Host immunity and HBV S gene mutation in HBsAg-negative HBV-infected patients.

Background: Clinically, some patients whose HBsAg becomes negative owing to antiviral therapy or spontaneously still show a low level of HBV DNA persistence in serum. T-lymphocyte subsets, cytokine levels and HBV S gene sequences were analyzed in this study. Methods: A total of 52 HBsAg-negative and HBV DNA-positive patients(HBsAg-/HBV DNA+ patients), 52 persistently HBsAg-positive patients(HBsAg+/HBV DNA+ patients) and 16 healthy people were evaluated. T-lymphocyte subsets of these patients were detected by flow cytometry, serum cytokines and chemokines were detected by the Luminex technique, and the HBV S region was evaluated by Sanger sequencing. T%, T-lymphocyte, CD8+ and CD4+T lymphocyte were lower in the HBsAg-negative group than in the HC group. Compared with the HBsAg-positive group, the HBsAg-negative group had lower levels in T lymphocyte %, CD8+T lymphocyte %, CD8+T lymphocyte and CD4/CD8. These difference were statistically significant (P<0.05). Serum IFN-γ, IFN-α and FLT-3L levels were significantly higher in the HBsAg-negative group than in the HBsAg-positive group (P<0.05). However, levels of many cytokines related to inflammation (i.e., IL-6, IL-8, IL10, IL-12, IL-17A) were lower in the HBsAg-negative group. Fifty-two HBsAg-negative samples were sequenced, revealing high-frequency amino acid substitution sites in the HBV S protein, including immune escape mutations (i.e., Y100C, S114T, C124Y, P127L, G130R, T131N, M133T, C137S, G145A) and TMD region substitutions (i.e., E2K/R/D, G7D/R, G10D, A17R, F20L/S, L21V, L22V). Conclusions: According to the results of T-lymphocyte subsets and serum cytokines, it can be deduced that the cellular immune function of HBsAg-negative patients is superior to that of HBsAg-positive patients, with attenuation of liver inflammation. HBsAg-negative patients may show a variety of mutations and amino acid replacement sites at high frequency in the HBV S region, and these mutations may lead to undetectable HBsAg, HBsAg antigenic changes or secretion inhibition.

Disease predisposition of human leukocyte antigen class II genes influences the gut microbiota composition in patients with primary biliary cholangitis.

Background: The human leukocyte antigen (HLA) susceptibility gene is the main genetic risk factor for primary biliary cholangitis (PBC). The prognosis of patients with PBC is linked to gut microbiota dysbiosis. However, whether the HLA alleles are associated with the gut microbiota distribution and disease severity remains unknown. Methods: A cohort of 964 Chinese patients with PBC was enrolled at Beijing YouAn Hospital, Beijing, China. High-resolution genotyping of the HLA class I and class II loci from 151 of these patients was performed using sequence-based PCR. Stool samples were collected from 43 of the 151 fully HLA-typed patients to analyze their microbiota compositions via 16S RNA gene sequencing. Results: Of the 964 patients, the male:female ratio was 114:850, and 342 of these patients (35.5%) had already developed liver cirrhosis (LC) before enrollment. Patients with PBC showed a significantly higher frequency of HLA DRB1*08:03 than did the controls (21.2% vs. 9.0%, P=0.0001). HLA-DRB1*03:01, DRB1*07:01, DRB1*14:05, and DRB1*14:54 frequencies were also increased but did not reach significance after Bonferroni's correction. Conversely, the DQB1*03:01 frequency was significantly lower in patients with PBC than in the controls (24.5% vs. 39.2%, P=0.0010). The patients' gut microbiota were analyzed from four perspectives. The microbial community abundances were significantly lower in FHRAC-positive patients (patients with a combination of five HLA DRB1 high-risk alleles) than in FHRAC-negative patients (P<0.05). Of the top 10 microbial genera, Lachnospiraceae_incertae_sedis was higher in the FHRAC-positive patients than in the FHRAC-negative patients (P<0.05). linear discriminant analysis (LDA) effect-size (LEfSe) analysis showed different microbes at different levels in the FHRAC-negative patients but not in the FHRAC-positive patients. DQB1*03:01-positive patients contained mostly Lactobacillaceae at the family level. A comparison of the FHRAC-positive patients with and without liver cirrhosis showed that the abundances of Veillonella were significantly higher in patients with cirrhosis and FHRAC than in those without cirrhosis and are FHRAC-negative. Conclusion: The HLA class II genes may influence the gut microbiota compositions in patients with PBC. Differential gut microbiota were expressed at different taxonomic levels. Some bacterial abundances may be increased in FHRAC-positive patients with PBC and cirrhosis.

