Selective Photoaffinity Probe for Monitoring Farnesoid X Receptor Expression in Cultured Cells.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, is a vital ligand-activated transcriptional factor, which is highly expressed in the liver, intestine, and adrenal gland. However, FXR homeostasis is influenced by many factors, such as diet and circadian rhythm, and the expression of FXR differs in diverse organs. Currently, there is no method to monitor the FXR homeostasis in real time, which restricts us from further investigating the function of FXR under physiological and pathological conditions. In this project, classic FXR agonists were selected to be modified to targeting FXR. The photo-cross-linking diazirine group and alkynyl, a click reaction group, were incorporated to the ligands. Through biorthogonal reaction, fluorophore was linked to the ligands to realize the monitoring of FXR expression in cells.

Gasdermin E-derived caspase-3 inhibitors effectively protect mice from acute hepatic failure.

Programmed cell death (PCD), including apoptosis, apoptotic necrosis, and pyroptosis, is involved in various organ dysfunction syndromes. Recent studies have revealed that a substrate of caspase-3, gasdermin E (GSDME), functions as an effector for pyroptosis; however, few inhibitors have been reported to prevent pyroptosis mediated by GSDME. Here, we developed a class of GSDME-derived inhibitors containing the core structure of DMPD or DMLD. Ac-DMPD-CMK and Ac-DMLD-CMK could directly bind to the catalytic domains of caspase-3 and specifically inhibit caspase-3 activity, exhibiting a lower IC50 than that of Z-DEVD-FMK. Functionally, Ac-DMPD/DMLD-CMK substantially inhibited both GSDME and PARP cleavage by caspase-3, preventing apoptotic and pyroptotic events in hepatocytes and macrophages. Furthermore, in a mouse model of bile duct ligation that mimics intrahepatic cholestasis-related acute hepatic failure, Ac-DMPD/DMLD-CMK significantly alleviated liver injury. Together, this study not only identified two specific inhibitors of caspase-3 for investigating PCD but also, more importantly, shed light on novel lead compounds for treating liver failure and organ dysfunctions caused by PCD.

The predictive role of CD4+ cell count and CD4/CD8 ratio in immune reconstitution outcome among HIV/AIDS patients receiving antiretroviral therapy: an eight-year observation in China.

BACKGROUND: The immune reconstitution after initiation of highly active antiretroviral therapy (HAART) among HIV-infected individuals substantially affects patients' prognosis. However, the dynamic characteristics and predictors of reconstitution outcome remain unclear. METHODS: In this study, the HIV/AIDS patients with sustained virological suppression (viral load < 50 copies/ml) after HAART were enrolled. The patients were subgrouped into immunological non-responders (INRs) (< 200 cells/µl), immunological inadequate responders (IIRs) (200 ~ 500 cells/µl) and immunological responders (IRs) (> 500 cells/µl) according to the CD4 cell count after two-year HAART. The immune reconstitution data based on the CD4+ and CD8+ cell counts with 8-year follow-up were collected for analysis. RESULTS: The CD4+ cell counts in the immunological responders (IRs) were significantly higher than in the immunological non-responders (INRs) and immunological inadequate responders (IIRs) (P <  0.001). The overall CD4+ cell count and CD4/CD8 ratio in the IRs increased faster than the IIRs and INRs. The CD4+ cell count growth at 0.5 year and 1 year after HAART in the IRs was significantly higher than the IIRs and INRs. The ROC curve demonstrated that 1 year CD4+ cell count had the highest predictive value, with the best cut-off value of 188 cells/µl, the predictive sensitivity was 81.0%, the predictive specificity was 85.2%, false positive rate was 14.8%, false negative rate was 19.0%, positive predictive value (IR) was 63.0%, negative predictive value (INR) was 93.5%. CONCLUSIONS: Taken together, our findings suggest that early initiation of HAART can reduce the immune reconstitution failure. The combination of baseline CD4+ cell count and baseline CD4/CD8 ratio may serve as a valid predictor of immune reconstitution prognosis after HAART.

[Preliminary study on standardization of production and processing of Scutellaria baicalensis pieces].

Decoction pieces are important raw materials in the production of traditional Chinese medicine( TCM),and their quality could directly affect the clinical efficacy and medication safety. Research on the production and processing technology of TCM is the basis for the normalization and standardization of Chinese medicine decoction pieces. At present,the production and processing standards for Scutellaria baicalensis pieces are non-regulated,lacking data foundation. In this study,with baicalin,baicalein,wogonoside and wogonin contents as evaluation indicators,single factor experiment was designed to optimize the softening,drying and cutting processes of S. baicalensis,providing a basis for the standardization of their production and processing. The effects of different softening,drying and cutting processes on the contents of the main components in S. baicalensis were comprehensively analyzed by the summation of relative differences. RESULTS:: showed that the contents of the four components and comprehensive indexes were affected by different softening methods and drying temperatures. The content of wogonin in boiling method was higher than that in boiling with cold water,and the content of glycosides in 70 ℃ drying condition was higher than that in other groups. The content of baicalin was significantly affected by different cutting thicknesses,but not by comprehensive index. Eventually,the optimal preparation process for S. baicalensis was determined as follows: boiled in boiling water for 20 min,cut into thin slices( 1-2 mm),and then dried at 70 ℃ in blast drier. This process was close to the actual production,practical and feasible and meanwhile,it was of great significance to improve the quality of S. baicalensis pieces.

