Exploration of nanofiltration conditions for separation of alcohol precipitation liquid during preparation of Reduning Injection by Box-Behnken design combined with flux attenuation / 中国中药杂志

This study employed Box-Behnken design combined with flux attenuation to explore the nanofiltration conditions for separation of alcohol precipitation liquid during the preparation of Reduning Injection and discussed the applicability of nanofiltration in the separation of the liquid with high-concentration ethanol. The effects of nanofiltration molecular weight cut-off(MWCO) and pH on the rejection of chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were consistent with the principles of pore size sieving and charge effect, respectively. The rejection of the three phenolic acids was reduced by concentration polarization effect caused by trans-membrane pressure(TMP). The swelling of membrane surface decreased the pore size and membrane flux for effective separation. Chlorogenic acid and 4,5-dicaffeoylquinic acid were more sensitive to pH and ethanol concentration than 3,5-dicaffeoylquinic acid. A certain correlation existed between the compound structure and the separation factors of nanofiltration, and the separation rules were associated with the comprehensive effect of charge effect, pore size sieving, concentration polarization, steric hindrance and so on.

Analysis of quality difference based on Astragalus membranaceus var. mongholicus in genuine region / 中国中药杂志

This work is to establish the fingerprint of Astragalus membranaceus var. mongholicus by HPLC-ELSD method, and to analyze the simulated wildness degree of A. membranaceus var. mongholicus in the genuine region of Inner Mongolia, Ningxia and Gansu. Compared with wild A. membranaceus var. mongholicus, the quality differences of A. membranaceus var. mongholicus in the genuine region were analyzed by identification of chromatographic peaks and similarity evaluation, cluster analysis(CA), principal components analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). HPLC fingerprints of A. membranaceus var. mongholicus in different genuine regions are established. The qualitative analysis of mass spectrometry identified 18 components. The similarity evaluation shows that the similarity of 32 batches of A. membranaceus var. mongholicus samples was 0.688-0.993. Among them, the similarity of samples in Shanxi, Inner Mongolia, Ningxia is 0.688-0.993, 0.835-0.989, 0.934-0.988, respectively and the similarity of samples in Gansu is 0.729-0.876 except No. 25 sample. The results of CA show that the samples of A. membranaceus var. mongholicus can be grouped into four categories according to the production area except the No. 11 and No. 25 samples. The results of PCA indicate that 32 batches of A. membranaceus var. mongholicus samples can be clustered according to quality and origin, and the quality of A. membranaceus var. mongholicus in Inner Mongolia is the closest to the wild breed. The results of OPLS-DA indicate that there are six components that can distinguish the wild and domestic A. membranaceus var. mongholicus, which are malonylastragaloside Ⅰ, astragaloside Ⅰ, calycosin-7-O-β-D-glycoside-6″-O-malonate, calycosin-7-O-β-D-glycoside, formononetin-7-O-β-D-glycoside-6″-O-malonate, and astrapterocarpan-3-O-β-D-glycoside-6″-O-malonate. The established method can be used to analyze differences between A. membranaceus var. mongholicus origin and planting environment, and can provide references for the protection and replacement of wild A. membranaceus var. mongholicus resources, and the cultivation, processing and production of A. membranaceus var. mongholicus.

Textual Research on Origin of Alismatis Rhizoma in China Pharmacopoeia / 中国实验方剂学杂志

