[MicroRNA-206-3p targeting heat shock protein 90αA1 in neuroinflammation and mitochondrial dysfunction in a rat model...

To investigate the molecular mechanism of microRNA-206-3p (miR-206-3p) regulating heat shock protein 90αA1 (HSP90αA1) in neuroinflammation and mitochondrial dysfunction.

(1) In vivo experiment: male Wistar rats (7 weeks old) were randomly divided into normal control group (n=10) and heat stroke model group (n=10). The rats in the normal control group were routinely fed and received no treatment. In the heat stroke model group, the heat stroke model was established by exposing rats to a controlled environment [temperature (40.0±0.5)centigrade, humidity (40±1)%]. Neurological function was assessed using modified Neurological Severity Score (mNSS) and brain water content was measured. Neuronal morphology was observed by Golgi staining. Inflammatory factors in the brain were detected by enzyme-linked immunoadsordent assay (ELISA). The localization of miR-206-3p and HSP90αA1 mRNA in the brain was determined using fluorescence in situ hybridization (FISH). (2) In vitro experiment: rat hippocampal neuronal cell line HT-22 was cultured in vitro, and the cells were divided into an empty vector control group [co-transfected with miR-206-3p mimic negative control (NC) and HSP90αA1 overexpressing empty virus (HSP90αA1 OE-NC)], a miR-206-3p overexpressing group (co-transfected with miR-206-3p mimic and HSP90αA1 OE-NC), a HSP90αA1 overexpressing group (co-transfected with miR-206-3p mimic-NC and HSP90αA1 OE), and a co-overexpressing group (co-transfected with miR-206-3p mimic and HSP90αA1 OE). Cleaved caspase-3/caspase-3 ratio and protein expressions of Bax and Bcl-2 were detected by Western blotting. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial DNA (mtDNA) copy number was quantified by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Reactive oxygen species (ROS) levels were measured using fluorescent probe method. Adenosine triphosphate (ATP) levels were determined by bioluminescence assay.

(1) In vivo experiment results: with prolonged heat exposure, mNSS scores, brain water content and levels of interleukins (IL-1β, IL-6, IL-10, IL-13, IL-17A) and tumor necrosis factor-α (TNF-α) in brain tissue were gradually increased in the heat stroke model group with peaking at 6 hours, and there was a statistically significant difference compared with the normal control group [mNSS score: 12.70±1.57 vs. 2.00±0.67, brain water content: (82.37±1.88)% vs. (70.69±1.75)%, IL-1β (ng/L): 5.56±0.64 vs. 1.00±0.13, IL-6 (ng/L): 6.70±0.84 vs. 1.00±0.10, IL-10 (ng/L): 4.92±0.58 vs. 1.00±0.11, IL-13 (ng/L): 4.55±0.53 vs. 1.00±0.09, IL-17A (ng/L): 3.83±0.44 vs. 1.00±0.11, TNF-α (ng/L): 6.70±0.72 vs. 1.00±0.09, all P<0.05]. Golgi staining revealed that with prolonged heat stroke injury, the number of dendritic branches in rat neurons gradually decreased. FISH experiment showed that miR-206-3p and HSP90αA1 were co-localized in neurons of the rat hippocampal CA3 region, with opposite expression trends. Furthermore, the longer the duration of heat stroke injury, the more significant the difference between the two. (2) In vitro experiment results: the neuronal apoptosis pathway was activated in the miR-206-3p overexpression group, accompanied by decreased mitochondrial number and ATP levels, and elevated ROS levels and mtDNA damage; however, the aforementioned trends were reversed in the HSP90αA1 overexpression group. Compared with miR-206-3p overexpression group, neuronal apoptosis was inhibited in the co-overexpressing group [cleaved caspase-3/caspase-3 ratio: 0.52±0.05 vs. 0.94±0.08, Bax protein (Bax/GAPDH): 0.64±0.07 vs. 1.18±0.11, Bcl-2 protein (Bcl-2/GAPDH): 0.74±0.08 vs. 0.42±0.04, all P<0.05], mitochondrial numbers and ATP levels were restored [ATP (μmol/g): 46.88±5.15 vs. 24.42±2.09, P<0.05], and ROS content and mtDNA damage were simultaneously reduced [ROS (fluorescence intensity): 33.05±3.73 vs. 59.72±6.29, mtDNA copy number (relative fold): 1.05±0.12 vs. 1.65±0.13, both P<0.05].

miR-206-3p participates in heat stroke-induced central nervous system injury through targeting HSP90αA1, which inhibits promotes apoptosis, and impairs mitochondrial function.