The mechanism by which miR-494-3p regulates PGC1-α-mediated inhibition of mitophagy in cardiomyocytes and alleviation of myocardial ischemia-reperfusion injury.

OBJECTIVE: The purpose of this study was to explore whether miR-494-3p inhibits the occurrence of mitochondrial autophagy in cardiomyocytes by inhibiting the expression of PGC1-α and to supplement the theoretical basis for the role of autophagy in cardiac injury induced by hypoxia/reperfusion (H/R). METHODS: The expression of miR-494-3p was detected by RT‒qPCR, and the expression of PGC1-α, autophagy-related proteins (LC3, Beclin 1), apoptosis-related proteins (Bax and Bcl-2), PINK1/Parkin signaling pathway-related proteins (PINK1, Parkin) and mitochondrial change-related proteins (Mfn1, Mfn2, OPA1) was detected by Western blotting. The changes in mitochondrial membrane potential were detected by JC-1 staining (ΔΨm). The formation of autophagosomes was observed by transmission electron microscopy. Cell proliferation activity was detected by CCK-8, and cell apoptosis was detected by flow cytometry. A dual-luciferase gene reporter assay identified a targeted binding site between miR-494-3p and PGC1-α. RESULTS: The results showed that miR-494-3p and PGC1-α were differentially expressed in H/R cardiomyocytes; that is, the expression of miR-494-3p was downregulated, and the expression of PGC1-α was upregulated. In addition, mitochondrial autophagy occurred in H/R cardiomyocytes. That is, LC3-II/LC3-I, Beclin 1, PINK1, and Parkin expression was upregulated, Mfn1, Mfn2, and OPA1 expression was downregulated, and the mitochondrial membrane potential was decreased. The transfection of miR-494-3p mimic can significantly improve the cell proliferation activity of cardiomyocytes and inhibit the occurrence of cardiomyocyte apoptosis and autophagy, while the transfection of miR-494-3p inhibitor has the opposite result. After transfection of the miR-494-3p mimic, treatment with autophagy inhibitors and activators changed the effects of miR-494-3p on cardiomyocyte proliferation and apoptosis. At the same time, the overexpression of PGC1-α reversed the promoting effect of miR-494-3p on cardiomyocyte proliferation and the inhibitory effect on apoptosis and autophagy. CONCLUSION: MiR-494-3p can target and negatively regulate the expression of PGC1-α to inhibit mitophagy in cardiomyocytes.

COX5A Plays a Vital Role in Memory Impairment Associated With Brain Aging via the BDNF/ERK1/2 Signaling Pathway.

Cytochrome c oxidase subunit Va (COX5A) is involved in maintaining normal mitochondrial function. However, little is known on the role of COX5A in the development and progress of Alzheimer's disease (Martinez-Losa et al., 2018). In this study, we established and characterized the genomic profiles of genes expressed in the hippocampus of Senescence-Accelerated Mouse-prone 8 (SAMP8) mice, and revealed differential expression of COX5A among 12-month-aged SAMP8 mice and 2-month-aged SAMP8 mice. Newly established transgenic mice with systemic COX5A overexpression (51% increase) resulted in the improvement of spatial recognition memory and hippocampal synaptic plasticity, recovery of hippocampal CA1 dendrites, and activation of the BDNF/ERK1/2 signaling pathway in vivo. Moreover, mice with both COX5A overexpression and BDNF knockdown showed a poor recovery in spatial recognition memory as well as a decrease in spine density and branching of dendrites in CA1, when compared to mice that only overexpressed COX5A. In vitro studies supported that COX5A affected neuronal growth via BDNF. In summary, this study was the first to show that COX5A in the hippocampus plays a vital role in aging-related cognitive deterioration via BDNF/ERK1/2 regulation, and suggested that COX5A may be a potential target for anti-senescence drugs.

Bone Marrow Mesenchymal Stem-Cell Transplantation Promotes Functional Improvement Associated with CNTF-STAT3 Activation after Hemi-Sectioned Spinal Cord Injury in Tree Shrews.

Hemi-sectioned spinal cord injury (hSCI) can lead to spastic paralysis on the injured side, as well as flaccid paralysis on the contralateral side, which can negatively affect a patient's daily life. Stem-cell therapy may offer an effective treatment option for individuals with hSCI. To examine the role of bone marrow mesenchymal stem cells (BMSCs) transplantation on hSCI and explore related mechanisms in the tree shrews, here, we created a model of hSCI by inducing injury at the tenth thoracic vertebra (T10). Hoechst 33342-labeled BMSCs derived from adult tree shrews were isolated, cultured, and implanted into the spinal cord around the injury site at 9 days after injury. The isolated BMSCs were able to survive, proliferate and release a variety of neurotrophic factors (NTFs) both in vitro and in vivo. At 28 days after injury, compared with the sham group, the hSCI group displayed scar formation and dramatic elevations in the mean interleukin 1 beta (IL-1ß) density and cell apoptosis level, whereas the expression of signal transducer and activator of transcription 3 (STAT3) and ciliary neurotrophic factor (CNTF) mRNA was reduced. Following BMSC transplantation, motoneurons extent of shrinkage were reduced and the animals' Basso, Beattie, and Bresnahan (BBB) locomotion scale scores were significantly higher at 21 and 28 days after injury when compared with the injured group. Moreover, the hSCI-induced elevations in scar formation, IL-1ß, and cell apoptosis were reduced by BMSC transplantation to levels that were close to those of the sham group. Corresponding elevations in the expression of STAT3 and CNTF mRNA were observed in the hSCI + BMSCs group, and the levels were not significantly different from those observed in the sham group. Together, our results support that grafted BMSCs can significantly improve locomotor function in tree shrews subjected to hSCI and that this improvement is associated with the upregulation of CNTF and STAT3 signaling.

