Atrase A, a P-III class metalloproteinase purified from cobra venom, exhibits potent anticoagulant activity by inhibiting coagulation pathway and activating the fibrinolytic system.

Snake venoms, comprising a complex array of protein-rich components, an important part of which are snake venom metalloproteinases (SVMPs). These SVMPs, which are predominantly isolated from viperid venoms, are integral to the pathology of snakebites. However, SVMPs derived from elapid venoms have not been extensively explored, and only a handful of SVMPs have been characterized to date. Atrase A, a nonhemorrhagic P-III class metalloproteinase from Naja atra venom, exhibits weak proteolytic activity against fibrinogen in vitro but has pronounced anticoagulant effects in vivo. This contrast spurred investigations into its anticoagulant mechanisms. Research findings indicate that atrase A notably extends the activated partial thromboplastin time, diminishes fibrinogen levels, and impedes platelet aggregation. The anticoagulant action of atrase A primarily involves inhibiting coagulation factor VIII and activating the endogenous fibrinolytic system, which in turn lowers fibrinogen levels. Additionally, its effect on platelet aggregation further contributes to its anticoagulant profile. This study unveils a novel anticoagulant mechanism of atrase A, significantly enriching the understanding of the roles of cobra venom metalloproteinases in snake venom. Furthermore, these findings underscore the potential of atrase A as a novel anticoagulant drug, offering insights into the functional evolutions of cobra venom metalloproteinases.

Icariin ameliorates LPS-induced acute lung injury in mice via complement C5a-C5aR1 and TLR4 signaling pathways.

Acute lung injury (ALI) is an acute respiratory-related progressive disorder, which lacks specific pharmacotherapy. Icariin (ICA) has been shown to be effective in treating ALI. However, the targets and pharmacological mechanisms underlying the effects of ICA in the treatment of ALI are relatively lacking. Based on network pharmacology and molecular docking analyses, the gene functions and potential target pathways of ICA in the treatment of ALI were determined. In addition, the underlying mechanisms of ICA were verified by immunohistochemistry, immunofluorescence, quantitative Real-time PCR, and Western blot in LPS-induced ALI mice. The biological processes targeted by ICA in the treatment of ALI included the pathological changes, inflammatory response, and cell signal transduction. Network pharmacology, molecular docking, and in vivo experimental results revealed that ICA inhibited the complement C5a-C5aR1 axis, TLR4 mediated NF-κB, MAPK, and JAK2-STAT3 signaling pathways related gene and protein expressions, and decreased inflammatory cytokine, chemokine, adhesion molecule expressions, and mitochondrial apoptosis in LPS-induced ALI.

Salvianolic acid A alleviates lipopolysaccharide-induced disseminated intravascular coagulation by inhibiting complement activation.

INTRODUCTION: Disseminated intravascular coagulation (DIC) is a syndrome characterized by coagulopathy, microthrombus, and multiple organ failure. The complement system in DIC is overactivated, and the functions of complement and coagulation pathways are closely related. Our previous screening revealed that salvianolic acid A (SAA) has anti-complement activity. The hyper-activated complement system was involved in the lipopolysaccharide (LPS) induced DIC in rats. The effects of SAA anti-complement action on LPS-induced DIC in rats were investigated. METHODS: The complement activity of the classical pathway and alternative pathway was detected through an in vitro hemolysis assay. The binding sites of SAA and complement C3b were predicted by molecular docking. LPS-induced disseminated coagulation experiments were performed on male Wistar rats to assess coagulation function, complement activity, inflammation, biochemistry, blood routine, fibrinolysis, and survival. RESULTS: SAA had an anti-complement activity in vivo and in vitro and inhibited the complement activation in the classical and alternative pathway of complement. The infusion of LPS into the rats impaired the coagulation function, increased the plasma inflammatory cytokine level, complemented activation, reduced the clotting factor levels, fibrinogen, and platelets, damaged renal, liver, and lung functions, and led to a high mortality rate (85%). SAA treatment of rats inhibited complement activation and attenuated the significant increase in D-dimer, interleukin-6, alanine aminotransferase, and creatinine. It ameliorated the decrease in plasma levels of fibrinogen and platelets and reversed the decline in activity of protein C and antithrombin III. The treatment reduced kidney, liver, and lung damage, and significantly improved the survival rate of rats (46.2 and 78.6% for the low- and high-dose groups, respectively). CONCLUSION: SAA reduced LPS-induced DIC by inhibiting complement activation. It has considerable potential in DIC treatment.

[Pharmacokinetic study on combined application of gastrodin and puerarin in rats].

