The lncRNA Malat1 functions as a ceRNA to contribute to berberine-mediated inhibition of HMGB1 by sponging miR-181c-5p in poststroke inflammation.

Long non-coding RNAs (lncRNAs) have been identified as essential mediators in neurological dysfunction. Our previous study shows that berberine (BBR) hampers the nuclear-to-cytosolic translocation of high-mobility group box 1 (HMGB1) in the process of poststroke inflammation. In this study, we explored the role of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) in the process of BBR-induced inhibition of HMGB1 in ischemic brain. Before the 60-min MCAO surgery, the mice were pretreated with BBR (50 mg· kg-1 per day, ig) for 14 days or ICV injected with specific lentiviral vector or shRNA. We showed that MCAO caused marked increase in the expression Malat1 and HMGB1 in the ipsilateral cortex, which was significantly attenuated by pretreatment with BBR. Knockdown of Malat1 attenuated the inflammatory injury after brain ischemia, whereas overexpression of Malat1 exacerbated ischemic brain inflammation. Overexpression of Malat1 also reversed BBR-induced reduction of HMGB1 and proinflammatory cytokines. The above results suggested a potential correlation between Malat1 and stroke inflammation. Based on informatics analysis we predicted that HMGB1 was a direct downstream target of miR-181c-5p, whereas Malat1 acted as a competitive endogenous RNA (ceRNA) for miR-181c-5p targeted the 3'-UTR of HMGB1 to promote inflammation after ischemic stroke. Knockdown of Malat1 significantly decreased HMGB1 level, which could be abrogated by transfection with miR-181c-5p inhibitors. Taken together, our results demonstrate for the first time that Malat1/miR-181c-5p/HMGB1 axis may be a key pathway of BBR-induced antiinflammation effects in stroke, and they may provide a novel avenue for targeted therapy.

Berberine Facilitates Angiogenesis Against Ischemic Stroke Through Modulating Microglial Polarization via AMPK Signaling.

Evidence suggests that microglia/macrophages can change their phenotype to M1 or M2 and participate in tissue damage or repair. Berberine (BBR) has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are elusive. Here, we investigated the effects of BBR on angiogenesis and microglial polarization through AMPK signaling after stroke. In the present study, C57BL/6 mice were subjected to transient middle cerebral artery occlusion (tMCAO), intragastrically administrated with BBR at 50 mg/kg/day. Neo-angiogenesis was observed by 68Ga-NODAGA-RGD micro-PET/CT and immunohistochemistry. Immunofluorescent staining further exhibited an increase of M2 microglia and a reduction of M1 microglia at 14 days after stroke. In vitro studies, the lipopolysaccharide (LPS)-induced BV2 microglial cells were used to confirm the AMPK activation effect of BBR. RT-PCR, Flow cytometry, and ELISA all demonstrated that BBR could inhibit M1 polarization and promote M2 polarization. Furthermore, treatment of human umbilical vein endothelial cells (HUVEC) with conditioned media collected from BBR-treated BV2 cells promoted angiogenesis. All effects stated above were reversed by AMPK inhibitor (Compound C) and AMPK siRNA. In conclusion, BBR treatment improves functional recovery and promotes angiogenesis following tMCAO via AMPK-dependent microglial M2 polarization.

Protective effects of ginsenoside Rb(3) on oxygen and glucose deprivation-induced ischemic injury in PC12 cells.

AIM: To investigate the protective effects of ginsenoside Rb(3), a triterpenoid saponin isolated from the leaves of Panax notoginseng, on ischemic and reperfusion injury model of PC12 cells and elucidate the related mechanisms. METHODS: PC12 cells exposed to oxygen and glucose deprivation (OGD) and restoration (OGD-Rep) were used as an in vitro model of ischemia and reperfusion. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) leakage were used to evaluate the protective effects of ginsenoside Rb(3). Cellular apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Intracellular calcium ion concentration ([Ca(2+)](i)) was detected using fluorophotometer system. Caspase-3, -8, and -9 activities were measured using assay kits with an ELISA reader. Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2 and Bax proteins. RESULTS: It was shown that ginsenoside Rb(3) (0.1-10 micromol/L) significantly increased cell viability and inhibited LDH release in a dose-dependent manner on the ischemic model. In addition, ginsenoside Rb(3) also significantly inhibited ischemic injury-induced apoptosis, [Ca(2+)](i) elevation, and decrease of MMP. Meanwhile, pretreatment with ginsenoside Rb(3) significantly induced an increase of Bcl-2 protein expression and a decrease of cytosolic cytochrome c, cleaved-caspase 3 and Bax protein expression, the caspase-3, -8, and -9 activity were also inhibited. CONCLUSION: The results indicated that ginsenoside Rb(3) could markedly protected OGD-Rep induced ischemic injury and the mechanisms maybe related to its suppression of the intracellular Ca(2+) elevation and inhibition of apoptosis and caspase activity. Ginsenoside Rb(3) could be a promising candidate in the development of a novel class of anti-ischemic agent.

