Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity

Objective Exposure to microgravity results in postflight cardiovascular deconditioning in astronauts.Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process.To elucidate the mechanism for this condition,we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted(HU)rat cerebral arteries.Methods Three-week HU was used to simulate microgravity in rats.The contractile responses to vasoconstrictors,mitochondrial fission/fusion,Ca^(2+) distribution,inositol 1,4,5-trisphosphate receptor(IP3 R)abundance,and the activities of voltage-gated K+channels(KV)and Ca^(2+)-activated K+channels(BKCa)were examined in rat cerebral vascular smooth muscle cells(VSMCs).Results An increase of cytoplasmic Ca^(2+) and a decrease of mitochondrial/sarcoplasmic reticulum(SR)Ca^(2+) were observed in HU rat cerebral VSMCs.The abundance of fusion proteins(mitofusin 1/2[MFN1/2])and fission proteins(dynamin-related protein 1[DRP1]and fission-mitochondrial 1[FIS1])was significantly downregulated and upregulated,respectively in HU rat cerebral VSMCs.The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats,and IP3 R protein/mRNA levels were significantly upregulated.The current densities and open probabilities of KV and BKCa decreased and increased,respectively.Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1.It also decreased IP3 R expression levels and restored the activities of the KV and BKCa channels.MitoTEMPO restored the Ca^(2+) distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries.Conclusion The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

Self-shrinking supramolecular nanoparticles syndicate energy suppression and NIR-II mild photothermal amplification of mitochondrial oxidative stress for breast cancer therapy

Photothermal therapy(PTT)may lead to healthy tissue damage,tumor metastasis,and recurrence,which makes mild photothermal therapy(mild PTT)stand out.However,overcoming heat resistance,insufficient therapeutic effect,and poor photothermal conversion efficiency has become new challenge.Herein,we report a dynamic supramolecular nanocarrier formed from amide-sericin and aldehyde-polyhydroxy glucan(denoted as SDA),the loose cavity of which can be filled by using the pharmaceutical combination of lonidamine(LND)and NIR-II photothermal agent of IR-1061,producing SDLI with a tighter inner hole,smaller and uniform particle size and excellent stability due to multiple pulling forces.Moreover,the intricate internal network structure prevents the hydrophobic IR-1061 from forming aggregates in the small cavity,and the photothermal conversion efficiency(PCE)can reach 48.9%.At the acidic tumor microenvironment of pH 6.5,the controlled release of LND can solve the problem of heat resistance of NIR-II mild PTT and significantly improve the therapeutic effect of NIR-II mild PTT.Meanwhile,SDLI also shows a reasonable tumor inhibition rate,so the synergistic strategy of inhibiting tumor energy metabolism and NIR-II mild PTT to magnify mitochondrial oxidative stress,continuous cell stress state-induced immunogenic cell death to promote the induction of tumor apoptosis is proposed to achieve more effective cancer treatment.

Attenuation of myocardial apoptosis by alpha-lipoic acid through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy

Background Cardiac failure is a leading cause of the mortality of diabetic patients. In part this is due to a specific cardiomyopathy, referred to as diabetic cardiomyopathy. Oxidative stress is widely considered to be one of the major factors underlying the pathogenesis of the disease. This study aimed to test whether the antioxidant α-lipoic acid （α-LA） could attenuate mitochondrion-dependent myocardial apoptosis through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy. Methods A rat model of diabetes was induced by a single tail intravenous injection of streptozotocin （STZ） 45 mg/kg. Experimental animals were randomly assigned to 3 groups： normal control （NC）, diabetes （DM） and DM treated with α-LA （α-LA）. The latter group was administered with a-LA （100 mg/kg ip per day）, the remainder received the same volume vehicle. At weeks 4, 8, and 12 after the onset of diabetes, cardiac apoptosis was examined by TUNEL assay. Cardiomyopathy was evaluated by assessment of cardiac structure and function. Oxidative damage was evaluated by the content of malondialdehyde （MDA）, reduced glutathione （GSH） and the activity of manganese superoxide diamutase （Mn-SOD） in the myocardial mitochondria. Expression of caspase-9 and caspase-3 proteins was determined by immunohistochemistry and mitochondrial cytochrome c release was detected by Western blotting Results At 4, 8, and 12 weeks after the onset of diabetes, significant reductions in TUNEL-positive cells, caspase-9,-3 expression, and mitochondrial cytochrome c release were observed in the α-LA group compared to the DM group. In the DM group, the content of MDA in the myocardial mitochondria was significantly increased, and there was a decrease in both the mitochondrial GSH content and the activities of Mn-SOD. They were significantly improved by α-LA treatment. HE staining displayed structural abnormalities in diabetic hearts, while α-LA reversed this structural derangement. The index of cardiac function （±dp/dtmax） in the diabetes group was aggravated progressively from 4 weeks to 12 weeks, but α-LA delayed deterioration of cardiac function （P 〈0.05）. Conclusions Our findings indicate that the antioxidant α-LA can effectively attenuate mitochondria-dependent cardiac apoptosis and exert a protective role against the development of diabetic cardiomyopathy. The ability of α-LA to suppress mitochondrial oxidative damage is concomitant with an enhancement of Mn-SOD activity and an increase in the GSH content of myocardial mitochondria.