This paper origin studies the origin of Alismatis Rhizoma in Chinese pharmacopoeia， and puts forward some suggestions for modification. Through the changes in the records of the source of Alismatis Rhizoma in the various versions of the Chinese Pharmacopoeia and the records of Flora of China and Materia Medica of China，it is found that the source of Alismatis Rhizoma in the Chinese Pharmacopoeia is confused. Specifically， the Chinese name of Alismatis Rhizoma does not correspond to the Latin name. As a common Chinese herbal medicine，Alismatis Rhizoma has a large market circulation. Many classic Chinese medicine prescriptions released by China Food and Drug Administration contain Alismatis Rhizoma. The development of the classic Chinese medicine prescriptions will further increase the market circulation of Alismatis Rhizoma. As a major national move to promote the development of traditional Chinese medicine， the study for classic Chinese medicine prescriptions requires defining the origin of the medicinal materials used，and the confused origin of Alismatis Rhizoma recorded in the Chinese Pharmacopoeia seriously hinder the development of the classics. Therefore，in order to regulate the origin of Alismatis Rhizoma， ensure the clinical efficacy and promote the development of classic Chinese medicine prescriptions，the confused origin of Alismatis Rhizoma in the Chinese Pharmacopoeia has to be resolved as soon as possible. Based on the analysis of the changes of Alismatis Rhizoma's producing areas in the past dynasties， it is found that the producing areas of Alismatis Rhizoma have continuous changed from Wei and Jin dynasties to present， and finally formed the current situation of Sichuan as the main producing area. In comparison of chemical composition，origin and market circulation of Alismatis Rhizoma in Sichuan Province that is the most productive， and Fujian Province that is the best quality， it is found that the two species are different in every aspects. Nowadays，Alisma plantago-aquatica occupies the majority of the market， which doesn't conform to Alisma orientale as specified in the 2015 edition of the Chinese Pharmacopoeia. Therefore， through textual research and analysis， it is suggested that both A. plantago-aquatica and A. orientale. Shall be used as the origin of Alismatis Rhizoma. In the 2015 edition of Chinese Pharmacopoeia，Cassiae Semen，Schizonepetae Herba，Aisaematis Rhizoma，Fibraureae Caulis and Ajugae Herba have the same problem. This paper provides ideas for the revision of sources of traditional Chinese medicine.

The correlation of molecular structure and nanofiltration mass transfer behavior of alkaloids / 药学学报

Based on the charge repulsion and solution-diffusion effect in nanofiltration separation, the correlation among mass transfer behavior, solution environment and molecular structure of three typical alkaloids from medicine was analyzed by nanofiltration mass mathematical model. The experiment revealed a linear relationship between ln[(1-Ro)·Jv/Ro] and Jv, and the regression coefficients were all greater than 0.9. Compared with the ultrafiltration separation behavior conforming to molecular sieve, the mass transfer coefficient of three alkaloids under different pH was pH 3.00 < pH 7.00 < pH 10.00. As the pH changed, the state of alkaloid transit from ionic state to a free state, the alkaloid could easily approach the membrane surface and pass through the nanofiltration membrane with charge repulsion and solution-diffusion effects, and the results were verified by the membrane adsorption tendency. The nanofiltration mass transfer of alkaloids is related to the state and molecular weight. In the ionic state, the charge effect produces separation behavior, and the molecular state is related to the molecular weight. The separation mechanism of nanofiltration for alkaloids was clarified further by analyzing the correlation of nanofiltration mass transfer behavior and molecular structure. The results of nanofiltration technology provide references for separation of alkaloids at room temperature with fast separation and low energy consumption.

Enhanced separation behavior of ephedrine in organic solution by nanofiltration mass transfer model / 中国中药杂志

Based on the nanofiltration mass transfer model, the enhanced separation behavior of ephedrine in organic solution was studied. In the experiment, the sensitive region of ethanol concentration and pH on the rejection of ephedrine was screened out by Box-Behnken central composite experiment design. Furthermore, to analyze the separation regularity of ephedrine and organic solution, the correlation between mass transfer coefficient and concentration of organic solvent was fitted with the changed organic solution by nanofiltration mass transfer mathematical model. Experiments showed the enhanced separation behavior, the decrease in the mass transfer coefficient while the increase in ethanol concentration from 20% to 40%, MWCO at 450 and pH 6.0. Under the same conditions, the enhanced separation behavior was appeared as the solvent changed into methanol and acetonitrile, the enhanced effect was positively correlated with the concentration of the three common organic solvents, and the effect order was acetonitrile>ethanol>methanol. This study took ephedrine as an example, and explored the mechanism of nanofiltration separation in the environment of organic solution, so as to provide references for nanofiltration separation for heat-sensitive traditional Chinese medicine of alkaloid.

