The mesencephalic astrocyte-derived neurotrophic factor (MANF) has been recently identified as a neurotrophic factor, but its role in hepatic fibrosis is unknown. Here, we found that MANF was upregulated in the fibrotic liver tissues of the patients with chronic liver diseases and of mice treated with CCl₄. MANF deficiency in either hepatocytes or hepatic mono-macrophages, particularly in hepatic mono-macrophages, clearly exacerbated hepatic fibrosis. Myeloid-specific MANF knockout increased the population of hepatic Ly6C^high macrophages and promoted HSCs activation. Furthermore, MANF-sufficient macrophages (from WT mice) transfusion ameliorated CCl₄-induced hepatic fibrosis in myeloid cells-specific MANF knockout (MKO) mice. Mechanistically, MANF interacted with S100A8 to competitively block S100A8/A9 heterodimer formation and inhibited S100A8/A9-mediated TLR4-NF-κB signal activation. Pharmacologically, systemic administration of recombinant human MANF significantly alleviated CCl₄-induced hepatic fibrosis in both WT and hepatocytes-specific MANF knockout (HKO) mice. This study reveals a mechanism by which MANF targets S100A8/A9-TLR4 as a "brake" on the upstream of NF-κB pathway, which exerts an impact on macrophage differentiation and shed light on hepatic fibrosis treatment.

【Objective】 To research the effect of the Fc, Fab and F(ab′)2 fragments of immunoglobulin G, the main components of Human Immunoglobulin(pH4) for Intravenous Injection(IVIG), on the phagocytic function of macrophages derived from THP-1 cells. 【Methods】 First of all, IVIG was digested with papain and pepsin to obtain Fc, Fab and F(ab′)2, and these components were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, propylene glycol monomethyl ether acetate (PMA) was used to induce THP-1 cells to differentiate into M0 macrophages. Finally, the sensitized erythrocytes were labeled with carboxy fluorescein succinimidyl ester (CFSE), and the effect of the above components on the phagocytic ability of M0 macrophages to engulf sensitized erythrocytes was detected by flow cytometry. 【Results】 The identification results of SDS-PAGE showed that the prepared IgG fragments met the requirements of subsequent experiments. Flow cytometry performs showed that the phagocytosis model of M0 macrophages had been successfully established. When the concentration of Fc increased from 0.1μg/ mL to 10μg/ mL, the phagocytosis rate of erythrocytes sensitized by M0 macrophages decreased from (24.21±0.58) % to (12.27±0.19) %. When the concentration of IVIG protein increased from 0.1 μg/ml to 10 μg/ml, the phagocytosis rate decreased from (20.57±0.39) % to (0.20±0.03) %. Meanwhile, at the same protein concentration (10 μg/ml), the inhibitory effect of Fc on phagocytosis was only half that of IVIG. In addition, Fab, F(ab′)2, and human serum albumin could not inhibit phagocytosis of M0 macrophages. 【Conclusion】 IVIG can effectively inhibit the phagocytosis of THP-1 derived M0 macrophages, which is mainly dependent on the Fc, but not related to the Fab of IgG and F (ab′)2.

【Objective】 To evaluate the efficacy and safety of human coagulation factor Ⅷ developed by Shenzhen Weiguang Biological products Co, Ltd in the treatment of patients with hemophilia A. 【Methods】 A prospective, multi-center, open, single-group clinical study was conducted. A total of 65 subjects with hemophilia A were enrolled, and human coagulation factor Ⅷ(FⅧ) was injected according to the patients’ bleeding severity. The improvement score of bleeding symptoms and signs after the first infusion of the first bleeding event and the transfusion efficiency of FⅧ activity at 10 min and 1 hour after infusion were taken as the main efficacy indexes. The improvement scores of bleeding symptoms and signs after the first infusion and the increase of FⅧ activity at 10 min and 1 hour after infusion were the secondary efficacy indexes. 【Results】 The 65 subjects were enrolled in safety analysis set (SS) and full analysis set (FAS), and 58 of them were enrolled in protocol analysis set (PPS). Ten minutes and one hour after the first infusion, the level of factor Ⅷ activity in the subjects increased significantly, and the FⅧ activity increased by 100% or more in more than 79% of the subjects. The average infusion efficiency of FⅧ activity in all subjects was more than 100%. In 70% of the subjects, the pain was relieved rapidly and /or the bleeding symptoms were significantly improved 8 hours after each bleeding infusion, and the improvement rate of bleeding symptoms and signs reached 100% 72 hours after infusion. 【Conclusion】 After infusion of human coagulation factor Ⅷ, the activity level of factor Ⅷ in patients with hemophilia A significantly increased. The infusion efficiency can reach a optimal level, and the bleeding symptoms can be significantly improved.