Characterization of the Clinical Features in HBV-Related Acute-on-Chronic Liver Failure.

OBJECTIVE: Acute-on-chronic liver failure (ACLF) is a type of liver failure commonly found in China, and currently the mechanism of the disease remains unknown. This study aimed to investigate the epidemiology, clinical features and prognostic factors in ACLF. METHODS: This study retrospectively included 170 patients with ACLF admitted to Beijing Friendship Hospital in Beijing, China from November 2017 to May 2019. Patients were divided into 2 groups: the improved group and the deteriorated group, according to the severity of their disease. Patients' demographic data; clinical manifestations; complications; laboratory indicators including platelets (PLT), alanine aminotransferase (ALT), aspartate amino transferase (AST), total bilirubin (TBIL), prothrombin time (PT), activated partial thromboplastin time (APTT), prothrombin activity (PTA), international normalized ratio (INR), and alkaline phosphatase (ALP) were collected. The relationship between these factors and the patients' prognosis were analyzed by logistic multivariate regression analysis. RESULTS: The highest morbidity rate was in the age group 40 to 49 years (29.41%). The age group with the second highest morbidity was between 50 and 59 years (25.29%), followed by >60 (21.18%), 30 to 39 (20.59%), 20 to 29 (2.94%) and <20 years (0.59%). A total of 53 patients (31.18%) had a family history of hepatitis B virus infection. The patients' main clinical manifestations were ascites (77.65%) and weakness (68.23%). The most common complications were hypoalbuminemia (80%), infection (67.65%) and electrolyte imbalance (44.12%). In addition, the PTA (P = .009), hepatorenal syndrome (P = .005) and hepatic encephalopathy (level IV) (P = .005) were independently related to the prognosis of ACLF. There is a significant relationship between complications and prognosis (χ2 = 8.502; P = .004). CONCLUSION: This study showed that prothrombin activity, hepatorenal syndrome and hepatic encephalopathy were independently related to the prognosis of ACLF. This outcome provided more options for reducing patient mortality in clinic.

Using two-step cluster analysis to classify inpatients with primary biliary cholangitis based on autoantibodies: A real-world retrospective study of 537 patients in China.

Background: A variety of autoantibodies have been detected in primary biliary cholangitis (PBC), while the presence of autoantibody clusters and their clinical significance have not been fully understood. We aimed at defining autoantibody clusters and to better understand the clinical features and prognosis of PBC patients based on autoantibody clusters under real-world conditions. Methods: We retrospectively analyzed 788 inpatients with PBC evaluated between October 2008 and July 2019, and included 537 patients. Nineteen autoantibodies which were measured routinely were investigated for cluster analysis. Two-step clustering, Kaplan-Meier survival, and Cox regression analyses were used. Results: Five clusters were defined. A cluster of antinuclear antibodies (ANA) and anti-gp210 positive patients were identified with a high rate of cirrhosis at baseline and low survival rate; a cluster of ANA, anti-centromere antibodies (ACA) and/or anti-CENP-B female dominant patients with older disease onset, low level of platelet count at baseline, high rate of hepatic decompensation, and low survival rate was also characterized; and another cluster of anti-mitochondrial antibodies (AMA) and/or AMA-M2, anti-Ro52 and a high rate of anti-gp210 positive patients were identified with a high proportion of male patients and low survival rate. A subgroup of patients with anti-SSA and/or anti-SSB coexists with SjS was also identified; patients with only AMA and/or AMA-M2-positive with a benign clinical outcome and relatively high complication of non-alcoholic fatty liver disease (NAFLD) were also identified. Only anti-gp210 was considered as a significant predictor for poor outcomes especially in patients with cirrhosis. Conclusion: Clustering methods allow the identification of distinct autoantibody profiles of PBC that form clinical subsets and can be useful for personalized approaches to diagnosis, clinical management, and the prediction of clinical outcomes. Anti-gp210 was the strongest predictive factor for poor outcomes especially in PBC patients with cirrhosis under real-world conditions.