HPLC characteristic fingerprints of Xinshenghua Keli / 中国中药杂志

Xinshenghua Keli is known as the "preferred prescription of postpartum", with large demand in the field of gynecologic medicine. However, the quality of the preparation is uneven in the market, so its clinical efficacy cannot be guaranteed. In order to improve and establish its quality control standard, high performance liquid chromatography (HPLC) was used to establish the fingerprint of Xinshenghua Keli. The detection was performed on Agilent 5 HC-C₁₈ (2) column(4.6 mm×250 mm, 5 microns) with methanol-0.1% formic acid solution as mobile phase for gradient elution, at a flow rate of 1 mL·min⁻¹ with column temperature of 25 °C. The injection volume was 10 μL and detection wavelength was set at the maximum value between 210.0 nm and 400.0 nm by Photo-Diode Array (PDA) detector. The fingerprint of 12 batches of high-quality Xinshenghua Keli was established and 43 common peaks were identified. The similarities of crowned products, 10 batches of ordinary ones made by Jiangsu Rongyu Pharmaceutical and 10 batches produced by different manufacturers were evaluated. The composition identification and source analysis for the common peaks were performed by comparing the retention time of herbal medicines and ultraviolet absorption spectrum, along with high performance liquid chromatography-mass spectrometry (HPLC-MS) technology. The established fingerprint of Xinshenghua Keli, has proven to have good precision, stability and repeatability through the methodology validation, so it can be used to comprehensively evaluate the quality of Xinshenghua Keli.

Design and implementation of Xinshenghua granule traceability system based on data authenticity / 中国中药杂志

The quality of traditional Chinese medicine affects the clinical efficacy and the market research at home or abroad, which is closely related to the standardization of production. At present, Chinese market still exist in drug quality and nonstandard production phenomenon. In addition to the existing GMP production standards, it is still necessary to ensure the authenticity and reliability of traditional Chinese medicine by means of retrospect. Based on the Xinshenghua granule, starting from the authenticity of the whole process of production, this study designs and constructs the traditional Chinese medicine traceability system which is equipped with the Internet platform by using two-dimensional code as a trace tool. By making authentic record of the production process and quality transfer, it is convenient for consumers to know the drug information. The production traceability system provides guarantee for the standardization of traditional Chinese medicine development, in order to obtain the production source，the testing quality, whereabouts and responsibility.

Correlation of molecular weight and nanofiltration mass transfer coefficient of phenolic acid composition from Salvia miltiorrhiza / 中国中药杂志

Based on the molecular sieving and solution-diffusion effect in nanofiltration separation, the correlation between initial concentration and mass transfer coefficient of three typical phenolic acids from Salvia miltiorrhiza was fitted to analyze the relationship among mass transfer coefficient, molecular weight and concentration. The experiment showed a linear relationship between operation pressure and membrane flux. Meanwhile, the membrane flux was gradually decayed with the increase of solute concentration. On the basis of the molecular sieving and solution-diffusion effect, the mass transfer coefficient and initial concentration of three phenolic acids showed a power function relationship, and the regression coefficients were all greater than 0.9. The mass transfer coefficient and molecular weight of three phenolic acids were negatively correlated with each other, and the order from high to low is protocatechualdehyde >rosmarinic acid> salvianolic acid B. The separation mechanism of nanofiltration for phenolic acids was further clarified through the analysis of the correlation of molecular weight and nanofiltration mass transfer coefficient. The findings provide references for nanofiltration separation, especially for traditional Chinese medicine with phenolic acids.

