Promethazine inhibits neuronal apoptosis via PI3K/Akt signaling pathway in rats with cerebral infarction.

The article "Promethazine inhibits neuronal apoptosis via PI3K/Akt signaling pathway in rats with cerebral infarction, by X.-D. Pan, X.-L. Chen, S.-F. Ding, D. Kou, H.-L. Hu, L. Li, published in Eur Rev Med Pharmacol Sci 2019; 23 (3 Suppl): 126-134-DOI: 10.26355/eurrev_201908_18639-PMID: 31389591" has been withdrawn from the authors stating that they "have not yet studied their work completely and have some difficulties answering questions and quickly providing solutions in the paper". The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/18639.

Promethazine inhibits neuronal apoptosis via PI3K/Akt signaling pathway in rats with cerebral infarction.

OBJECTIVE: To study the effect of promethazine on neuronal apoptosis in rats with cerebral infarction (CI) through the phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/Akt) signaling pathway. MATERIALS AND METHODS: A total of 36 Sprague-Dawley rats were randomly divided into the sham group (n=12), model group (n=12), and promethazine group (n=12). The external carotid artery was only exposed in the model group, and the ischemia-reperfusion model after CI was established using the suture method in the other two groups. After modeling, the normal saline was intraperitoneally injected in the sham group and model group, while promethazine was intraperitoneally injected in the promethazine group. The rats were sampled after 1 week of intervention. The neurological deficits of rats were evaluated using the Zea-Longa score, and the cognitive function, the spatial learning, and memory of rats were detected via the water maze test. Moreover, the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in brain tissues were detected via immunohistochemistry, and the relative protein expressions of PI3K p85, PI3K p110, and p-Akt were detected via Western blotting. The mRNA expressions of Bax and Bcl-2 were detected via quantitative Polymerase Chain Reaction (qPCR), and the apoptosis was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: The Zea-Longa score was significantly increased in the model group and promethazine group compared with that in the sham group (p<0.05), while it significantly declined in the promethazine group compared with that in the model group (p<0.05). The escape latency was significantly prolonged and the times of crossing platform were significantly reduced in the model group and promethazine group compared with those in the sham group (p<0.05), while the escape latency was significantly shortened and the times of crossing platform were significantly increased in the promethazine group compared with those in the model group (p<0.05). Compared with those in the sham group, the positive expression of Bax was significantly increased, while the positive expression of Bcl-2 was remarkably decreased in the model group and promethazine group (p<0.05). Compared with those in the model group, the positive expression of Bax was significantly decreased, while the positive expression of Bcl-2 was remarkably increased in the promethazine group (p<0.05). Besides, the model group and promethazine group had evidently higher relative protein expressions of PI3K p85, PI3K p110, and p-Akt than the sham group (p<0.05), while the promethazine group also had evidently higher relative protein expressions of PI3K p85, PI3K p110, and p-Akt than the model group (p<0.05). Compared with the sham group, model group, and promethazine group had remarkably increased relative mRNA expression of Bax, and remarkably decreased relative mRNA expression of Bcl-2 (p<0.05). Compared with those in the model group, the relative mRNA expression of Bax was remarkably decreased, while the relative mRNA expression of Bcl-2 was remarkably increased in the promethazine group (p<0.05). Finally, the apoptosis rate was significantly higher in the model group and promethazine group than that in the sham group (p<0.05), while it was significantly lower in the promethazine group than that in the model group (p<0.05). CONCLUSIONS: Promethazine inhibits neuronal apoptosis in CI rats by upregulating the PI3K/Akt signaling pathway, thereby exerting a protective effect.

[Impacts of early metoprolol intervention on connexin 43 and phosphorylated connexin 43 expression in rabbits with experimental myocardial infarction].