Berberine inhibits macrophage M1 polarization via AKT1/SOCS1/NF-κB signaling pathway to protect against DSS-induced colitis.

Berberine has been reported to have protective effects in colitis treatment. However, the detailed mechanisms remain unclear. Herein, we demonstrated that berberine could protect against dextran sulfate sodium (DSS)-induced colitis in mice by regulating macrophage polarization. In the colitis mouse model, berberine ameliorated DSS-induced colon shortening and colon tissue injury. Moreover, berberine-treated mice showed significant reduction in the disease activity index (DAI), pro-inflammatory cytokines expression and macrophages infiltration compared with the DSS-treated mice. Notably, berberine significantly reduced the percentage of M1 macrophages. In vitro analysis also confirmed the inhibitory effects of berberine on macrophages M1 polarization in RAW267.4 cells. Further investigation showed that berberine promoted AKT1 expression in mRNA and protein level. Silence of AKT1 abolished the inhibitory effect of berberine on macrophages M1 polarization. The berberine-induced AKT1 expression promoted suppressers of cytokine signaling (SOCS1) activation, which inhibited nuclear factor-kappa B (NF-κB) phosphorylation. In addition, we also found that berberine activated AKT1/SOCS1 signaling pathway but inhibited p65 phosphorylation in macrophages in vivo. Therefore, we concluded that berberine played a regulatory role in macrophages M1 polarization in DSS-induced colitis via AKT1/SOCS1/NF-κB signaling pathway. This unexpected property of berberine may provide a potential explanation for its protective effects in colitis treatment.

A logistic equation to determine the validity of tramadol from related gene polymorphisms and psychological factors.

AIM: This study was performed to develop an algorithm using polymorphisms of CYP2D6, p-gp, OPRM1, COMT and psychological variables to predict tramadol response in Chinese patients recovering from upper limb fracture internal fixation surgery. METHODS: A total of 250 Han Chinese patients recovering from fracture in the upper limb were enrolled. CYP2D6*10, p-gp G2677T, p-gp C3435T, OPRM1 A118G and COMT Val158Met were detected by the ligase detection reaction (LDR) method. The algorithm was developed with binary logistic regression in cohort 1 (200 patients) and assessed with Wilcoxon signed-rank test in cohort 2 (50 patients). RESULTS: According to cohort 1, the predictive equation was calculated with the following logistic regression parameters: Logit (1) = 2.304-4.841 × (anxiety I) - 23.709 × (anxiety II) + 2.823 × (p-gp 3435CT) + 5.737 × (p-gp 3435 TT) - 1.586 × (CYP2D6*10 CT) - 4.542 × (CYP2D6*10 TT). The cutoff point for the prediction was defined as a probability value ≥0.5. The equation's positive predictive value is 90%. When applied to a new sample, the equation's positive predictive value is 86%. The Nagelkerke R² of the model is 0.819, the results of the Hosmer and Leme test show a value of 0.981. The nonparametric correlations between predicted and observed response showed significant correlation (coefficient = 0.879; p < 0.001). CONCLUSION: The algorithm we have developed might predict tramadol response in Chinese upper limb fracture patients.

A new algorithm to predict warfarin dose from polymorphisms of CYP4F2 , CYP2C9 and VKORC1 and clinical variables: derivation in Han Chinese patients with non valvular atrial fibrillation.