Sangtongjian Mixture ameliorates glucose and lipid metabolism in type 2 diabetic rats / 中成药

AIM To observe the effects of Sangtongjian Mixture (STJ) on glucose and lipid metabolism,insulin resistance and fat cytokines in type 2 diabetic rats,and their mechanisms of action.METHODS One hundred and forty rats fed on the combination of STZ and high fat diet were established as the type 2 diabetic models.Fasting blood glucose (FBG) level reached more than 16.7 mmol/L and then the rats were randomly divided into model group,metformin (180 mg/kg) group,STJ (73.5,147 and 294 mg/kg) groups.Ten rats were set as the blank group.Each treatment group was intragastrically given the corresponding agents for twelve weeks.The fasting blood glucose levels of rats were measured once every two weeks after the administration.After a 12-week administration period,glycosylated serum protein (GSP),glycosylated hemoglobin (GHb) and lipid profile indices (TC,TG,HDL-C and LDL-C) were determined.The serum insulin level was measured by radioimmunoassay,and homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated.The levels of serum adiponectin and leptin were detected by ELISA.RESULTS STJ remarkably decreased the levels of FBG,GSP,GHb,TC,TG,LDL-C,leptin and HOMR-IR in type 2 diabetic rats.Furthermore,STJ also significantly increased the levels of HDL-C,adiponectin and ISI.CONCLUSION STJ can improve glucose and lipid metabolism in type 2 diabetic rats by ameliorating insulin resistance and regulating fat cytokine levels.

Exploring correlation between molecular state and nanofiltration mass transfer process of synephrine from Citrus aurantium / 中国中药杂志

Based on the solution-diffusion effect and the charge effect theory in nanofiltration separation, the correlation between initial concentration and mass transfer coefficient was constructed to establish a mathematic model of synephrine in mass transfer process and verify its applicability. The experimental results showed that there was a linear relationship between operation pressure and membrane flux. Meanwhile, the membrane flux was gradually decayed with the increase of solute concentration. Besides, mass transfer coefficient and initial concentration of synephrine showed power function correlation with each other by solution-diffusion effect and the charge effect, and the regression coefficients were greater than 0.9. The mass transfer coefficient of dissociation synephrine was less than that in the state of free and free-dissociation. Moreover, on the basis of power function relationship between mass transfer coefficient and initial concentration, the results showed that the predicted rejections of synephrine from Citrus aurantium water extract by use of the mathematical model approximated well to real ones, verifying that the model was practical and feasible. The unclear separation mechanism of nanofiltration for alkaloids was clarified preliminary by the predicted model of nanofiltration separation with synephrine as the example, providing theoretical and technical support for nanofiltration separation, especially for traditional Chinese medicine with alkaloids.

Analyze nanofiltration separation rule of chlorogenic acid from low concentration ethanol by Donnan effect and solution-diffusion effect / 中国中药杂志

To separate chlorogenic acid from low concentration ethanol and explore the influence of Donnan effect and solution-diffusion effect on the nanofiltration separation rule. The experiment showed that solution pH and ethanol volume percent had influences on the separation of chlorogenic acid. Within the pH values from 3 to 7 for chlorogenic acid in 30% ethanol, the rejection rate of chlorogenic acid was changed by 70.27%. Through the response surface method for quadratic regression model, an interaction had been found in molecule weight cut-off, pH and ethanol volume percent. In fixed nanofiltration apparatus, the existence states of chlorogenic acid determinedits separation rules. With the increase of ethanol concentration, the free form chlorogenic acid was easily adsorbed, dissolved on membrane surface and then caused high transmittance due to the solution-diffusion effect. However, at the same time, due to the double effects of Donnan effect and solution-diffusion effect, the ionic state of chlorogenic acid was hard to be adsorbed in membrane surface and thus caused high rejection rate. The combination of Box-Behnken design and response surface analysis can well optimize the concentrate process by nanofiltration, and the results showed that nanofiltration had several big advantages over the traditional vacuum concentrate technology, meanwhile, and solved the problems of low efficiency and serious component lossesin the Chinese medicines separation process for low concentration organic solvent-water solution.

