AT1R knockdown confers cardioprotection against sepsis-induced myocardial injury by inhibiting the MAPK signaling pathway in rats.

Myocardial dysfunction is an important manifestation of sepsis. In addition, inactivation of the mitogen-activated protein kinase (MAPK) signaling pathway has been reported to be beneficial in sepsis. The current study used gene expression profiling to demonstrate the overexpression of angiotensin II type 1 receptor (AT1R) and activation of the MAPK signaling pathway in sepsis. In this study, we used a rat model of sepsis established by cecal ligation and puncture to explore the mechanism of AT1R silencing in relation to the MAPK signaling pathway on myocardial injury. Various parameters including blood pressure, heart rate, and cardiac function changes were observed. Enzyme-linked immunosorbent assay was used to measure the concentration of cardiac troponin T (TnT), cardiac troponin I (cTnI), and creatine kinase isoenzyme muscle/brain (CK-MB). Myocardial enzyme, tissue antioxidant capacity, mitochondria swelling, and membrane potential were also detected. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining was applied to measure cell apoptosis, and messenger RNA and protein levels of apoptosis-related proteins (Fas ligand [Fasl], B-cell CLL/lymphoma [Bcl-2], p53) were also detected. Initially, sepsis rats exhibited decreased survival rate, but increased ejection fraction (EF), heart rate, and concentrations of TnT, cTnI, and CK-MB. Furthermore, decreased AT1R expression inactivated the MAPK signaling pathway (shown as decreased extracellular signal-regulated kinase and cyclic adenosine 3',5'-monophosphate response element binding protein expression), decreased EF, heart rate, and concentrations of TnT, cTnI, and CK-MB, but increased sepsis rat survival rate. Eventually, decreased AT1R expression inhibited myocardial cell apoptosis (shown as decreased apoptosis rate and p53 and Fasl expression as well as increased Bcl-2 expression). These findings indicated that AT1R silencing plays an inhibitory role in sepsis-induced myocardial injury by inhibiting the MAPK signaling pathway.

Synthetic innate defense regulator peptide combination using CpG ODN as a novel adjuvant induces long­lasting and balanced immune responses.

Vaccines are critical tools for the prevention and treatment of several diseases. Adjuvants have been traditionally used to enhance immunity to vaccines and experimental antigens. In the present study, the adjuvant combination of CpG oligodeoxynucleotides (CpG ODN) and the innate defense regulator (IDR) peptide, IDR­HH2, was evaluated for its ability to enhance and modulate the immune response when formulated with alum and the recombinant hepatitis B surface antigen (HBsAg). The CpG­HH2 complex enhanced the secretions of tumor necrosis factor­α, monocyte chemotactic protein 1 and interferon­Î³ by human peripheral blood mononuclear cells and promoted murine bone marrow dentritic cell maturation. In addition, the present study demonstrated that IDR­HH2 was chemotactic for human neutrophils, THP­1 cells and RAW264.7 cells at concentrations between 2.5 and 40 µg/ml. The present study also observed that significantly higher anti­HBs antibody titers, which were sustained at high levels for as long as 35 weeks following the boost immunization, were induced by the combination adjuvant, even when co­administered with a commercial hepatitis B vaccine at a low antigen dose (0.1 µg HBsAg). Notably, the level of IgG2a was almost equal to the level of IgG1, indicating that a balanced T helper (Th)1/Th2 immune response was elicited by the novel vaccine, which was consistent with the ELISpot results. These data suggest that the CpG­HH2 complex may be a potential effective adjuvant, which facilitates a reduction in the dose of antigen and induces long­lasting, balanced immune responses.

Ad-endostatin treatment combined with low-dose irradiation in a murine lung cancer model.

Radiation therapy is a conventional strategy for treating advanced lung cancer yet is accompanied by serious side-effects. Its combination with other strategies, such as antiangiogenesis and gene therapy, has shown excellent prospects. As one of the potent endogenous vascular inhibitors, endostatin has been widely used in the antiangiogenic gene therapy of tumors. In the present study, LL/2 cells were infected with a recombinant adenovirus encoding endostatin (Ad-endostatin) to express endostatin. The results showed that LL/2 cells infected with the Ad-endostatin efficiently and longlastingly expressed endostatin. In order to further explore the role of Ad-endostatin combined with irradiation in the treatment of cancer, a murine lung cancer model was established and treated with Ad-endostatin combined with low-dose irradiation. The results showed that the combination treatment markedly inhibited tumor growth and metastasis, and prolonged the survival time of the tumor-bearing mice. Furthermore, this significant antitumor activity was associated with lower levels of microvessel density and anoxia factors in the Ad-Endo combined with irradiation group, and with an increased apoptotic index of tumor cells. In addition, no serious side-effects were noted in the combination group. Based on our findings, Ad-endostatin combined with low-dose irradiation may be a rational alternative treatment for lung cancer and other solid tumors.

[Effects of Lin28a and Lin28b on let-7 family activity].

In this report, we study the effects of over-expression of Lin28a and Lin28b on let-7 family activity in HeLaS3. Firstly, we constructed pAAV2neo-Lin28a and pAAV2neo-Lin28b to express Lin28a and Lin28b, respectively. Then, pAAV2neo-Lin28a and pAAV2neo-Lin28b were transfected into HeLaS3, selected with G418 and obtained cell lines, HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b, to express Lin28a and Lin28b stably. Thereafter, we constructed eight plasmid vectors for detection of let-7 family activity based on pAAV2neo-Gluc-(Fluc). These vectors were further packaged into recombinant adeno-associated viral vectors (rAAV) which were used as sensors, nominated as Asensors, to detect inhibition activity of miRNA at post-transcriptional level. Subsequently, with HeLaS3 as a control, we assayed expression levels of Lin28a and Lin28b by Western blot, detected expression levels of let-7 family by QRT-PCR, and tested let-7 family activity by Asensors in HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b. Results demonstrated that both HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b could express Lin28a and Lin28b effectively. Compared with HeLaS3, the expression level of let-7 family except let-7e declined in HeLaS3/pAAV2neo-Lin28a. But declining extent among members of let-7 family was different. The let-7 family activity also decreased while the decreasing extent varied among members. Furthermore, the activity level was not consistent with its expression level for the same member in let-7 family. Compared with HeLaS3, both expression level and activity level of let-7 family in HeLaS3/ pAAV2neo-Lin28b were decreased. However, the decreasing extent of let-7 family expression changes was larger than that of HeLaS3/pAAV2neo-Lin28a while the decreasing extent of activity changes was similar. In this study, we established a method to detect and compare post-transcriptional inhibition level mediated by miRNA complementary targets. We firstly clarified the effect of Lin28a and Lin28b on let-7 family activity profile and found that this effect was not the same as that at expression level of let-7 family, suggesting that it was more comprehensive to understand miRNA regulation roles to detect both miRNA expression and activity. This paves a way for further research on mechanism of regulation of let-7 family.