Few pharmacogenomic dosing regimens of warfarin have been developed for Chinese patients with non valvular atrial fibrillation (NVAF). The objective of this study was to develop a new algorithm by polymorphisms of CYP2C9, VKORC1 and CYP4F2 to predict the daily stable dose of warfarin in Chinese patients with NVAF. A total of 325 Chinese NVAF patients on stable dose of warfarin with a target international normalised ratio of 1.5 to 3.0 were recruited and divided randomly into two cohorts. CYP2C9*3, VKORC1-1639, VKORC1 1173 and CYP4F2 were detected by ligase detection reaction method. The new algorithm was developed with multivariate linear regression in cohort 1 (260 patients) and assessed with Pearson Correlation Analysis (PCA) in cohort 2 (65 patients). From 260 enrolled patients, the model (R2 = 51.7%) was developed as: Dose = 3.47 - 0.022 (AGE) + 0.017 (WT) + 0.189 (PTE) - 0.283 (ß-blocker) - 0.471 (AMIO) - 0.586 (CYP2C9 *1/*3) - 0.296 (VKORC1 CT) - 0.648 (VKORC1 TT) + 0.219 (CYP4F2 TT). PCA displayed that the algorithm was good (r = 0.658). The residual plots revealed that the predicted doses by the algorithm tend to be overestimated when lower doses were administered to patients and to be underestimated in higher doses. The algorithm developed by us might predict warfarin dose used by Chinese NVAF patients.

Effects of the multidrug resistance modulator HZ08 on the apoptosis pathway in human chronic leukaemia cell line K562/A02.

oancer falls to respond to chemotherapy by acquiring multidrug resistance in over 90% of patients. A previous study revealed that multidrug resistance modulator HZ08 had great multidrug resistance reversal effect in vitro and in vivo. It could enhance adriamycin (doxorubicin) induced intrinsic apoptosis pathway and rectify cell cycle and some apoptosis related proteins in human breast resistant cancer MCF-7/ADM cells. This study detected Rh123 accumulation to assess the effect of HZ08 on P-glycoprotein function in human chronic leukaemia cell line K562/A02. Moreover, mitochondria membrane potential, cytochrome c release and caspase-3 activity were analyzed for HZ08 treatment with or without vincristine. Since pretreatment with HZ08 could also reverse the multidrug resistance to vincristine in K562/A02 cells, the individual influence of HZ08 was further detected on apoptotic regulator like Bcl-2, Bax, p53, cell cycle checkpoints and proliferation regulatory factors like survivin, hTERT, c-Myc, c-Fos, c-Jun. Finally, it revealed that HZ08 increased vincristine induced activation in intrinsic apoptosis pathway by inhibition of P-gp mediated efflux. In addition, the outstanding reversal effect of HZ08 should also attribute to its individual effect on apoptosis and proliferation related regulatory factors. It renders HZ08 possibility of application in pretreatment to reverse multidrug resistance while avoiding unexpected drug interactions and accumulative toxicity.

Bioequivalence and pharmacokinetic comparison of two mycophenolate mofetil formulations in healthy Chinese male volunteers: an open-label, randomized-sequence, single-dose, two-way crossover study.