Optimize concentrate process of alkaloid from Leonurus japonicus by ultrafultration-nanofiltration coupling technology / 中国中药杂志

To optimize the concentrate process of alkaloid from Leonurus japonicus by nanofiltration-ultrafiltration coupling technology with response surface methodology. The experiment showed that after ultrafiltration pre-treatment, the total protein removal rate was 94.38% in aqueous extract from L. japonicus, and the nanofiltration technology had obvious advantages over the conventional concentrate process. The optimal concentrate conditions were as follows：molecular weight cut-off 450, pH 3.07, concentration of stachydrine hydrochloride 80.15 mg•L⁻¹, and concentration of the total alkaloid 285.73 mg•L⁻¹. The cut-off rate was 93.37% and 95.85% respectively for stachydrine hydrochloride and the total alkaloid under the optimum conditions, with a relative error of 0.79% and 1.16% respectively. The combination of Box-Behnken design and response surface analysis can well optimize the concentrate process of L. japonicus by nanofiltration, and the results provide the basis for nanofiltration concentrate for heat-sensitive traditional Chinese medicine.

Multi-index determination and optimization of liquirtin separated from polyamide resin / 中国中药杂志

To optimize the separation process of liquirtin from glycyrrhiz by static, dynamic adsorption and desorption experiments on polyamide resin, with liquirtin, isoliquiritin and glycyrrhizic acid as the study index. The optimum process conditions were that the pH of solution was regulated to be 7.0, the concentration of liquirtin was 1.296 g x L(-1), the volume of loading buffer was 3 BV. After absorption, efforts shall be made to elute resin with water, 10%, 20%, 30% ethanol (3 BV for each), collect 20% ethanol eluted fraction, and recover solvents. The results showed lower contents of such impurities as isoliquiritin and isoliquiritin in extracts sepaprated under this process conditions, as well as an increase in purity of liquirtin from 4.86% to 88.5%. The method was simple and feasible, it could obtain a higher purity in extracts from liquirtin and provide basis for industrialized separation and preparation of liquirtin.

Subtype and sequence analysis of the gag genes for HIV-1 strains isolated in Hubei province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the characteristic of subtypes and genetic diversity of HIV-1 circulating in Hubei province and its molecular epidemiological linkages with regard to risk factors of viral transmission.</p><p><b>METHODS</b>plasma samples of 80 diagnosed individuals was characterized. The gene fragments of gag were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and HIV-1 genotypes were determined based on the nucleotide sequences of gag region.</p><p><b>RESULTS</b>Seven HIV-1 group M subtypes or CRF including B, B', G, CRF01-AE, CRF07-BC, CRF08-BC and CRF15-01B were identified. CRF01-AE was found to be the most dominant subtype (48.4%) followed by CRF7-BC (22.6%) and B' (12.9%).</p><p><b>CONCLUSION</b>The data from this study indicate the existence of multiple HIV-1 subtypes or CRFs in Hubei province and the surveillance of HIV-1 gene variation should be paid more attention to.</p>

Interaction of microRNA-338 and its potential targeting protein eiF4E3 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To analyze the interaction between the microRNA-338 and its targeting proteins during the cerebral ischemia and reperfusion injury.</p><p><b>METHODS</b>TargetScan was used to predict the targets of microRNA-338. The potential targeting proteins were then selected according to their secondary structures using RNA structure 4.6 software and their involvement in cerebral ischemia and reperfusion injury was studied. Dual-luciferase reporter assay was used to testify whether microRNA-338 can recognize the 3'UTR of target protein. Western blot was applied to analyze the expression of eiF4E3 in both experimental group and control group.</p><p><b>RESULT</b>EiF4E3 was the most likely potential targeting protein of microRNA-338. The secondary structure of local region of eiF4E3 recognizing microRNA-338 was conservative. The ratio of firefly to renilla luciferase activity in the experimental group was much higher than that of control group. However, there was no significant difference in the expression of eiF4E3 between these two groups.</p><p><b>CONCLUSION</b>MicroRNA-338 can recognize the 3'UTR of eiF4E3 while it has no significant effect on the expression of eiF4E3. The post-target-recognizing regulation for miRNA do exist and this mechanism is possibly related to the tertiary structure of target mRNA.</p>