To establish a HPLC method for simultaneously determining plasma concentrations of gastrodin (Gas) and its metabolites hydroxybenzyl alcohol (HBA), puerarin (Pur) and internal standard (IS) p-hydroxyphenylethanol (Tyr) in rats and studying the pharmacokinetic process and interactions of gastrodin and puerarin after single and combined intravenous injection and oral administration. With Tyr as the internal standard, plasma samples were processed with methanol for protein precipitation, supernatant was dried with N2, and residues were re-dissolved with acetonitrile-0.05% phosphoric acid (20: 80). Chromatography was carried out on an Agilent ZORBAX SB-Aq C18 column (4.6 mm x 250 mm, 5 µm), with acetonitrile-0.05% phosphoric acid as the gradient mobile phase for the gradient elution. The UV detector wavelength was set at 221 nm for Gas HBA and IS and 250 nm for Pur. After the single or combined administration of Gas and Pur, their plasma concentrations in rats were detected. WinNonlin 5.2 pharmacokinetic software and SPSS 17. 0 software were used to respectively calculate pharmacokinetic parameters of each group, make a statistical analysis and compare the pharmacokinetic processes of Gas and Pur after the single or combined administration. According to the results, the absolute recoveries between low, media and high concentrations of Gas, HBA and Pur and IS as well as Tyr were more than 77.20%, with a good linearity (r > 0.999 6, n = 5) for Gas, HBA and Pur within concentration ranges of 0.10-101, 0.03-7.58 and 0.05-5.98 mg xL ('1) respectively. The lower limits of quantification for Gas, HBA and Pur were 0.10, 0.03, 0.05 mg x L(-1), respectively. Their in-ra-day and inter-day precisions were less than 12% with the accuracy between 85. 1% -1 10. %. All of the three substances and IS were stable during the whole analysis process. The findings showed significant differences in the main in vivo pharmacokinetic parame-ers in rats (AUC, C.(max) T,½ T.(max) MRT) after the single and combined administration of Gas and Pur. Either after the oral adminis-ration or after the intravenous injection, parameters showed a lower clearance rate ( L) longer mean residence time ( RT) and higher relative bioavailability, especially after the oral administration. Specifically, the relative bioavailability of the combined oral ad-inistration of Pur was 10. 7 times of that of the single administration, while that of Gas was 1. times of that of the single administra-ion. The combined administration of Gas and Pur can promote the absorption, decrease the elimination rate and prolong the mean resi-ence time. The method is simple and accurate and can be applied in the simultaneous determination of plasma concentrations of Gas, HBA and Pur in rats and the pharmacokinetic studies.

[Polymerase region mutations and hepatitis B virus genotypes in chronic hepatitis B patients with poor response to lamivudine].

OBJECTIVE: To investigate the mutations in Polymerase region and hepatitis B virus (HBV) genotypes in chronic hepatitis B patients with poor response to Lamivudine treatment. METHODS: 631 chronic hepatitis B patients with poor response to Lamivudine were recruited in this study. Real-time PCR and DNA sequencing were used to determine HBV genotypes; direct sequencing was performed to detect mutations, and real-time PCR was used to quantify HBV DNA load. Mutations in polymerase region were investigated in different HBV genotypes. RESULTS: 272 patients were infected with HBV of genotype B, and 359 patients were infected with HBV of genotype C. The mean age of patients infected with HBV of genotype C (39.1+/-11.4 years old) were significant higher than that of patients infected with HBV of genotype B (33.7+/-9.7 years old) (t = -6.55, P less than 0.01). The patients infected with HBV of genotype C had relatively higher HBV DNA load [(5.96+/-1.22) log10 copies/ml] than the patients infected with HBV of genotype B [(5.58+/-1.21) log10 copies/ml] (t = -2.01, P less than 0.05). The overall incidence rate of A181V/T mutation in genotype C (5.3%) was significantly higher than that in genotype B (0.4%) (x2=12.23, P less than 0.01), but the incidence rate of M204I/V, L180M, T184A/G/I/S, S202G/I and V173L mutations was not significantly different between genotype B and C (each P more than 0.05). M204I mutation in genotype B (20.6%) was more frequent than that in genotype C (13.9%) (x2=4.91, P less than 0.05). The Lamivudine resistance mutations were not significantly different between genotype B and genotype C (x2 = 0.00, P more than 0.05). CONCLUSIONS: The incidence rate of lamivudine resistance mutation is not significantly different between genotype B and genotype C, but patients infected with HBV of genotype C have higher HBV DNA load than patients infected with HBV of genotype B.