【Objective】 To establish and evaluate a nephelometric assay for the determination of immunoglobulin A (IgA) residues in human intravenous immunoglobulin(IVIG). 【Methods】 BN ProSpec^© automatic protein analyzer and its supporting immunoglobulin A determination kit (nephelometry) produced by German Siemens and the national standard of human IgA were used to establish the nephelometric assay to determine IgA residue in test products and verify the methodology. The test products include IVIG (pH4) prepared by low-temperature ethanol protein separation process and a novel IVIG prepared by chromatography. 【Results】 The average deviation of three calibration curves for IgA residues determination by the nephelometric assay were 1.08%, 0.95% and 1.54%,, and the three deviations of the quality control were 4.00%, -2.30% and -0.20%, respectively, which indicated good calibration and quality control. In the specificity test, the average recovery rates of IgA for reference substance 1 containing 100g/L maltose and reference substance 2 containing 20g/L glycine were 102.7% and 105.8%, respectively. The relative standard deviation (RSD) values of the repeatability tests of the two test products were 3.9% and 1.9%, and the RSD values of the intermediate precision test were 3.6% and 2.3%, respectively.The difference values at each time point in the durability test of test products′ storage time were all less than 10%, and the RSD values of the two test products in the durability test of kits of different batches were 2.8% and 2.2%, respectively. In the accuracy test, the average recovery rates of IVIG (pH4) added to the standard were 94.2%, 101.7% and 96.2%, respectively, and the average recovery rates of the novel IVIG added to the standard were 102.8%, 106.3% and 99.7%, respectively. The average recovery rate of the limit quantification test was 101.0%, and the RSD was 4.0%. 【Conclusion】 Nephelometric assay has the advantages of strong specificity, high precision and accuracy, good repeatability, simple and rapid operation, and automation, and can be used for the determination of IgA residue in IVIG (pH4) and novel IVIG products.

Objective To investigate the protective effect of Carbachol on hyperoxia-induced acute lung injury (HALI) in mice and its related mechanisms. Methods Thirty-two healthy male ICR mice were randomly divided into four groups:control group, hyperoxia exposure three days group (HO3d group), hyperoxia exposure three days + Carbachol group (HO3d+Carba group), and Carbachol group (Carba group), eight mice in each group. The pathological changes of lung tissue in each group were observed under light microscope after the models were completed in each group.The expression of TLR4 and NF-κB protein in lung tissues were detected by Western blot, and the expression of HMGB-1 and TNF-α mRNA in lung tissues by RT-PCR. LSD-t test was used for sample pairwise comparison, and one-way ANOVA for intergroup comparison. P<0.05 was considered statistically significant. Results There was no statistical difference between the control group and the Carba group (P> 0.05), and no obvious abnormal changes in lung tissue structure. The expression of TLR4, NF-κB protein and HMGB-1 and TNF-α mRNA in the HO3d group were significantly higher than those in the control group (P<0.01), and there were obvious bleeding on the surface of the lung tissue and severe pathological damage. The expression of TLR4,NF-κB protein and HMGB-1 and TNF-α mRNA in the HO3d+Carba group were significantly lower than those in the HO3d group(P<0.01), while lung tissue damage degree was also lower than that in the HO3d group. Conclusions Hyperoxia can increase the expression of TLR4 and NF-κB in lung tissues, and cause inflammatory injury in lung tissue. Carbachol can reduce the release of HMGB-1 and TNF-α inflammatory factors in hyperoxia-induced acute lung injury, and its mechanism is related to the inhibition of TLR4/NF-κB signal pathway, which has a protective effect on HALI.

Objective To investigate both in mechanism of hyperoxia-induced acute lung injury (HALI) by vivo experiment,to observe the Bruton' s tyrosine kinase (Btk) and nuclear factor kappa B (NF-κB) signals expression level.Methods Total of 72 healthy male Kunming mice were randomly (random number) divided into four groups:air control group,hyperoxia exposure 3 days group (H3d group),hyperoxia exposure 3 days + inhibitor group (H3d + Ⅰ group) and inhibitor groups.Then the pathological changes of lung tissues were observed under light microscope;The total protein content (TP) of bronchoalveolar lavage fluid (BALF) and wet/dry weight ratio (W/D) of lung were detected;The protein expression of Btk,p-Btk,pNF-κB p65 were mersured by Western blot;tlhe mRNA level of IL-6 was determined by real-time polymerase chain reaction (qRT-PCR);the level of monocyte chemoattractant protein-1 (MCP-1) in serum was detected by enzyme-linked immunosorbent assay (ELISA).Statistcal significance was determined by 1-way ANOVA.Results There were no significant difference in the data between the control group and the inhibitor group (P ＞ 0.05).The pathological injury in light microscope,content of total protein in BALF,W/D ratio of lung tissues in H3d group were significantly higher than H3d + Ⅰ group (Respectively P =O.002,P =0.000).Western blot analysis showed that expression of Btk,p-Btk,pNF-κB p65 in H3d group were significantly higher than those in H3d + Ⅰ group (Respectively P =0.002,P =0.013,P =0.000).RT-qPCR results showed that the expression of IL-6 mRNA in H3d group were significantly higher than control group (P =0.004),inhibitor group (P =0.000) and H3d + Ⅰ group (P =0.021).In addition,The serum MCP-1 levels in H3d group were higher markely than the control group (P =0.002),inhibitor group (P =0.000) and H3d + Ⅰ group (P =0.009).The correlation analysis showed that pNF-κB p65 were positively correlated wiht Btk and p-Btk (r =0.902 and 0.954,P ＜ 0.01).Conclusions Btk may trigger the release of IL-6 and MCP-1 by mediating the signaling pathway of NF-κB in vivo study,which was most important in the occurrence of HALI.Therefore,inhibiting the Btk activity would alleviate the severity of lung injury effectively.