Telomerase expression is not involved in aging process of human keratinocytes induced by UVB irradiation / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the mechanism involved in aging process of immortalized human keratinocyte (HaCaT) and primary human epidermis keratinocyte of adults (HEKa) irradiated by ultraviolet B(UVB).</p><p><b>METHODS</b>HEKa and HaCaT were repeatedly exposed to UVB at a subcytotoxic level. SA-beta-Gal staining was performed to evaluate the senescence state; flow cytometry was applied to detect the changes of apoptosis, necrosis and cell cycle. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by ELISA method. Western blot was performed to detect the expression pattern of redox protein p66Shc and RT-PCR was performed to determine the mRNA level of human telomerase reverse transcriptase (hTERT).</p><p><b>RESULT</b>Strong positive SA-beta-Gal staining was observed in both HEKa cell and HaCaT cells after UVB irradiation. Apoptosis rate increased from (1.81 +/-0.25)% to (4.43 +/-0.28)% and necrosis rate increased from (0.05 +/-0.01)% to (0.10 +/-0.03)% in HaCaT cell, but no marked arrest of cell cycle was observed during UVB irradiation. As a contrast, apoptosis rate of in HEKa cells significantly increased from (0.65 +/-0.05)% to (59.53 +/-2.35)%, and the necrosis rate in HEKa cells also reached (3.89 +/-0.24)%(P<0.05). Growth arrest in G0/G1 phase was also found in HEKa cells. In both cell lines, intracellular level of SOD decreased and MDA increased remarkably after UVB exposure, and an increased expression of p66Shc protein was also observed. High level of hTERT mRNA was detected in HaCaT cells and UVB exposure had little effect on its expression.</p><p><b>CONCLUSION</b>The stress-induced premature senescence (SIPS) in HaCaT and HEKa cell lines by UVB irradiation might be closely associated with increased intracellular levels of oxidative stress, not related to the telomerase expression.</p>

Survival analysis of 530 HIV infected former unsafe commercial blood and plasma donors / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate HIV survival time and it's influencing factors among former commercial blood and plasma donors engaged in unsafe blood donation practices in China.</p><p><b>METHODS</b>HIV/AIDS cases from 8 counties (districts) in 4 provinces confirmed prior to January 24, 2006 related with former commercial blood and plasma donors were selected and data regarding infection, AIDS progression, death, and influencing factors were retrospectively collected.</p><p><b>RESULTS</b>In 530 cases of HIV infection, 334 (63.0%) cases had developed AIDS, 168 (50.3%) had received antiretroviral therapy (ART), and 152 (29.0%) had died. For the 530 cases, there was an average (10.1 +/- 1.8) years of observation from time of infection. Among 166 AIDS patients not receiving ART, average survival was 9.1 years (95% CI: 9.1 - 9.4), with an 8 year survival rate of 52.0%. Among 168 AIDS patients receiving ART, average survival was 12.1 years (95% CI: 11.9 - 12.3), with a 12-year survival rate of 80.0%. In 3 years of ART, average survival was longer in the treatment group as compared to the no treatment group with a hazard ratio for death of 12.2. Univariate analysis showed a significant difference (P < 0.05) in AIDS patient average survival based on gender, age, location, ART status, and baseline CD(4)(+) T cells count. Results from multivariate COX-regression showed that highly active ant iretroriral therapy (HAART) was the strongest protective factor for prolonging AIDS patients' survival (HR = 13.3, P = 0.00).</p><p><b>CONCLUSION</b>Although there are many factors influencing AIDS patients survival, intervention with HAART is the principle measure to prolong survival and decrease the risk of death.</p>

