Involvement of Autophagy in the Protective Effects of Ginsenoside Rb1 in a Rat Model of Traumatic Brain Injury.

BACKGROUND AND OBJECTIVES: No treatment modalities have been identified to prevent neuron damage induced by traumatic brain injury (TBI). The objective of this study was to investigate whether ginsenoside Rb1 (GS-Rb1) could be utilized to exert neuroprotective effects in TBI. METHODS: Lateral fluid percussion injury (LFPI) was used to induce an experimental TBI model. Lewis rats were divided into a GS-Rb1 group (5, 10, 20 mg/kg, intraperitoneally injected daily), a sham group, and a vehicle group. Neurological impairments were assessed with brain water content, Evans blue extravasation, neurological deficit scores, and Morris water maze test. TUNEL and NeuN staining were utilized to detect neuron apoptosis. The relative expression of apoptosis- and autophagy-relevant molecules were assayed with real-time PCR and western blot. RESULTS: GS-Rb1 inhibited TBI-induced brain edema and Evans blue extravasation in a dose-dependent manner. Furthermore, GS-Rb1 improved neurological impairments with diminished neurological deficit scores, decreased escape latencies, increased time in the target quadrant, and increased number of platform site crossings. GS-Rb1 protected against neuron apoptosis with downregulated Bax expression and upregulated Bcl-2 expression. It was worth noting that TBI increased the LC3II/LC3I ratio and upregulated the relative expression of Beclin-1, Atg-7, and Atg-3; moreover, TBI downregulated the relative expression of P62. The administration of GS-Rb1 further strengthened the relative expression of autophagy-related molecules. CONCLUSIONS: GS-Rb1 alleviates neurological impairments induced by TBI with upregulated autophagy.

Neonatal microglia and proteinase inhibitors-treated adult microglia improve traumatic brain injury in rats by resolving the neuroinflammation.

Microglia participate in the regulation of neuroinflammation caused by traumatic brain injury (TBI). This research aimed to explore the repair effects of intracranial injection of neonatal microglia or protease-treated adult microglia on TBI in rat model. Lateral fluid percussion injury was used to establish rat brain injury model. E64 and serpinA3N were employed for the treatment of adult microglia. Cleaved caspase-3 level was analyzed through immunoblotting assay. Enzyme-linked immunosorbent assay was employed to analyze cytokine and chemokine levels. Astrocytosis and microgliosis were shown by immunofluorescence. The cognitive function of rats was analyzed by water maze. The injection of neonatal microglia inhibited cell apoptosis, reduced astrocytosis and microgliosis, decreased the level of chemokines and cytokines in cortex and ipsilateral hippocampus, and improved cognitive function of TBI rat model. The transplantation of peptidase inhibitors-treated adult microglia also inhibited cell apoptosis, reduced astrocytosis and microgliosis, and improved cognitive function of rats with TBI. The transplantation of either neonatal microglia or peptidase inhibitors-treated adult microglia significantly inhibited the pathogenesis of TBI in rat model, while untreated adult microglia showed no significant effect.

GJA1-20K Enhances Mitochondria Transfer from Astrocytes to Neurons via Cx43-TnTs After Traumatic Brain Injury.

Astrocytes are crucial in neural protection after traumatic brain injury (TBI), a global health problem causing severe brain tissue damage. Astrocytic connexin 43 (Cx43), encoded by GJA1 gene, has been demonstrated to facilitate the protection of astrocytes to neural damage with unclear mechanisms. This study aims to explore the role of GJA1-20K/Cx43 axis in the astrocyte-neuron interaction after TBI and the underlying mechanisms. Primarily cultured cortical neurons isolated from embryonic C57BL/6 mice were treated by compressed nitrogen-oxygen mixed gas to simulate TBI-like damage in vitro. The transwell astrocyte-neuron co-culture system were constructed to recapitulate the interaction between the two cell types. Quantitative PCR was applied to analyze mRNA level of target genes. Western blot and immunofluorescence were conducted to detect target proteins expression. GJA1-20K overexpression significantly down-regulated the expression of phosphorylated Cx43 (p-Cx43) without affecting the total Cx43 protein level. Besides, GJA1-20K overexpression obviously enhanced the dendrite length, as well as the expression levels of function and synthesis-related factors of mitochondria in damaged neurons. GJA1-20K up-regulated functional Cx43 expression in astrocytes, which promoted mitochondria transmission from astrocytes to neurons which might be responsible to the protection of astrocyte to neurons after TBI-like damage in vitro.

Overexpression of Astrocytes-Specific GJA1-20k Enhances the Viability and Recovery of the Neurons in a Rat Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a devastating actuality in clinics worldwide. It is estimated that approximately 10 million people among the world suffer from TBI each year, and a considerable number of patients will be temporarily or permanently disabled or even die due to this disease. Astrocytes play a very important role in the repair of brain tissue after TBI, including the formation of a neuroprotective barrier, inhibition of brain edema, and inhibition of normal nerve cell apoptosis. However, the detailed mechanism underlying this protective effect is still unclear. To investigate the regulatory factors of astrocytes to other neurons post-TBI, we established a TBI rat model and used the AAV to mediate the overexpression of GJA1-20k in astrocytes of rats. And functionally, the specific overexpression of GJA1-20k in astrocytes promoted the viability and recovery of neurons in TBI. Mechanistically, the astrocytes-specific upregulation of GJA1-20k protected the function of mitochondria in neurons of FPI rats, thus suppressing the apoptosis of the damaged neurons. We hereby reported that astrocytes-specific overexpression of GJA1-20k enhanced the viability and recovery of the neurons in TBI through regulating their mitochondrial function.

Astrocytes-derived exosomes induce neuronal recovery after traumatic brain injury via delivering gap junction alpha 1-20 k.

Astrocytes are more resistant to ischemia and hypoxia in the acute phase of brain injury after traumatic brain injury (TBI). Previous study showed that gap junction alpha 1 (GJA1) phosphorylation can increase the survival of damaged astrocytes. The GJA1-20 k expression in neurons co-culture with astrocytes was positively correlated with exosomes uptake. This study aims to explore the effect of exogenous GJA1-20 k carried by astrocyte-derived exosomes on neurons apoptosis and mitochondrial function after TBI. Astrocytes were co-cultured with the neuron with/without damage from air pressure. Exosomes were isolated, extracted from the culture medium by differential ultra-centrifugation, and verified by electron microscopy. Immunofluorescence staining, tunnel, western blot were employed to detect exosomes marker CD60, apoptosis, and mitochondrial function related protein expression and GJA1-20 k in cell culture. A rat model of hydraulic injury TBI was built, and exosomes was transferred. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and immunohistochemistry staining of Nissl and microtubule associated protein 2 were used to detect the brain damage. A transwell stereo culture model of astrocytes and TBI-like injured neuron was constructed. The exosomes derived from astrocytes promoted the recovery of damaged neuron by in vitro exosome treatment. Compared with GJA1-20 k knockout exosome control group, GJA1-20 k exosomes were uptaken by neuron and downregulated the apoptosis rate and upregulated mitochondrial function to promote neuronal recovery. Finally, the results were validated by TTC staining and damaged tissue sections of rat TBI model. This study contributes to a better understanding of the astrocyte-neuron protection mechanism in TBI and provides a potential new target for the treatment of TBI.