Clinical characteristics and HLA genotypes in Chinese patients with anti-SLA/LP-positive autoimmune hepatitis.

BACKGROUND: Anti-soluble liver antigen/liver pancreas (anti-SLA/LP) is a highly specific serological marker for the diagnosis of autoimmune hepatitis (AIH). The aim of the present study was to define the clinical characteristics and human leucocyte antigen (HLA) genotypes of Chinese patients with anti-SLA/LP positive AIH. METHODS: Ninety-one AIH patients who were anti-SLA/LP positive were enrolled in this case control study. Clinical information was obtained through reviewing patients' clinical notes. High-resolution genotyping of HLA-A, B, C, DRB1, and DQB1 alleles was performed by sequence-based typing polymerase chain reaction on 62 of the 91 patients. Data from 500 healthy patients were used as baseline controls. RESULTS: Anti-SLA/LP-positive AIH patients were characterized as follows: adults (age 20-80 years), female (88%), and frequent anti-nuclear antibody positivity (91%). Genetically, compared with the controls, HLA-B*35:01 and C*08:01 were significantly more frequent in patients. The frequencies of HLA-B*08:01, B*40:02, DRB1*04:01, DRB1*04:05, DRB1*14:01, and DRB1*16:02 increased, and the frequency in DRB1*15:01 decreased in patients, but did not reach significance after Bonferroni's correction. Patients with other autoimmune diseases had a higher DRB1*04:05 and DQB1*04:01 allele carrier frequency than those without. DRB1*04:05 and DQB1*04:01 alleles were found at increased frequency in patients with decompensated liver disease than those with compensated liver disease. CONCLUSIONS: Chinese anti-SLA/LP-positive AIH patients have some distinct clinical characteristics than other populations reported in the literature. The presence of certain specific HLA alleles could potentially increase the risk of developing anti-SLA/LP-positive AIH or other autoimmune disease and decompensated liver disease in the Chinese population.

The prevalence and immunological features of anti-glomerular basement membrane antibody in patients with HIV.

BACKGROUND: Anti-glomerular basement membrane disease (GBM) is an autoimmune disease caused by the deposition of circulating anti-GBM antibodies. Non-collagen region of α3 chain of type IV collagen (α3(IV)NC1) is one of the main target antigens, in which EA and EB are the most classical antigen epitopes. It has been reported that anti-GBM antibodies can be detected in HIV patients; however, its immunological characteristics are still unclear. OBJECTIVES: In this study, the positive rate of the anti-GBM antibodies in HIV and the immunological characteristics of the target antigens were clarified. METHODS: A total of 93 HIV patients diagnosed in Beijing Youan Hospital from November 2017 to January 2018 were included. Enzyme-linked immunosorbent assay was used to measure the serum IgG autoantibodies specifically against GBM in these patients, as well as their subtypes and antigen spectra. RESULTS: It was found that five out of the 93 patients with HIV had low to moderate levels of anti-GBM antibodies. However, these patients presented with no clinical manifestation of any kidney injury or pulmonary hemorrhages. Compared with HIV patients with negative antibodies, there were no significant differences in gender, age, CD4+T cell count and HIV viral load. All sera of five patients recognized non-collagenous domain1 (NC1) of alpha 3 chain of type IV collagen [(α3(IV)NC1] as classic anti-GBM patients, followed by α5(IV)NC1. The antibodies against α3(IV)NC1 were IgG3 predominant, while these antibodies did not react with either of the classic epitopes on α3 (EA and EB). CONCLUSION: These data suggest a distinct immunological profile of anti-GBM antibodies in patients with HIV, and might explain the non-pathogenic features of HIV associated anti-GBM antibodies.

