±0.44) pA/pF (P<0.01, n=5) and inhibited that of I(to) from (18.69±2.42) pA/pF to (11.73±1.67) pA/pF (P<0.01, n=5). Furthermore, the effects of naltrindole, a highly selective antagonist of δ-opioid receptors, on I(Ca-L) and I(to) were also observed. The results showed that naltrindole alone had no effects on I(Ca-L) and I(to), while it abolished the inhibitory effects of SNC162 on I(Ca-L) and I(to). In conclusion, SNC162 concentration-dependently inhibited I(Ca-L) and I(to) in rat ventricular myocytes via activation of the δ-opioid receptors, which may be a fundamental mechanism underlying the antiarrhythmic effect of activating δ-opioid receptors.

Construction of recombinant plasmid human imprinted gene PEG10 and the primary functional identification in transfected cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a plasmid expressing human imprinted gene PEG10 and to study the effect of overexpression of PEG10 in a stable transfected human normal liver cell line L02 and in non-liver derived cell line 293.</p><p><b>METHODS</b>Full length cDNA of PEG10 open reading frame 1 was amplified and subcloned into a mammalian expression vector pcDNA3.1hisC. Recombinant plasmid was stably transfected into L02 cells and control cells via Lipofectamine 2000. The expression and the function of PEG10 in L02 cells and control group cells were examined using RT-PCR, Western blot, MTT and TUNEL.</p><p><b>RESULTS</b>Recombinant plasmid was successfully constructed and confirmed through DNA sequencing and restriction digesting. PEG10 gene accelerated the growth of L02 cells and inhibited their apoptosis but it had no conspicuous effect on the non-liver derived cells.</p><p><b>CONCLUSION</b>The constructed expressing vector pcDNA3.1hisC-PEG10 provides a useful tool for further study on the effects and mechanisms of PEG10. Over-expression of PEG10 may promote L02 cells' proliferation and inhibit their apoptosis, but not in the non-liver derived cell line 293.</p>

The deletion of La protein binding site in HBV RNA leads to instability of S gene mRNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of deletion of the La protein binding site of HBV RNA, caused by its mutation, on the HBV S-mRNA stability of S gene, to study the role of the site in hepatitis B virus life cycle, and to try to find a new anti-HBV target in the future.</p><p><b>METHODS</b>A HBV vector with mutation related to the La protein binding site was constructed using molecular cloning and PCR based site directed mutagenesis, and the vector was named pHBV-mLa. The HBV RNA secondary structure of the site was calculated using a computer. Normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamine 2000. HBV S-mRNA levels in the two groups were analyzed using semi-quantitative RT-PCR, and HBsAg secretion into the culture media was tested using ELISA.</p><p><b>RESULTS</b>A HBV vector with mutation related to the La protein binding site was successfully constructed, and it was identified and confirmed using restriction analysis and sequencing. The HBV RNA secondary structure of the mutant vector was completely different to the stem-loop structure of the normal HBV vector. Semi-quantitative RT-PCR and ELISA analyses showed that the level of HBV S-mRNA in the mutant vector group was significantly lower than that in the normal HBV vector group (t'=12.703, P<0.05), and the expression efficacy of HBsAg was reduced in the mutant vector group (t= 44.648, P<0.01).</p><p><b>CONCLUSIONS</b>The change of La protein binding site in the HBV RNA caused by the mutation in HBV DNA disorganizes the stem-loop structure in the HBV RNA site. With the structural change, the La protein cannot bind the site and stabilize the HBV RNA (HBV S-mRNA), as the cleavage site in the upstream of the stem-loop structure is exposed to endoribonuclease. This results in HBV S-mRNA decay and affects the expression of the S gene. This study shows that only the sequence of this site in the HBV DNA is reserved, then the stem-loop structure in the La protein binding site will remain intact, and the disorganization of the stem-loop structure affects the stability of the transcripted HBV RNA. The La protein binding site in HBV RNA and the special secondary structure of the site are crucial to the life cycle of the hepatitis B virus.</p>

Construction and expression of EGFP controlled HBV promoters in eukaryotic cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct four expression vectors carrying enhanced green fluorescent protein (EGFP) gene under the control of different HBV promoters, and to detect and analyze their expressions in hepatoma cell lines.</p><p><b>METHODS</b>Four HBV promoters were amplified using PCR, and they were inserted into the T-vector and identified using restriction enzymes and sequencing, then cloned into the expression vector pEGFP-1. The four recombinant plasmids were transfected into human hepatoma cell line HepG2 by lipofectamine2000, and the positive cell clones were detected using fluorescence microscopy.</p><p><b>RESULTS</b>All target fragments were separately obtained and successfully cloned into the expression vector. The expressions of EGFP under the control of the four promoters were detected. The expressions of EGFP controlled by different promoters had some differences.</p><p><b>CONCLUSIONS</b>Reporter gene EGFP under the control of four HBV promoters can be specifically expressed in hepatoma cell line HepG2, and different promoters give different results; this may provide another option in gene therapy of liver diseases.</p>