BACKGROUND: Mycophenolate mofetil (MMF) is an ester prodrug of mycophenolic acid (MPA), so clinical studies measure the circulating plasma MPA concentration instead of MMF. MPA is extensively glucuronidated by several uridine diphosphate glycosyltransferases into an inactive 7-O-glucuronide and a pharmacologically active acylglucuronide. Considering the effect of racial differences and genetic factors on the pharmacokinetic (PK) properties of drugs, it is necessary to study them in Chinese populations. OBJECTIVES: The aim of this study was to compare the clinical bioequivalence and PK properties of a test (dispersible tablets) and reference (capsules) formulation of MMF 1.0 g in healthy Chinese volunteers. We also established a validated HPLC method for the determination and quantification of MPA in human plasma. The study was required to obtain Chinese regulatory approval for the test formulation. METHODS: This open-label, randomized-sequence, single-dose, 2-way crossover study was conducted at the First Hospital of Nanjing Medical University, Nanjing, China. Eligible subjects were healthy male volunteers who were randomly assigned at a 1:1 ratio to receive a single 1.0-g dose of the test or reference formulation, followed by a 1-week washout period and administration of the alternate formulation. The plasma concentration of MPA, which is the active metabolite of MMF, was determined using a validated HPLC method. For analysis of PK properties, blood samples were collected at 0, 10, 20, 30, and 45 minutes, and 1, 1.5, 3, 5, 8, 11, 18, 36, and 48 hour(s). The PK parameters, including C(max), T(max), t((1/2)), AUC(0-48), and AUC(0-infinity), were determined from the plasma concentrations of the 2 formulations by noncompartmental analysis. Tolerability was assessed at baseline (be- fore administration) and at 30 minutes and 1, 5, 18, and 48 hours after administration by monitoring vital signs. Laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) were performed for the identification of adverse events (AEs) (eg, leukopenia, thrombocytopenia, anemia). Patient interviews were conducted to assess the occurrence of AEs such as diarrhea, abdominal pain, nausea, vomiting, and secondary infections. The formulations were considered to meet the regulatory requirements of bioequivalence if the log-transformed ratios of C(max) and AUC were within the predetermined equivalence range (80%-125%) as established by the US Food and Drug Administration (FDA). RESULTS: Eighteen healthy Chinese male volunteers (mean [range] age, 23.5 [22-30] years; weight, 63.3 [56-68] kg; height, 171 [165-184] cm) were enrolled and completed the study. The main PK parameters of the MMF test and reference formulations were as follows: mean (SD) T(max), 0.68 (0.21) and 0.81 (0.18) hour, respectively; C(max), 25.58 (4.79) and 26.47 (3.67) mg/L; AUC(0-48), 59.19 (9.23) and 58.32 (9.28) mg/L/h; t((1/2)), 15.12 (3.17) and 16.04 (4.22) hours; AUC(0-infinity)), 63.28 (10.23) and 62.41 (10.28) mg/L/h. The mean (SD) relative bioavailability was 101.5% (10.3%). No statistically significant differences were found based on ANOVA. The ratios of C(max) (0.97) and AUC (1.01) for the test and reference formulations were within the FDA bioequivalence definition intervals of 80% to 125%. No AEs were reported by subjects or found on analysis of vital signs or laboratory tests. CONCLUSIONS: In this study in healthy Chinese male volunteers, results from the PK analysis suggested that a single dose of the test and reference formulations of MMF 1.0 g met the regulatory requirements of bioequivalence, based on the FDA regulatory definition (rate and extent of absorption). Both formulations were well tolerated.

HZ08, a great regulator to reverse multidrug resistance via cycle arrest and apoptosis sensitization in MCF-7/ADM.

In early studies, it was demonstrated that R-HZ08, S-HZ08 and the racemate had strong reverse efficacy of multidrug resistance in vitro and in vivo (Yan et al., 2008b). The effect was supposed to have direct interaction with multidrug resistance-associated protein (MRP1) in MCF-7/ADM and P-glycoprotein in K562/A02. According to our latest study, we found HZ08 could enhance chemotherapy induced apoptosis by synergistic action on reactive oxygen species generation, GSH depletion, mitochondrial membrane potential depolarization, cytochrome c release and caspase activation. Moreover, the potential selective effect of HZ08 on resistant cells suggested that HZ08 have specific targets for resistance reversal via apoptosis regulation. Therefore, we traced individual influence of HZ08, not only on apoptosis pathway per se but also on apoptosis related intracellular regulation systems. Then we found HZ08 could increase cells in G(0)/G(1) phase and regulate apoptosis related proteins (Bcl-2, Bax) as well as upstream functional molecules (c-Myc and c-Fos), which are usually abnormal in malignancy and responsible for multidrug resistance in MCF-7/ADM. Thereby, chemotherapy induced apoptosis was promoted. R-HZ08 showed better effect than S-HZ08 or the racemate did in most of targets above. Furthermore, HZ08 did not change the concentration of intracellular Ca(2+) which means it would not have side effect as verapamil does. Considering multidrug resistance is multifactorial, HZ08, especially R-HZ08, which could sensitize apoptosis by multiple improvements of upstream malignant characters, will be a promising drug to enhance the effect of chemotherapy in the treatment of multidrug resistant tumor.