Objective: To investigate the early intervention effects of metoprolol on connexin 43(Cx43) and phosphorylated Cx43 (p-Cx43) expression in rabbits with post myocardial infarction. Methods: A total of 24 adult male New Zealand white rabbits were divided into sham group (n=6), early treatment group(n=6), routine treatment group(n=6), and myocardial infarction group(n=6) with a randomized block design blocked by weight. Myocardial infarction was induced by left anterior descending coronary artery (LAD) ligation. Rabbits in sham group received similar surgical procedure without LAD ligation. Metoprolol (12.5 mg/kg dissolved in 2 ml distilled water) was applied to rabbits in early treatment group and routine treatment group per gavage immediately after recovery from anesthesia and at 24 hours after myocardial infarction, respectively, then treated daily for 40 days. Rabbits in sham group and myocardial infarction group received 2 ml distilled water per gavage daily for 40 days. Plasma lactate dehydrogenase (LDH) and creatine kinase (CK) level were detected by automatic biochemistry analyzer after 6 hours in all rabbits. Ventricular fibrillation threshold (VFT) was measured in vivo by bipolar pacing electrodes at 40 days. Cx43 and p-Cx43 distribution in ventricular tissue was detected by immunofluorescence analyses. Cx43 and p-Cx43 protein level in ventricular tissue was determined by Western blot. Results: (1) Plasma LDH ((851.7±85.9)U/L vs. (332.3±39.6)U/L, P<0.01) and CK ((1 192.7±105.3)U/L vs. (462.3±65.6)U/L, P<0.01) were significantly higher in myocardial infarction group than in sham group (both P<0.01). (2) VFT was significantly lower in myocardial infarction group than that in sham group ((470.0±91.0) beats per minute vs. (683.3±60.9) beats per minute, P<0.05), and VFT was significantly higher in early treatment group ((633.3±43.2) beats per minute) and routine treatment group ((645.0±30.8) beats per minute) than in the myocardial infarction group (both P<0.05). (3) Immunofluorescence analyses showed that Cx43 was mainly localized in the intercalated disk, which was perpendicular to the cell long axis with linear arrangement, and less lateral distribution in sham group, early treatment group and routine treatment group, which was significantly different as the case in the myocardial infarction group. The expression of p-Cx43 in myocardial infarction group was less than in sham group, which was significantly upregulated in in early treatment group and routine treatment group when compared with myocardial infarction group, and expression of p-Cx43 was significantly higher in early treatment group than in routine treatment group. (4)The p-Cx43/Cx43 ratio of protein was significantly lower in myocardial infarction group than in sham group (0.165±0.011 vs. 0.363±0.046, P<0.05), and significantly higher in early treatment group (0.720±0.063) and routine treatment group (0.364±0.030) than in myocardial infarction group (both P<0.05), and this ratio was significantly higher in early treatment group than in routine treatment group (P<0.05). Conclusion: Metoprolol treatment, especially the early metoprolol treatment (within 24 hours after LAD ligation), could significantly improve VFT by ameliorating the distribution and dephosphorylation of myocardial Cx43 in rabbits with experimental myocardial infarction.

Impact of citrate pretreatment on ventricular arrhythmia and myocardial capase-3 expression in ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury often triggers ventricular arrhythmia. Citrate binds calcium ions, forming a soluble calcium citrate complex that may reduce I/R injury by affecting calcium ion concentration. We tested the effects of citrate pretreatment on ventricular heart rate and related factors in a rat I/R model. Fifty male Sprague Dawley rats weighing 350-400 g were randomly divided into equally sized control (A), model (B), and 0.1 M (C), 0.05 M (D), and 0.025 M (E) citrate groups. An I/R model was established by ligating the left anterior descending coronary artery. Serum calcium ion concentration was measured before and after citrate treatment. Triphenyltetrazolium chloride staining and spectrophotometry were used to determine infarction area and caspase-3 protein levels in myocardial tissue, respectively. Polymerase chain reaction was performed to test myocardial calmodulin (CAM) expression. The frequency of ventricular arrhythmia in group B was significantly higher than in the sham surgery group (P < 0.05). Citrate pretreatment resulted in lower and higher frequencies than those observed in the model and control groups, respectively, in a dose-independent manner. The most obvious reduction in ventricular arrhythmia was seen in Group D. Serum calcium ion concentration decreased markedly after citrate treatment (P < 0.05), with a specific pattern emerging over time. Infarction area and caspase-3 and CAM levels were significantly lower in the citrate groups compared with the model group (P < 0.05). Citrate can reduce myocardial cell apoptosis, alleviating ventricular arrhythmia and protecting the myocardium by reducing serum calcium ion concentration and downregulating caspase-3 and CAM expression.

[Predictive value of early lactate area for mortality in elderly patients with septic shock].