Ginsenosides synergize with mitomycin C in combating human non-small cell lung cancer by repressing Rad51-mediated DNA repair.

The use of ginseng extract as an adjuvant for cancer treatment has been reported in both animal models and clinical applications, but its molecular mechanisms have not been fully elucidated. Mitomycin C (MMC), an anticancer antibiotic used as a first- or second-line regimen in the treatment for non-small cell lung carcinoma (NSCLC), causes serious adverse reactions when used alone. Here, by using both in vitro and in vivo experiments, we provide evidence for an optimal therapy for NSCLC with total ginsenosides extract (TGS), which significantly enhanced the MMC-induced cytotoxicity against NSCLC A549 and PC-9 cells in vitro when used in combination with relatively low concentrations of MMC. A NSCLC xenograft mouse model was used to confirm the in vivo synergistic effects of the combination of TGS with MMC. Further investigation revealed that TGS could significantly reverse MMC-induced S-phase cell cycle arrest and inhibit Rad51-mediated DNA damage repair, which was evidenced by the inhibitory effects of TGS on the levels of phospho-MEK1/2, phospho-ERK1/2 and Rad51 protein and the translocation of Rad51 from the cytoplasm to the nucleus in response to MMC. In summary, our results demonstrate that TGS could effectively enhance the cytotoxicity of MMC against NSCLC cells in vitro and in vivo, thereby revealing a novel adjuvant anticancer mechanism of TGS. Combined treatment with TGS and MMC can significantly lower the required concentration of MMC and can further reduce the risk of side effects, suggesting a better treatment option for NSCLC patients.

The RNA-binding protein QKI suppresses cancer-associated aberrant splicing.

Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.

[Establishment of biological assess for quality control of Fuzi based on determination of premature ventricular contractions in rats].

Aconiti Lateralis Radix (Fuzi) is a toxic traditional Chinese medicine with definite efficacy. In order to improve the quality control of its different prepared products and ensure the security in clinic, it is significant to establish a method of quality evaluation related to clinic adverse effects. Aiming at the important biological marker of early cardiac toxicity reaction, there was no method to detect it. In this manuscript, a novel approach for measuring the minimal toxic dose (MTD) of premature ventricular contractions (PVC) poisoning of rats was established. Then, the determination methodology and conditions were optimized to meet the needs of the quality and biological assessment, including animal sex, weight, stability of standards and test solutions. Using this method, the MTD value of different Fuzi products were determined, such as Heishunpian, Baifupian, Zhengfupian, Baofupian, and Paotianxiong. The results showed that the MTD of Fuzi was significantly decreased after detoxification processed (P<0.05) and the MTD of Heishunpian, Zhengfupian, Baofupian and Baifupian was as much as 15.76, 22.36, 19.65 and 20.97 times to that of unprocessed Shengfuzi. In addition, Paotianxiong could not induce PVC in rats, which indicated that Paotianxiong was nontoxic and safe.This method could appropriately reflects the cardiotoxity of Fuzi and its prepared samples. Together with the chemical composition analysis, the contents of diester alkaloids were explored including aconitine, mesaconitine and hypaconitine as well as monoester alkaloids in Fuzi and its prepared products were significantly associated with PVC. Furthermore, there may be some components undetermined facilitating arrhythmia to be worth exploring. This research provides an overall and comprehensive approach to diagnose early clinical cardiotoxity and control the quality of Fuzi, which could not only be a complementary solution for the chemical evaluation, but a new method to ensure its efficacy and security of clinical application.

Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice.

AIM: In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury. METHODS: Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining. RESULTS: In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 µmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume. CONCLUSION: Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.

[The relationship of SIRT3 with cellular metabolism and cardiovascular diseases].

Protein deacetylases play an extremely crucial role in cellular biological processes and have been categorized into four families (HDACⅠ, HDACⅡ, HDACⅢ and HDACⅣ) in human. Of them, HDACⅢ, also known as the Sir2 (Silent information regulator 2) family, contains seven members, SIRT1-7, each exhibiting different cellular localization and biological function. As a major mitochondrial deacetylase, SIRT3 not only modulates cellular metabolism, but also plays important roles in apoptosis, tumor growth, aging and a number of other diseases. In this review, we summarize recent findings related to SIRT3 with an emphasis on its biological functions in regulating cell metabolism and its possible roles in cardiovascular diseases.

Research on contents of anthraquinones in Cassiae Semen by principal component analysis.

Cassiae Semen is a common traditional Chinese medicine, and contents of anthraquinones of Cassiae Semen different significantly from area to area. According to Chinese Pharmacopoeia (2010 edition), only contents of aurantio obtusin and chrysophanol were used to evaluate the quality of Cassiae Semen, another data could be added later. Ten batches of Cassiae Semen from different areas were determined, and total anthraquinones, total free anthraquinones and total combined anthraquinones contents were assessed by ultraviolet visible spectrophotometer, contents of aurantio obtusin, rhein, aloe emodin, emodin, chrysophanol and physcion were determined by HPLC. After that, principal components analysis was used to evaluate these data determined previous by dimension reduction analysis. At last, the result suggests that three main components were found out, it shows that content of aloe emodin could be used to evaluate the quality of Cassiae Semen as well as contents of aurantio obtusin and chrysophanol. And Cassiae Semen from Hebei province posseses higher quality than Cassiae Semen from other different areas. All these results can provide a good reference for quality evaluating of Cassiae Semen medicinal materials at a certain extent.