A multiple-center clinical evaluation of a new real-time reverse transcriptase PCR diagnostic kit for SARS-CoV-2.

Aim: The outbreak of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has had serious repercussions worldwide. This study was aimed to evaluate the accuracy of a new kit for detection of SARS-CoV-2 compared with similar detection kit. Materials & methods: A total of 500 subjects were included and tested with both the new test and control kits. Clinical diagnosis results were taken as the reference standard. Results: Compared with clinical diagnosis, the sensitivity of the test kit was 82.64%, specificity was 98.45% and total coincidence rate was 90.80%. The total coincidence rate, sensitivity and specificity between control kit and clinical diagnosis were 89.20%, 78.10% and 99.61%, respectively. Conclusions: The new kit was comparable to the similar detection kit for detection of SARS-CoV-2 in sensitivity, specificity and total coincidence rate.

Occult HBV infection in patients with autoimmune hepatitis: A virological and clinical study.

BACKGROUND/PURPOSE: Occult HBV infection (OBI) could have serious clinical consequences in patients receiving immunosuppressive therapy. We aimed to investigate the prevalence of OBI in Chinese patients with autoimmune hepatitis (AIH) and to analyze its clinical and virological features. METHODS: 103 AIH cases were enrolled. Hepatitis B virus (HBV) serological markers were screened by chemiluminescence. HBV-DNA were detected by nest-PCR and real-time PCR. HBV genotyping and mutation analysis were performed by Sanger sequencing. RESULTS: Twenty-four out of 103 (23.30%) AIH patients had OBI as evidenced by positive HBV-DNA and negative hepatitis B surface antigen (HBsAg). HBV genotype C is the predominant genotype (57.89%), which had more amino acid (AA) substitutions in S region than that of B-genotype group (P = 0.001). The distribution of AA substitution in the 'α' determinant region between genotype C and B were significantly different (P = 0.042). In addition to those already reported OBI-associated AA substitutions (e.g., sG145R and sV184A), some new OBI-associated AA substitutions (e.g., sV106A, sC137* and sL176P) were found in AIH patients in our study. Three out of 24 (12.50%) OBI patients were diagnosed as decompensated cirrhosis, one patient with S deletion mutation and two patients with HBV extensive AA substitutions. CONCLUSIONS: There was a higher proportion of AIH patients with OBI than the general population in China, which can be either seropositive or seronegative-OBI in AIH patients is associated with some specific AA substitutions. The presence of deletion mutations and the extent of AA substitutions in the HBV S region may have predictive clinical implications.

Coexistence of low levels of HBsAg and high levels of anti-HBs may increase risk of hepatocellular carcinoma in chronic hepatitis B patients with high HBV load.

OBJECTIVE: The clinical significance of coexistence of HBsAg/anti-HBs in chronic hepatitis B (CHB) patients remains controversial. This study was aimed to assess the association of this serological pattern with hepatocellular carcinoma (HCC) in patients with CHB. METHODS: In this cross-section study, 206 CHB patients with coexistence of HBsAg/anti-HBs and 206 CHB patients with HBsAg alone were included to evaluate the risk of HCC development by logistic regression analysis. In addition, a retrospective cohort of 260 patients with CHB was recruited to estimate the cumulative incidence of HCC by Kaplan-Meier analysis. RESULTS: The serological pattern of coexistence of HBsAg/anti-HBs, with high levels of ("High") HBsAg/low levels of ("Low") anti-HBs, were considered as independent risk factors for HCC. In particular, patients with "High" HBsAg/"High" anti-HBs [odds ratio (OR), 4.295; 95% confidence interval (CI), 1.104-16.699; p = 0.035] and "Low" HBsAg/ "High" anti-HBs (OR, 3.207; 95%CI, 1.299-7.919; p = 0.012) exhibited significantly higher risk for HCC development. However, only "Low" HBsAg /"High" anti-HBs might increase risk of HCC in CHB patients with high HBV load (logrank p < 0.001) in our cohort study. CONCLUSION: The coexistence of "Low" HBsAg /"High" anti-HBs might increase the risk of HCC development in CHB patients with high HBV load, which reflected that the long-term interaction between immune response and virus might lead to the development of HCC. The identification of the patients with poor prognosis will help clinicians to refine the therapeutic decisions and individualize follow-up strategies.