Objective: To investigate the predictive value of early lactate area for mortality in elderly patients with septic shock. Methods: From January 2012 to December 2013, a prospective study was conducted in the Department of Critical Care Medicine, Qilu Hospital of Shandong University. A total of 115 septic shock patients with age ≥65 years were included in the study. Serum lactate was measured every 6 hours, the lactate indicators, including early lactate area, APACHE Ⅱ score etc were recorded. Results: The overall 28-day mortality rate was 67.0%. The top three primary infection sources were lung, abdominal cavity and bloodstream. When compared to survivors, non-survivors had significantly elevated early lactate area and APACHE Ⅱ score and lowered lactate clearance[(27.4±7.6) vs ( 20.3±6.5)], they were significantly more likely to have undergone mechanical ventilation, renal replacement therapy and inotropic or vasopressor support for ≥3 d, and more frequently displayed signs of cardiovascular, respiratory, and renal and hepatic dysfunction (all P<0.05) .Receiver Operating Characteristic curves indicated the lactate area score displayed a strong predictive power for 28 day mortality as indicated by an AUC of 0.758 (P<0.01) and had significantly greater predictive power when compared to the initial lactate or lactate clearance (all P<0.05). Conclusions: In geriatric patients with septic shock, the early lactate area is a useful predictor for early death and showed better predictive value than other lactate indicators.

Ultrastructure changes induced by dry film formation of a trisodium phosphate blend, antimicrobial solution.

Trisodium phosphate (TSP) has been reported to have antimicrobial activity and is approved by the U.S. Department of Agriculture for use in food processing applications. A novel (U.S. Patent 6,184,198), antimicrobial solution containing a blend of TSP, sodium bicarbonate, and sodium carbonate (TSP blend) has demonstrated effective inhibition of microbial contamination in a broad spectrum of applications. This high-resolution cold field emission scanning electron microcopy (LVSEM) investigation details structural changes and dry film formation in various classes of microbes as a mechanism for antimicrobial activity of this solution. The results showed that this TSP blend solution completely inhibited the growth of pathogenic E. coli O157:H7, and Salmonella bacteria, the mold Cryptococcus, as well as a Norwalk virus surrogate-feline calicivirus. Results by LVSEM confirmed that the antimicrobial effect was induced when encapsulating the target microbes in a high lubricity film that is formed around these organisms as the solution dries. The thickness of the film was estimated to be approximately 60 nm.

Targeted RNases: a feasibility study for use in HIV gene therapy.

A targeted RNase would be ideal for gene therapy of several acquired and inherited disorders. Such an RNase may be engineered to contain a ribonucleolytic domain and a specific target RNA binding domain. To demonstrate the feasibility of this approach, an RNase targeted against human immunodeficiency virus (HIV) RNA--Tev-RNase T1--was designed and tested for its use in HIV-1 gene therapy. A human CD4+ T lymphoid (MT4) cell line and human peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors lacking or expressing the tevT1 gene. Expression of enzymatically functional Tev-RNase T1 protein and its lack of toxicity was demonstrated in stable MT4 transductants. Compared with control cells lacking this protein, both transduced MT4 cells and PBLs expressing Tev-RNase T1 delayed HIV-1 replication. Tev-RNase T1 was shown to act after integration, since HIV-1 proviral DNA could be detected, but the amount of HIV-1 RNA produced in MT4 cells and PBLs was significantly decreased. This study demonstrates the feasibility of a targeted RNase strategy for therapeutic use.

Co-packaging of sense and antisense RNAs: a novel strategy for blocking HIV-1 replication.

Retroviral vectors were engineered to express either sense (MoTiN-TRPsie+) or sense and antisense (MoTN-TRPsie+/-) RNAs containing the human immunodeficiency virus type-1 (HIV-1) trans -activation response (TAR) element and the extended packaging (Psie) signal. The Psie signal includes the dimer linkage structure (DLS) and the Rev response element (RRE). Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid (MT4) cell line. Stable transductants were then tested for sense and antisense RNA production and susceptibility to HIV-1 infection. HIV-1 production was significantly decreased in cells transduced with MoTiN-TRPsie+ and MoTN-TRPsie+/-vectors. Efficient packaging of sense and most remarkably of antisense RNA was observed within the virus progeny. Infectivity of this virus was significantly decreased in both cases, suggesting that the interfering RNAs were co-packaged with HIV-1 RNA. Vector transduction was not expected to occur and was not observed. Inhibition of HIV-1 replication was also demonstrated in human peripheral blood lymphocytes transduced with retroviral vectors expressing antisense RNA. These results suggest that (i) both sense and antisense RNAs were co-packaged with HIV-1 RNA, (ii) the co-packaged sense and antisense RNAs inhibited virus infectivity and (iii) the co-packaged sense and antisense RNAs were not transduced. Sense and antisense RNA-based strategies may also be used to co-package other interfering RNAs (e.g. ribozymes) to cleave HIV-1 virion RNA.