[Viral load test conducive to excluding negative subjects from suspects in HIV antibody detections].

OBJECTIVE: To study whether plasma viral load testing is helpful to exclude ones free from Human immunodeficiency virus (HIV) infections from suspects in HIV antibody detections. METHODS: 19 Specimens, which showed disconcordant results of the two HIV EIA testing (S/CO < 6) and indeterminated results of Western blot (WB) test, were selected. Viral load of the specimens were detected. A six-month follow up survey in detecting HIV antibody was conducted in these subjects. RESULTS: None of these 19 cases was observed to be positive HIV viral loads and there was no any progress in WB bands development during the follow-up period. The possibility of HIV infection could be excluded. CONCLUSION: When the specimens react with very low intensity in both EIA and WB, negative viral load result is conducive to exclude negative subjects from suspects in HIV antibody detections.

[Subtype and sequence analysis of gag and env genes among HIV-1 strains circulating in Beijing residents during 2006].

OBJECTIVE: To investigate the subtype distribution and the prevalence of sequence characteristics of HIV-1 strains in Beijing residents during 2006 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routines. METHODS: Blood samples from 32 new confirmed HIV-1 infected individuals from Beijing residents in 2006 and separated plasma specimens were collected. RNAs were extracted and the gag and env gene were amplified by RT-PCR and nest-PCR. PCR products were sequenced directly and phylogenetic analyses of gag and env gene were performed using the MEGA2 software. RESULTS: Among 32 HIV-1 plasma samples, 22 gag and 4 env gene fragments were amplified and analyzed. Five HIV-1 subtypes or circulating recombinant forms(CRFs) of HIV-1 including Thai B (2 strains), B (9 strains), C (2 strains), CRF07_BC (5 strains), CRF01 AE (4 strains) were identified being circulated in Beijing. The gene divergences of gag gene inside the subtypes were 6.6%, 4.3%, 6.8%, 4.9% and 3.0% in subtype B, Thai B, C, CRF01_AE and CRF07_BC respectively. Subtypes B were predominant in Beijing, accounted for 40.9% among 22 samples. CONCLUSION: Five HIV-1 subtypes were identified in Beijing and the surveillance of HIV-1 gene variation should be paid more attention to.

Field evaluation of alternative testing strategies for the detection of HIV infection in Beijing.

OBJECTIVE: To identify a cost-efficient alternative antibody testing strategy for screening and confirmation of HIV infection by rapid simple tests (RSTs) and enzyme-linked immunosorbent assays (ELISAs). METHODS: Four RSTs (RST1, RST2, RST3, and RST4) and five ELISAs (ELISA1, ELISA2, ELISA3, ELISA4, and ELISA5) were evaluated in two phases by using banked and serum specimens prospectively collected at regional hospitals and voluntary counseling and testing (VCT) centers in Beijing. A total of 200 banked serum specimens were included in the first phase, including 62 HIV-positive, 127 HIV-negative and 11 indeterminate specimens. All specimens were tested by four RSTs and five ELISAs respectively. The second phase involved prospective testing of 389 routine specimens, including 92 HIV-positive, 287 HIV-negative, and 10 indeterminate specimens. All the specimens were tested by two RSTs (RST2 and RST4) and three ELISAs (ELISA1, ELISA3, and ELISA4), which were selected for their respective excellent sensitivity and/or specificity. Western blot (WB) was used as a gold standard for confirming the reactivity of all the specimens. RESULTS: Sensitivity, specificity, and efficacy were calculated for each assay in two phases. In the first phase, four assays (ELISA4, RST2, RST3, and RST4) had a specificity of 100%. For the determination of efficacy, ELISA4, RST2, and RST4 were selected in the second phase. ELISA1 and ELISA3 which have a sensitivity of 95.9% and 93.2% respectively also entered this phase. In the second phase, all the five assays (ELISA1, ELISA3, ELISA4, RST2, and RST4) had a sensitivity and specifity of over 90%. ELISA1 had a sensitivity of 99% and ELISA4 a specificity of 99%. CONCLUSION: The sensitivity ELISA1 and the specificit of ELISA4 are comparable to ELISA/WB standard strategy. Application of this alternative testing strategy provides a cost-effective method for determining HIV prevalence in Beijing.

[Epidemiological study on the status of HIV/STDs and relative behaviors among MSM in Beijing].