Mitochondrial oxidative damage and apoptosis induced by high glucose through Rho kinase signal pathway in renal tubular epithelial cells

Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.

Targeting glutamine utilization to block metabolic adaptation of tumor cells under the stress of carboxyamidotriazole-induced nutrients unavailability

Tumor cells have unique metabolic programming that is biologically distinct from that of corresponding normal cells.Resetting tumor metabolic programming is a promising strategy to ameliorate drug resistance and improve the tumor microenvironment.Here,we show that carboxyamidotriazole(CAI),an anticancer drug,can function as a metabolic modulator that decreases glucose and lipid metabolism and increases the dependency of colon cancer cells on glutamine metabolism.CAI suppressed glucose and lipid metabolism utilization,causing inhibition of mitochondrial respiratory chain complex I,thus producing reactive oxygen species(ROS).In parallel,activation of the aryl hydrocarbon receptor(Ah R)increased glutamine uptake via the transporter SLC1A5,which could activate the ROS-scavenging enzyme glutathione peroxidase.As a result,combined use of inhibitors of GLS/GDH1,CAI could effectively restrict colorectal cancer(CRC)energy metabolism.These data illuminate a new antitumor mechanism of CAI,suggesting a new strategy for CRC metabolic reprogramming treatment.

Contribution of β-phenethylamine, a component of chocolate and wine, to dopaminergic neurodegeneration: implications for the pathogenesis of Parkinson's disease

While the cause of dopaminergic neuronal cell death in Parkinson＇s disease（PD）is not yet understood,many endogenous molecules have been implicated in its pathogenesis.β-phenethylamine（β-PEA）,a component of various food items including chocolate and wine,is an endogenous molecule produced from phenylalanine in the brain.It has been reported recently that long-term administration ofβ-PEA in rodents causes neurochemical and behavioral alterations similar to that produced by parkinsonian neurotoxins.The toxicity ofβ-PEA has been linked to the production of hydroxyl radical（.OH）and the generation of oxidative stress in dopaminergic areas of the brain,and this may be mediated by inhibition of mitochondrial complex-I.Another significant observation is that administration ofβ-PEA to rodents reduces striatal dopamine content and induces movement disorders similar to those of parkinsonian rodents.However,no reports are available on the extent of dopaminergic neuronal cell death after administration ofβ-PEA.Based on the literature,we set out to establishβ-PEA as an endogenous molecule that potentially contributes to the progressive development of PD.The sequence of molecular events that could be responsible for dopaminergic neuronal cell death in PD by consumption ofβ-PEA-containing foods is proposed here.Thus,long-term over-consumption of food items containingβ-PEA could be a neurological risk factor having significant pathological consequences.

Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis

Background Persistent inflammatory response in the brain can lead to tissue damage and neurodegeneration.In Alzheimer’s disease(AD),there is an aberrant activation of inflammasomes,molecular platforms that drive inflammation through caspase-1-mediated proteolytic cleavage of proinflammatory cytokines and gasdermin D(GSDMD),the executor of pyroptosis.However,the mechanisms underlying the sustained activation of inflammasomes in AD are largely unknown.We have previously shown that high brain cholesterol levels promote amyloid-β(Aβ)accumulation and oxidative stress.Here,we investigate whether these cholesterol-mediated changes may regulate the inflam-masome pathway.Methods SIM-A9 microglia and SH-SY5Y neuroblastoma cells were cholesterol-enriched using a water-soluble cholesterol complex.After exposure to lipopolysaccharide(LPS)plus muramyl dipeptide or Aβ,activation of the inflammasome pathway was analyzed by immunofluorescence,ELISA and immunoblotting analysis.Fluorescently-labeled Aβwas employed to monitor changes in microglia phagocytosis.Conditioned medium was used to study how microglia-neuron interrelationship modulates the inflammasome-mediated response.Results In activated microglia,cholesterol enrichment promoted the release of encapsulated IL-1βaccompanied by a switch to a more neuroprotective phenotype,with increased phagocytic capacity and release of neurotrophic factors.In contrast,in SH-SY5Y cells,high cholesterol levels stimulated inflammasome assembly triggered by both bacterial toxins and Aβpeptides,resulting in GSDMD-mediated pyroptosis.Glutathione(GSH)ethyl ester treatment,which recovered the cholesterol-mediated depletion of mitochondrial GSH levels,significantly reduced the Aβ-induced oxidative stress in the neuronal cells,resulting in lower inflammasome activation and cell death.Furthermore,using conditioned media,we showed that neuronal pyroptosis affects the function of the cholesterol-enriched microglia,lowering its phagocytic activity and,therefore,the ability to degrade extracellular Aβ.Conclusions Changes in intracellular cholesterol levels differentially regulate the inflammasome-mediated immune response in microglia and neuronal cells.Given the microglia-neuron cross-talk in the brain,cholesterol modulation should be considered a potential therapeutic target for AD treatment,which may help to block the aberrant and chronic inflammation observed during the disease progression.