Sodium Channel Voltage-Gated Beta 2 Plays a Vital Role in Brain Aging Associated with Synaptic Plasticity and Expression of COX5A and FGF-2.

The role of sodium channel voltage-gated beta 2 (SCN2B) in brain aging is largely unknown. The present study was therefore designed to determine the role of SCN2B in brain aging by using the senescence-accelerated mice prone 8 (SAMP8), a brain senescence-accelerated animal model, together with the SCN2B transgenic mice. The results showed that SAMP8 exhibited impaired learning and memory functions, assessed by the Morris water maze test, as early as 8 months of age. The messenger RNA (mRNA) and protein expressions of SCN2B were also upregulated in the prefrontal cortex at this age. Treatment with traditional Chinese anti-aging medicine Xueshuangtong (Panax notoginseng saponins, PNS) significantly reversed the SCN2B expressions in the prefrontal cortex, resulting in improved learning and memory. Moreover, SCN2B knockdown transgenic mice were generated and bred to determine the roles of SCN2B in brain senescence. A reduction in the SCN2B level by 60.68% resulted in improvement in the hippocampus-dependent spatial recognition memory and long-term potential (LTP) slope of field excitatory postsynaptic potential (fEPSP), followed by an upregulation of COX5A mRNA levels and downregulation of fibroblast growth factor-2 (FGF-2) mRNA expression. Together, the present findings indicated that SCN2B could play an important role in the aging-related cognitive deterioration, which is associated with the regulations of COX5A and FGF-2. These findings could provide the potential strategy of candidate target to develop antisenescence drugs for the treatment of brain aging.

Expressional difference, distributions of TGF-ß1 in TGF-ß1 knock down transgenic mouse, and its possible roles in injured spinal cord.

Transforming growth factor ß1 (TGF-ß1) is a multi-functional cytokine implicated in many aspects of mammalian wound healing and scar tissue formation. However, few experiments have so far addressed the potential biological effects of TGF-ß1 in the nervous system after injury, in addition to the immune system. In the present study, expressional silencing TGF-ß1 was achieved by selecting predesigning hairpins targeting mouse TGF-ß1 genes. Four homozygous transgenic offspring were generated and designed as Founder 90, Founder 12, Founder 41 and Founder 46. The down-regulated rates of TGF-ß1 in different transgenic mice were also determined. To investigate the potential roles of TGF-ß1, we observed changes in the neurological behavior of TGF-ß1-knockdown (TGF-ß1-kd) mice after spinal cord transection (SCT). Moreover, mRNA levels of inflammatory cytokines, including IL-1, IL-6, IL-10, NF-κB and TNF, were also detected in nucleate cells from blood by real-time PCR. Consequently, different TGF-ß1 expressions were detected in multiple tissues, and protein levels of TGF-ß1 decreased at different rates relative to that of wild type (WT) ones. The levels of TGF-ß1 proteins in TGF-ß1-kd mice decreased at most by 57% in Founder 90, which showed a significant recovery in Basso, Beattie, Bresnahan (BBB) scores after SCT compared with that of WT. However, expressions of immune relative genes showed no dramatic difference compared with WT ones. This study is the first to generate TGF-ß1 down regulated mice and determine the possible roles of TGF-ß1 in vivo in different conditions.

Neural stem cells grafts decrease neural apoptosis associated with caspase-7 downregulation and BDNF upregulation in rats following spinal cord hemisection.

Transplantation of neural stem cells (NSCs) into lesioned spinal cord demonstrated a beneficial effect for neural repair, the underlying mechanism, however, remains to be elusive. Here, we showed that NSCs, possessing the capacity to differentiate toward into neurons and astrocytes, exhibit a neuroprotective effect by anti-apoptosis mechanism in spinal cord hemi-transected rats despite it did not improve behavior. Intravenous NSCs injection substantially upregulated the level of BDNF mRNA but not its receptor TrkB in hemisected spinal cord, while caspase-7, a downstream apoptosis gene of caspase-3, has been largely down-regulated. TUNEL staining showed that the number of apoptosis cells in injured spinal cord decreased significantly, compared with seen in rats with no NSCs administration. The present finding therefore provided crucial evidence to explain neuroprotective effect of NSCs grafts in hemisected spinal cord, which is associated with BDNF upregulation and caspase-7 downregulation.

Newly developed TGF-ß2 knock down transgenic mouse lines express TGF-ß2 differently and its distribution in multiple tissues varies.

BACKGROUND: Transforming growth factor-betas (TGF-ßs), including beta2 (TGF-ß2), constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation and tissue remodeling. TGF-ß2 is thought to play important roles in multiple developmental processes and neuron survival. However, before we carried out these investigations, a TGF-ß2 gene down-regulated transgenic animal model was needed. In the present study, expressional silencing TGF-ß2 was achieved by select predesigning interference short hairpin RNAs (shRNAs) targeting mouse TGF-ß2 genes. RESULTS: Four homozygous transgenic offspring were generated by genetic manipulation and the protein expressions of TGF-ß2 were detected in different tissues of these mice. The transgenic mice were designated as Founder 66, Founder 16, Founder 53 and Founder 41. The rates of TGF-ß2 down-expression in different transgenic mice were evaluated. The present study showed that different TGF-ß2 expressions were detected in multiple tissues and protein levels of TGF-ß2 decreased at different rates relative to that of wild type mice. The expressions of TGF-ß2 proteins in transgenic mice (Founder 66) reduced most by 52%. CONCLUSIONS: The present study generated transgenic mice with TGF-ß2 down-regulated, which established mice model for systemic exploring the possible roles of TGF-ß2 in vivo in different pathology conditions.