IL-6 promotes pluripotency of mouse embryonic stem cells and regulates cardiac differentiation in a development-dependent manner.

Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.

[Effect of Mycobacterium tuberculosis higBA on Bacterial Stress Response and Intracellular Infection and Immunity].

Objective: To investigate the effect of Mycobacterium tuberculosis ( Mtb) higBA on bacterial stress response and intracellular infection and immunity. Methods: The target gene amplified from Mtb H37Rv genome was cloned to the vector and then transferred to Mycobacterium smegmatis ( Ms) to construct a recombinant strain. Stress response experiment and Raw264.7 mouse macrophage infection was carried out with Ms_higBA, the recombinant strain, and Ms_ vec, the vector strain. Tests were conducted to measure bacterial colony forming unit (CFU) and transcriptional levels of cytokines, including interleukin ( IL)-1ß, IL-6, IL-10, IL-12 p40, interferon ( IFN)- γ, tumor necrosis factor ( TNF)- α, and inducible nitric oxide synthase ( iNOS). Results: The recombinant strain, Ms_higBA, was constructed successfully. According to the findings of the stress response experiment, higBA could indeed enhance bacterial survival under certain conditions of in vitro culture. Intracellular infection experiment demonstrated that higBA enhanced bacterial survival in macrophages and influenced the transcriptional level of cytokines. Conclusion: The higBA genes from Mtb play a role in bacterial stress response and intracellular infection and immunity.

Cardiac fibroblast paracrine factors modulate mouse embryonic stem cells.

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.

[Preliminary Study on the Secretory Performance of Mycobacterium tuberculosis Hypoxic Response Protein 1 (Rv2626c)].

OBJECTIVE: To verify the secretory ability of the hypoxic response protein 1 (HRP1) encoded by Mycobacterium tuberculosis (Mtb) Rv2626c. METHODS: The target gene attached with His tag was amplified from the genome of Mtb standard virulence strain H37Rv. The recombinant plasmid contained the above amplified product was constructed and electroporated into Mycobacterium smegmatis (Ms) (MC 2155) to construct a recombinant strain. Protein expression was induced under heat condition, and the expression of protein from the culture filtrates and the bacterial lysates was detected afterward. The 10 kDa culture filtrate antigen (CFP-10) (Ms) and CFP-10 (Mtb) were used as positive controls, and the cytoplasmic protein heat shock protein 65 (GroEL2) (Mtb) was used as negative controls. RESULTS: The HRP1, GroEL2 (Mtb), CFP-10 (Mtb) and CFP-10 (Ms) were successfully amplified by PCR from recombinant plasmid, and sequencing results of the recombinant plasmid is right, confirming the successful construction of the recombinant plasmid. The recombinant Ms was successfully constructed and it could express the proteins GroEL2 (Mtb), HRP1, CFP-10 (Mtb) and CFP-10 (Ms). The target protein HRP1 was detected in both of the lysate and the culture filtrate of the recombinant strain by Western blot, which was consistent with the positive control CFP-10. The negative control GroEL2 (Mtb) was only detected in the bacterial lysate, but not detected in the culture filtrate. CONCLUSION: The protein HRP1 encoded by Mtb Rv2626c can be secreted out of Ms by the secretion system of Ms. It may be a secreted protein and play an important role in the pathogenesis of Mtb.

Diosgenin Glucoside Protects against Spinal Cord Injury by Regulating Autophagy and Alleviating Apoptosis.

Spinal cord injury (SCI) is a severe traumatic lesion of central nervous system (CNS) with only a limited number of restorative therapeutic options. Diosgenin glucoside (DG), a major bioactive ingredient of Trillium tschonoskii Max., possesses neuroprotective effects through its antioxidant and anti-apoptotic functions. In this study, we investigated the therapeutic benefit and underlying mechanisms of DG treatment in SCI. We found that in Sprague-Dawley rats with traumatic SCI, the expressions of autophagy marker Light Chain 3 (LC3) and Beclin1 were decreased with concomitant accumulation of autophagy substrate protein p62 and ubiquitinated proteins, indicating an impaired autophagic activity. DG treatment, however, significantly attenuated p62 expression and upregulated the Rheb/mTOR signaling pathway (evidenced as Ras homolog enriched in brain) due to the downregulation of miR-155-3p. We also observed significantly less tissue injury and edema in the DG-treated group, leading to appreciable functional recovery compared to that of the control group. Overall, the observed neuroprotection afforded by DG treatment warrants further investigation on its therapeutic potential in SCI.