Retraction Note: Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells.

[This retracts the article on p. 224 in vol. 23, PMID: 28127196.].

Protective Effect of Ganshuang Granules () on Liver Cirrhosis by Suppressing Regulatory T Cells in Mouse Model.

OBJECTIVE: To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules (, GSG) and to provide clinical therapeutic evidence of its effects. METHODS: A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl4 (40%) and oil (60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium- and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin (SMA) and Foxp3 in liver were detected by immunoflfluorescence. The regulatory T cell (Treg) frequency was determined through flflow cytometry analysis. Collagen-I, SMA, IL-6, tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and transforming growth factor ß1 (TGF-ß1) expression levels were measured using quantitative polymerase chain reaction (qPCR). RESULTS: Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment (ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-I and SMA in the cirrhotic livers were also attenuated by GSG treatment (collagen-I: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment (F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1ß increased, and TGF-ß levels decreased in the cirrhotic livers after GSG treatment (IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1ß: F=6.658, P=0.001; and TGF-ß1: F=11.239, P=0.000). CONCLUSIONS: GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.

Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells.

AIM: To prepare a Gpm6a/ReelinGFPCreERT2 construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC). METHODS: Gpm6a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreERT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit. RESULTS: A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with BamHI digestion. The GFPCreERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCreERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers. CONCLUSION: The construct of Gpm6aGFPCreERT2 or ReelinGFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

AIM: To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). METHODS: Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. RESULTS: Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. CONCLUSION: ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

[Effect of puerarin on liver injury in KKAy mice with type 2 diabetes mellitus].

OBJECTIVE: To study the possible pathogenic mechanism of liver injury in type 2 diabetes mellitus (T2DM) and the intervening effect of puerarin on it. METHODS: Mice with T2DM (KKAy) were randomly divided into two groups, the model group and the puerarin group. And the C57BL/J mice of the same age were set up as normal controls. They were sacrificed at 28 weeks old for observing serum fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) by automatic biochemistry; liver cell apoptosis by flow cytometry; pathomorphology by electron microscope; and mRNA expressions of bcl-2 and bax genes by RT-PCR; as well as the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), Na(+)-K(+)-ATPase; and content of malondialdehyde (MDA) in liver tissue by spectrophotometer. RESULTS: In KKAy mice, blood levels of FBG, TG, TC, ALT, AST and liver cell apoptosis rate were higher; the bax mRNA expression was higher and bcl-2 mRNA was lower markedly; the activities of SOD, GSH-Px, Na(+)-K(+)-ATPase in liver tissue were lower, and MDA content was higher than those in the normal control significantly (all P <0.01). Besides, mitochondria swelling and damage were found in liver tissue. While in the puerarin group after treatment, all the above-mentioned changes were alleviated to some extent. CONCLUSIONS: Obvious liver injury emerges in KKAy mice. Puerarin shows a protective effect on the T2DM caused oxidative damage by way of up-regulating bcl-2 to inhibit oxidative stress, and improving the energy metabolic dysfunction in liver of mice.

[Effect of Xinnao Shutong capsule on apoptosis after cerebral ischemia and reperfusion in rats].

OBJECTIVE: To study apoptosis-regulating cytokines and apoptosis on focal cerebral ischemia and reperfusion in rats treated with Xinnao Shutong capsule. METHOD: Rat models of focal cerebral ischemia and reperfusion were established by thread ligation in middle cerebral artery occlusions (MCAO). After 24 hours, the brains were removed to detect changes of protein expression of Bax, Bcl-2, Fas, Fas-L and caspase-3 by immuno-hisochemistry, and apoptosis of cortical neurons by TUNEL RESULT: Compared to control, brain cortex have decreasing the protein expression of Bcl-2 and the ratio of Bcl-2/Bax, increasing the protein expression of Bax, Fas, Fas-L and caspase-3 of ischemia and reperfusion models group (P < 0.01). Xinnao Shutong capsule group could increase the protein expression of Bcl-2 and the ratio of Bcl-2/Bax, and obviously decrease the protein expression of Bax, Fas, Fas-L and caspase-3, then reduce the number of apoptotic cells of cortex (P < 0.01). CONCLUSION: Xinnao Shutong capsule protect injured rat brain tissue, may be related to decrease neuronal apoptosis and adjusted protein expression of apoptosis-regulating cytokines.

[Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats].

OBJECTIVE: To observe the effects of Tribulus terrestris L. saponion (TTLS) on apoptosis in cortical neurons induced by hypoxia-reoxygenation in rats. METHODS: Primary culture of rat cortical neurons was performed in vitro. A model of apoptosis of cortical neurons was established by hypoxia and reoxygenation. Hypoxia for 3 h in neural cells was induced with mixture of 95% N(2) and 5% CO(2), and then reoxygenation in neural cells was induced with mixture of 95% O(2) and 5% CO(2) for 12 h. Different concentrations of TTLS were administered to traditional Chinese herbal medicine-treated group separately during hypoxia and reoxygenation. The apoptosis rate was analyzed quantitatively by flow cytometry with Annexin V-FITC and propidium iodide staining. Mitochondria membrane potential was observed by a confocal laser-scanning microscope with JC-1 fluorescence. Caspase-3/7 activity in cytoplasm was measured by fluorescent plate reader. Bax protein expression was observed by immunohistochemical technique. RESULTS: The percentage of apoptosis was significantly increased, mitochondria membrane potential was obviously decreased, fluorescence of caspase-3/7 activity was increased, and Bax protein was abundantly expressed followed by 3 h of hypoxia and 12 h of reoxygenation (P<0.01). TTLS could inhibit the depression of membrane potential induced by hypoxia and reoxygenation, decrease the activity of caspase-3/7, reduce the expression of Bax protein, and inhibit the apoptosis of the cortical neurons. CONCLUSION: Hypoxia and reoxygenation can induce apoptosis of rat cortical neurons. TTLS can decrease the apoptosis induced by hypoxia and reoxygenation. The mechanism might be related to stabilization of mitochondria membrane potential, inhibition of caspase activity and reduction of Bax protein expression.

[Protective effects of Xinnao Shutong capsule on acute cerebral ischemic injury of multiple infarcts in rats].

OBJECTIVE: To study the effect of Xinnao Shutong capsule (XNST) on energy metabolism dysfunction, free radical injury and inflammatic factors in the course of acute cerebral ischemic damage, and try to reveal the mechanism of the protection against ischemia. METHOD: 60 male Wistar rats weighing 280 - 320 g were randomly divided into five groups: normal, sham operation, model, XNST treatment( XNST-T) , and Western medicine treatment (WM-T) group. Acute multi-infarct model in rats was induced by injecting the embolus of blood powder through the right external carotid artery (ECA) into the internal carotid artery (ICA). At 72 hours after ischemia, morphologic change and the express of tumor necrosis factor-alpha (TNF-alpha) and interleukin -1beta ( IL-1beta) in hippocampus CAl section and cortex were observed, biochemical criterions including the activity of Na+ -K+ -ATPase, lactate dehydrogenase (LDH), superoxide dismutase (SOD), and the content of malondialdehyde (MDA) in hippocampus were examined. RESULT: The morphologic change of hippocampus and cortex in both XNST-T and WM-T groups was milder than that in model group. The activity of Na+ -K+ -ATPase, LDH and SOD in hippocampus were all significantly decreased in model group (P <0. 01), and elevated in XNST group (P <0. 01) as well as in WM-T group (P <0. 01). The content of MDA in hippocampus was significantly increased in model group (P <0. 05), and was reduced in XNST group (P <0. 05) as well as in WM-T group (P <0. 01). CONCLUSION: The results reveal that XNST has the protective effect against cerebral ischemic injury. And its possible mechanism is that XNST can prevent the upper pathological process.