PD-L1 expression in circulating dendritic cells of patients with chronic hepatitis B / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the PD-L1 expression levels in circulating dendritic cells(DCs) of patients with HBeAg positive chronic hepatitis B, and to investigate the effects of anti-PD-L1 antibody on DCs stimulating capacity of allogeneic lymphocytes.</p><p><b>METHODS</b>DCs were separated and induced from 22 HBeAg positive chronic hepatitis B patients (CHB), 8 acute resolved hepatitis B patients (AHB) and 10 healthy blood donors. PD-L1 and PD-L2 expression in DCs were determined using real-time RT-PCR and flow cytometry. The potential of circulating DCs on the proliferation of allogeneic T cells was detected and a specific monoclonal antibody against PD-L1 was used in alternative experiments. Serum HBV-DNA titers were measured using real-time PCR, and HBV markers and liver function were also evaluated.</p><p><b>RESULT</b>The expression of PD-L1 but not PD-L2 was upregulated in circulating DCs of CHB patients, compared to AHB patients and healthy controls (both P<0.01). CHB patients with greater than 106 copies /ml of serum HBV DNA loads had a higher level of PD-L1 in circulating DCs than those with less than 106 copies/ml (P<0.05), and the high expression of PD-L1 in DCs was positively correlated with the plasma viral load. Moreover, the potential of circulating DCs from CHB patients was significantly decreased compared with healthy controls or AHB patients, while the blockade of PD-L1 using anti-PD-L1 monoclonal antibody increased the ability of DCs on the proliferation of allogeneic T cells in vitro.</p><p><b>CONCLUSION</b>High expression of PD-L1 on circulating DCs may be associated with T cell exhaustion and persistent high levels of HBV DNA replication in chronic hepatitis B patients.</p>

PD-1 expression in peripheral T cells of patients with HBV infection and its significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To analyze the expression levels of PD-1 (program death factor-1) in peripheral T cells from patients infected with HBV, and to investigate its relationship with HBV serological markers.</p><p><b>METHODS</b>A total of 65 HLA-A2+ subjects, including 31 patients with chronic hepatitis B (CHB), 9 with acute resolved hepatitis B (AHB), 15 with HBV related liver cirrhosis (LC) and 10 healthy blood donators, were enrolled. The expression of PD-1 in peripheral T cells and PD-1 ligands PD-L1 and PD-L2 in PBMCs were determined by relative quantitative real-time PCR. The serum HBV markers, HBV DNA load and liver function were also measured.</p><p><b>RESULTS</b>Taken the PD-1 and PD-ligands expression in normal controls as a baseline level, the expression of PD-1 and PD-L1 from CHB patients was significantly increased, while the expression of PD-L2 was relatively low in all groups. In CHB patients, the PD-1 expression in peripheral T cells from patients with high viral load was much higher than that from those with low viral load or from normal controls. And the PD-1 expression level positively correlated with serum HBV DNA load (r=0.41, P<0.01) but not with serum ALT level.</p><p><b>CONCLUSION</b>Long-term exposure to HBV antigens in CHB patients may increase the expression of PD-1 in T cells and thus leads to the virus persistent infection.</p>

An improved quantitative method for evaluation of ischemic injury and neuroprotection in mouse brain slices / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices.</p><p><b>METHODS</b>An incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices, and formazan was isolated, purified and identified. Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD), the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects.</p><p><b>RESULT</b>The incubation instrument worked well for incubating brain slices and obtaining stable results efficiently. Standard formazan was biosynthesized and purified with a purity of 99.3%, and showed a linear range of 0.05 - 1 mg/ml in absorbance at 490 nm (r=0.9997). OGD decreased formazan production in the cortex and striatum in a duration-dependent manner. Edaravone (0.01 to 1 micromol/L) recovered OGD-induced decrease of formazan production, but ONO-1078 showed no effect.</p><p><b>CONCLUSION</b>The incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.</p>