Co-packaging of non-vector RNAs generates replication-defective retroviral vector particles: a novel approach for blocking retrovirus replication.

A Moloney murine leukemia virus (MoMuLV)-derived packaging retroviral vector, pUCMoTN-PR3, was previously developed in which the packaging (psi) signal was cloned within the 5'-long terminal repeat (LTR) U3-r and U5 sequences. The MoTN-PR3 vector particles released from a transfected packaging cell line contain RNAs with r-psi-U5 sequences at the 5'-end and U3-r sequences at the 3'-end. Upon infection, these vector particles can efficiently transduce the neomycin phosphotransferase (neo) gene to the target cells. The structure of the proviral DNA synthesized in these cells was shown to contain modified 5'- and 3'-LTRs with U3-r-psi-U5 sequences, indicating that this vector can undergo reverse transcription and integration. Analysis of psi signal-containing RNAs revealed that in addition to vector RNA transcribed from the MoMuLV 5'-LTR promoter, readthrough neo RNA transcribed from the internal herpes simplex virus (HSV) thymidine kinase (tk) promoter and cellular RNAs transcribed from the MoMuLV 3'-LTR promoter are produced. Of these, the downstream cellular RNAs are also packaged within the vector particles. These vector particles containing the vector and non-vector RNAs carrying the MoMuLV psi signal are non-infectious. It is proposed that intracellular expression of packageable non-viral RNAs may represent an effective strategy for inhibiting animal and plant virus replication.

Inhibition of HIV-1 replication by retroviral vectors expressing monomeric and multimeric hammerhead ribozymes.

Retroviral vectors were engineered to express monomeric and multimeric hammerhead ribozymes targeting one and nine highly conserved sites within the HIV-1 envelope (Env) coding region. In vitro, both the monomeric and multimeric ribozymes were shown to be active and cleave the target RNA containing the cleavage sites. A human CD4+ T lymphocyte-derived MT4 cell line was stably transduced with retroviral vectors expressing these ribozymes. Ribozyme expression in stably transduced cells was confirmed by Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR). As compared with the control cells lacking any ribozyme, HIV-1 replication was delayed in monomeric RzEnv-expressing cells. Virus replication was almost completely inhibited in multimeric RzEnv1-9-expressing cells as no viral RNA or protein could be detected in these cells and in their culture supernatants for up to 60 days after infection. The genomic DNA from RzEnv1-9-expressing cells was shown to contain HIV-1 proviral DNA sequences at days 3 and 60 after HIV infection. HIV-1 used in the challenge experiments was found to contain fully reverse transcribed '-' strand DNA which should have been able to infect complete proviral DNA synthesis and integrate within the cellular genome without being affected by pre-existing ribozymes. Therefore, the proviral DNA present at day 3 after infection may have originated from infection by such DNA-containing virus particles. The results obtained with the retroviral vector expressing RzEnv1-9 are very encouraging and we envisage its future use in anti-HIV-1 gene therapy.

Synthesis and HIV inhibition activity of 2',3'-dideoxy-3'-C-hydroxymethyl nucleosides.

A series if 2',3'-dideoxy-3'-C-hydroxymethyl purine nucleosides were prepared based on the photochemical ring expansion of a chiral cyclobutanone precursor, (2S)-trans-2,3-bis[(benzoyloxy)methyl]cyclobutanone, in the presence of a 6-substituted purine. Both alpha- and beta-anomers are produced in this transformation. Deprotection was effected by reaction of the photoadducts with saturated methanolic ammonia. Nine purine nucleosides were tested for their inhibitory effect of HIV IIIB virus on H9 cells. The 6-hexyloxy and adenine derivatives 4e,c, respectively, appeared to be most effective at inhibiting viral reproduction with 4c comparable in activity to ddI and AZT.

Preliminary report: the short-term effects of direct p53 DNA injection in primary hepatocellular carcinomas.

A pilot study, to assess the therapeutic potential of percutaneous injection of wild-type p53 (wt-p53) in five patients with primary hepatocellular carcinoma is reported. Three of the five patients showed objective tumor response with reduction of the tumor volume on computed tomographic (CT) scan measurements as well as a significant fall of serum alphafetoprotein. Much further work will be needed to elucidate the mechanism of action.