OBJECTIVE:To establish the method for simultaneous determination of 6 components in Lonicera japonica,and to study the content changes of them before and after before and after carbonized. METHODS:UPLC method was adopted. The deter-mination was performed on Agilent Eclipse Plus C18 RRHD column with mobile phase consisting of 0.1% phosphoric acid solu-tion-acetonitrile(gradient elution)at the flow rate of 0.2 mL /min. The detection wavelength was set at 350 nm,and column tem-perature was 25 ℃. The sample size was 1 μL. RESULTS:The linear ranges of chlorogenic acid,rutin,galuteolin,isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C were 21.2-424 μg(r=0.9993),1.17-23 μg(r=0.9995),2.18-43 μg(r=0.9998),5.10-102 μg(r=0.9993),2.60-52 μg(r=0.9991),4.95-99 μg(r=0.9998),respectively. RSDs of precision,stability and repeatability tests were all lower than 2.0%. Recoveries were 97.11%-99.76%(RSD=1.20%,n=6),95.20%-99.90%(RSD=2.20%,n=6),95.71%-100.30%(RSD=2.20%,n=6),95.00%-96.98%(RSD=0.88%,n=6),96.47%-103.00%(RSD=2.40%, n=6),95.78%-103.80%(RSD=3.20%,n=6). Compared with before processing,the contents of rutin,isochlorogenic acid B and isochlorogenic acid C in L. japonica were increased along with processing,the contents of chlorogenic acid and isochlorogenic acid A were decreased significantly,while the content of galuteolin had no significant change. CONCLUSIONS:The method is sim-ple,precise,stable and repeatable,and can be used for simultaneous determination of 6 components in L. japonica. Those chemi-cal components have certain changes before and after carbonized.

OBJECTIVETo correlate sperm nucleoprotein transition (SNT) with sperm morphology, DNA damage and embryo development, and assess its value for assisted reproductive technology (ART).

METHODSThe SNT of 437 infertile men underwent ART were assayed, and its correlation with sperm morphology, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, available embryo rate, D3 high quality embryo rate, blastocyst formation rate and high quality blastocyst rate were analyzed.

RESULTSThe normal morphology rate of sperms, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, embryo transfer rate (ETR), D3 high quality embryo rate, blastocyst formation rate (BFR) and high quality blastocyst in normal males (Group A, abnormal rate≤30%, 135 subjects) did not significantly differ from those with an abnormal rate between 30% and 60% (Group B, 170 subjects) (P>0.05). For those with an abnormal rate of above 60% (Group C, 132 subjects), the sperm normal morphology rate, DNA damage, normal fertilization rate, ETR, D3 high quality embryo rate, high quality blastocyst rate were significantly lower compared with Group A (P<0.01), while no significant difference was found in fertilization rate, cleavage rate and BFR between groups A and C (P>0.05).

CONCLUSIONSNT is related with sperm morphology rate, DNA damage and embryo development, and should be assessed before ART.

Adult ; Blastocyst ; metabolism ; DNA Damage ; Embryo Transfer ; Embryonic Development ; Female ; Fertilization in Vitro ; Humans ; Infertility, Male ; genetics ; metabolism ; Male ; Nucleoproteins ; genetics ; metabolism ; Spermatozoa ; metabolism

Objective Infrared spectroscopy was used to study on both raw material and different degree water processing varieties extract of Radix Aconiti kusnezoffii,to observe the changes of main toxic components in processing,and thus to improve the quality of Radix Aconiti kusnezoffii processed products.Methods Fu Liye transform infrared spectroscopy was adopted to study the infrared spectrum characteristic of raw materials and different degree water processing varieties of.Radix Aconiti kusnezoffii.Results ①Aconitine and hypaconitine infrared spectrum showed that 1717 cm-1,1727 cm-1 and 1711 cm-1 is the C=O stretching vibration peak.It is a diester alkaloids characteristic peaks; ② although absorption peak of all vesicular samples had a certain change,it still existed diester alkaloids absorption peak,indicating the incomplete hydrolysis; ③ boiled aconite processing methods demonstrated diester alkaloids absorption peak shift in the water sample.Conclusion Diester alkaloids in Radix Aconiti kusnezoffii shows a positive relation with its time soaked in water.

OBJECTIVE: To establish a quality standard for Jieyinling lotion. METHODS: Cortex Phellodendri and Epimedium Herb were identified by TLC, and the content of Icariin was determined by HPLC. RESULTS: The TLC spots were fairly clear and a good reproducibility was obtained. The linear range of Icariin was 2.0 ～10.0?g(r=0.999 6),with average recovery at 100.1%,RSD =1.92%. CONCLUSION: The established method is reliable and accurate, and suitable for the quality control of Jieyinling lotion.