OBJECTIVE: To understand the infected status of human immunodeficiency virus (HIV), sexually transmitted disease (STD) and relative risk behaviors of men who have sex with men (MSM) in Beijing. METHODS: Respondent-driven sampling (RDS) was adopted to recruit target population and to conduct behavior and serological studies among MSM. RDSAT 5.5 was used to analyze data. RESULTS: Among 427 MSM, the age distribution was as follows: below 30 and less than 35 were 65.4% and 81.0% respectively. 69.2% (95% CI: 63.9-75.5) of them had 12 years or less of education. 73.3% (95% CI: 66.7-79.8) of them were non-Beijing registered residents. The urban and rural registered residents almost accounted for half of all the recruits. HIV positive rate was 4.6% (95% CI: 2.2-7.6) while 56.3% (95% CI: 50.9-62.5) of them reported having had bisexual sex preference. Only 14.8% (95% CI: 10.8-19.6) of them had ever had HIV test, while 22.8% (95% CI: 18.7-27.8) reported ever having had STDs. 55.3% (95% CI: 49.3-61.9) and 55.1% (95% CI: 48.7-61.3) of them had unprotected insert and receptive anal sex over the last 6 months. CONCLUSION: Data from the HIV positive rate showed that there was a trend of increase among MSM in Beijing which called for urgent care to them.

Direct detection of nitric oxide in human blood serum by use of 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)difluoroboradiaza-s-indacene with HPLC.

To study the relationship between amounts of nitric oxide (NO) in blood and the development of ischemic cardio-cerebrovascular diseases, trace NO in human blood serum has, for the first time, been determined by use of a 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)difluoroboradiaza-s-indacene (TMDABODIPY)-based HPLC method. The proposed method is simple, rapid, and efficient, owing to its high sensitivity and good selectivity for NO. TMDABODIPY and its NO derivative are separated to baseline in 4 min, with simple separation conditions, on a C(18) column eluted with 50 mmol L(-1) ethanolamine in methanol. The derivative is detected by fluorescence at an emission wavelength of 507 nm with excitation at 498 nm. The response is a linear function of concentration in the range 0.8-800 nmol L(-1) NO. The detection limit can reach 2 x 10(-11) mol L(-1) (signal-to-noise ratio=3). The method has been used to detect NO in the serum of patients with five kinds of ischemic cardio-cerebrovascular disease and two diseases closely connected with ischemic cardio-cerebrovascular diseases. Recoveries of NO from spiked serum samples were between 96.58 and 105.71% and concentrations of NO observed in real samples were at 10(-7) mol L(-1) levels. Our studies indicate that the proposed TMDABODIPY-based HPLC technique can be developed into a sensitive and new method for clinical assay and pathology research.

[IgM to recombinant antigen E2 of HEV envelope protein is a sensitive and specific serological marker in diagnosis of acute hepatitis E].

OBJECTIVES: To evaluate the sensitivity and specificity of IgM to recombinant antigen E2 of HEV envelope protein in the diagnosis of acute sporadic hepatitis E. METHODS: anti-E2 IgM was detected in sera samples from 176 cases of acute sporadic hepatitis E and 191 cases of acute non A-E hepatitis by ELISA and was compared with HEV IgM detected by some domestic and Genelabs (GL) kits. HEV RNA was also detected in sera positive for anti-E2 IgM. Logistic regression was used to analyze factors associated with the detection of anti-E2 IgM and HEV RNA. RESULTS: Anti-E2 IgM was found in 68.75% of the serum samples from the 176 patients of acute hepatitis E and the positive rate of HEV IgM detection by domestic kits was 56.25% (chi2 IgM = 6.49, P < 0.05). There were 37 (19.37%) anti-E2 IgM positive cases in those 191 sera of the acute non A-E hepatitis, out of which 11 cases were also detected as positive by the GL kit. Of the 158 anti-E2 IgM positive sera, HEV RNA was found in 81 (51.27%), among which 57.02% was positive in acute hepatitis E and 32.43% in acute non A-E hepatitis. No HEV RNA was found in the anti-E2 IgM negative sera from the cases of acute hepatitis E. By logistic regression analysis, no variance relative to the detection of anti-E2 IgM was found with the time interval from onset to hospitalization, age, levels of bilirubin, ALT and AST of the serum. Only the level of serum ALT was found being significantly associated with the detection of HEV RNA (P = 0.024). CONCLUSIONS: (1) anti-E2 IgM is a sensitive and specific serological maker for diagnosing an acute infection of HEV. (2) HEV is still the pathogen of some cases diagnosed clinically as non-A-E hepatitis. (3) Persistent HEV viremia is possibly an important factor influencing the severity of acute hepatitis E.