Abnormalities of the RB1 and DCC tumor suppressor genes: uncommon in human pancreatic adenocarcinoma.

Evidence of RB1 allele loss was found in only 6% of pancreatic cancers, and we found no significant sequence abnormalities nor loss of RB protein expression in a panel of tumors and cell lines. Using reverse transcription-polymerase chain reaction and Southern blot analysis, we found no evidence for loss of DCC expression in pancreatic cancer cell lines, and allele loss only rarely in tumor biopsies. These findings suggest that abnormalities of RB1 and DCC are unlikely to play a major role in pancreatic carcinogenesis.

Expression of hepatocyte growth factor mRNA, and c-met mRNA (hepatocyte growth factor receptor) in human liver tumours.

We have quantified mRNA for the hepatocyte growth factor and its putative receptor the c-met proto-oncogene protein product, in a series of human primary and secondary liver tumours and adjacent non-neoplastic liver. In all hepatocellular cancers, hepatocyte growth factor 6 kb mRNA expression was less (mean 23.93% +/- 6.33% S.E.M. n = 7) in the tumours than in the adjacent normal liver. Both relative over- and under-expression of c-met transcripts were found in tumour tissue compared to non-neoplastic liver. Thus hepatocellular cancer tissue does not over-express mRNA for hepatocyte growth factor, though this growth factor might play a role in hyperproliferative states leading to liver cancer.

Chromosome allele loss in colorectal liver metastases and its association with clinical features.

Molecular genetic changes are better characterized in colorectal carcinoma than in other common adult tumours. Consistent allele losses, or loss of heterozygosity (LOH), on chromosomes 5q, 17p and 18q have been well established. These changes are associated with the prognosis of the disease. Little is known of such changes in liver metastases of colorectal origin. The extent of allelic loss and its association with clinical features were investigated in 19 patients with colorectal liver metastases by using 24 probes to detect restriction fragment length polymorphism. A high frequency of LOH on chromosomes 5q, 17p and 18q was found in these secondary tumours. No consistent loss has so far been shown in any other chromosome. The frequency of allele loss correlated significantly with prognostic features such as the number and size of liver secondaries (P < 0.005), metastasis to the lymph nodes (P < 0.01) and curative or palliative operation (P < 0.02).

Partial characterization of a cDNA for human stearoyl-CoA desaturase and changes in its mRNA expression in some normal and malignant tissues.

A segment of 712 bases coding for part of the human stearoyl-CoA desaturase gene was made by polymerase chain reaction (PCR) using primers based on published rat cDNA sequences. The human PCR product was confirmed by DNA sequencing. It was next cloned into a vector from which anti-sense, highly radioactive RNA transcripts were made in vitro using T7 polymerase. The transcripts were used to probe desaturase mRNA in a number of human tumour and control tissues, using a very sensitive solution hybridization/RNase protection assay. Increased desaturase mRNA levels were found in colonic and oesophageal carcinomas and in hepatocellular adenoma; however, no consistent trend was seen in hepatocellular carcinoma. It is suggested that certain classes of tumour may exhibit increased levels of desaturase mRNA.

Loss of constitutional heterozygosity on chromosomes 5 and 17 in cholangiocarcinoma.

It has been established that loss of tumour suppressor genes is crucial in carcinogenesis. There has been no reported study on searching for tumour suppressor genes in cholangiocarcinomas as yet. In order to investigate the loss of heterozygosity (LOH), which may represent such gene loss, in cholangiocarcinoma, we studied 14 patients with this tumour using restriction fragment length polymorphism analysis. Twenty-two probes assigned to chromosomes 1, 5, 7, 9, 11, 12, 13, 14, 16, 17 and 18 were used. Allelic losses were found in chromosomal regions 5q35-qter and 17p13. Loss of genetic material in these regions in cholangiocarcinoma was shared with hepatocellular carcinoma. Probes for other chromosomes have as yet shown no consistent LOH. In conclusion, this study for the first time showed LOH on chromosomes 5 and 17 in cholangiocarcinoma.

Chromosome 17 allele loss in hepatocellular carcinoma but not in synchronous liver adenoma.

A 51-year-old female underwent resection of two synchronous liver tumours, a hepatocellular carcinoma and an adenoma. DNA analysis revealed allele loss on chromosome 17 (17p13, near the locus of p53 tumour suppressor gene) in the hepatocellular carcinoma but not in the adenoma. This finding may support the view that loss of p53 tumour suppressor gene is associated with tumour progression.