This study aims to investigate the ameliorating effect of Corni Fructus(CF) on the myocardial ischemic injury and the pharmacokinetic properties of characteristic components of CF. The mouse model of isoproterenol-induced myocardial ischemia was established and administrated with the aqueous extract of CF. The general efficacy of CF in ameliorating the myocardial ischemic injury was evaluated based on the cardiac histopathology and the levels of myocardial injury markers: creatine kinase isoenzyme(CK-MB) and cardiac troponin I(cTn-I). The metabolomics analysis was carried out for the heart and serum samples of mice to screen the biomarkers of CF in ameliorating the myocardial ischemic injury and then the predicted biomarkers were submitted to metabolic pathway enrichment. The pharmacokinetic analysis was performed for morroniside, loganin, and cornuside Ⅰ in mouse heart and serum samples to obtain the pharmacokinetic parameters of these components. The pharmacokinetic parameters were then integrated on the basis of self-defined weighting coefficients to simulate an integrated pharmacokinetic profile of CF iridoid glycosides in the heart and serum of the mouse model of myocardial ischemia. The results indicated that CF reduced the pathological damage to cardiac cells and tissue(hematoxylin-eosin staining) and lowered the levels of CK-MB and cTn-I in the serum of the mouse model of myocardial ischemia(P<0.01). Metabolomics analysis screed out 31 endogenous metabolites in the heart and 35 in the serum as biomarkers of CF in ameliorating the myocardial ischemic injury. These biomarkers were altered by modeling and restored by CF. Six metabolic pathways in the heart and 5 in the serum were enriched based on these metabolic markers. The main integrated pharmacokinetic parameters of CF iridoid glycosides were T_(max)=1 h, t_(1/2)=(1.52±0.05) h in the heart and T_(max)=1 h, t_(1/2)=(1.56±0.50) h in the serum. Both concentration-time curves showed a double-peak phenomenon. In conclusion, CF demonstrated the cardioprotective effect by regulating metabolic pathways such as taurine and hypotaurine metabolism, and pantothenic acid and coenzyme A biosynthesis. The integrated pharmacokinetics reflect the general pharmacokinetic properties of characteristic components in CF.
Animals ; Cornus/chemistry* ; Mice ; Metabolomics ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Myocardial Ischemia/metabolism* ; Humans ; Troponin I/metabolism* ; Myocardium/pathology* ; Disease Models, Animal ; Biomarkers/metabolism* ; Creatine Kinase, MB Form/metabolism*

Objective To explore the effects of Shuanglu Tongnao Formula on neuroinflammation in ischemic stroke (IS) rats via the P2X purinoceptor 7 receptor (P2X7R)/NLR family pyrin domain-containing 3 (NLRP3) pathway. Methods The rats were divided into five groups: the IS group, control group, Shuanglu Tongnao Formula group, P2X7R inhibitor brilliant blue G (BBG) group, and Shuanglu Tongnao Formula combined with P2X7R activator adenosine triphosphate (ATP) group, with 18 rats in each group. Except for the control group, rats in all other groups were used to construct an IS model using the suture method. After successful modeling, the drug was given once a day for 2 weeks. Neurological function scores and cerebral infarction volume ratios were measured in rats. Pathological examination of the ischemic penumbra brain tissue was performed. Immunofluorescence staining was used to quantify the proportions of microglia co-expressing both inducible nitric oxide synthase (iNOS) and ionized calcium-binding adapter molecule 1 (Iba1), as well as arginase 1 (Arg1) and Iba1, in the ischemic penumbra brain tissue. ELISA was used to detect tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interleukin 6 (IL-6) and IL-10 in the ischemic penumbra brain tissue. Western blotting was used to measure P2X7R, NLRP3, and IL-1β proteins in the ischemic penumbra brain tissue. Results Compared with the control group, the IS group showed disordered neuronal arrangement, nuclear condensation, and obvious infiltration of inflammatory cells in the ischemic penumbra; significantly elevated neurological function scores, cerebral infarction volume ratios, proportions of microglia co-expressing iNOS and Iba1, and levels of TNF-α, IL-6, and P2X7R, NLRP3, IL-1β proteins; along with reduced proportions of microglia co-expressing Arg1 and Iba1 and levels of TGF-β and IL-10. Compared with the IS group, the Zhuang medicine Shuanglu Tongnao Formula and BBG groups demonstrated alleviated brain tissue damage; reduced neurological function scores, cerebral infarction volume ratios, proportions of microglia co-expressing iNOS and Iba1, and levels of TNF-α, IL-6, and P2X7R, NLRP3, IL-1β proteins; along with increased proportions of microglia co-expressing Arg1 and Iba1 and levels of TGF-β and IL-10. ATP reversed the effects of Zhuang medicine Shuanglu Tongnao Formula on microglial polarization and neuroinflammation in IS rats. Conclusion Zhuang medicine Shuanglu Tongnao Formula may promote the transformation of microglia from M1 type to M2 type by inhibiting the P2X7R/NLRP3 pathway, thereby improving neuroinflammation in IS rats.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Receptors, Purinergic P2X7/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Ischemic Stroke/pathology* ; Rats, Sprague-Dawley ; Disease Models, Animal ; Signal Transduction/drug effects* ; Neuroinflammatory Diseases/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Interleukin-10/metabolism* ; Brain Ischemia/drug therapy* ; Microglia/metabolism*

Objective To investigate the protective effect of artesunate on hypoxic-ischemic brain damage (HIBD) and its mechanism in neonatal rats. Methods 7-day-old neonatal SD rats were randomly divided into sham operation group, model group, artesunate 5 mg/kg group, artesunate 10 mg/kg group, artesunate 20 mg/kg group and dexamethasone 6 mg/kg group, with 18 rats in each group. HIBD models were established in groups except for the sham operation group. The sham operation group only needed to separate the left common carotid artery without ligation and nitrogen-oxygen mixed gas ventilation. Each group was injected with drug intraperitoneally right after surgery and the rats in the sham operation group and the model group were injected with an equal volume of normal saline (once a day for a total of 5 times). One hour after the last injection, the rats in each group were scored for neurological defects. After the rats were sacrificed, the brain water content was measured and the pathological changes of the brain tissues of rats were observed. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to detect the neuronal cell apoptosis, and ELISA was applied to detect the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood of each group of rats. Western blot analysis was adopted to detect the protein expression levels of NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 in the rats brain tissues of each group. Results Compared with the model group, the neurological deficit score was decreased; the pathological damage of brain tissues was relieved; the brain water content was significantly reduced; the apoptosis number of hippocampal neurons was decreased significantly; the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood were significantly reduced; the protein expression levels of NLRP3, ASC and caspase-1 were significantly lowered in the middle-dose and high-dose artesunate groups and the dexamethasone group. Conclusion Artesunate can improve the neurological function, relieve the brain damage, and alleviate the brain edema in neonatal rats with HIBD. It can protect the HIBD, which may be related to the inhibition of NLRP3 inflammasome activation and reduction of inflammatory cytokine secretion.
Animals ; Rats ; Animals, Newborn ; Artesunate/pharmacology* ; Brain/metabolism* ; Caspases/metabolism* ; Dexamethasone ; Hypoxia-Ischemia, Brain/pathology* ; Inflammasomes ; Interleukin-6/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Water/metabolism*

OBJECTIVES: Acute kidney injury (AKI) can be caused by ischemia/reperfusion (I/R), nephrotoxin, and sepsis, with poor prognosis and high mortality. Leptin is a protein molecule that regulates the body's energy metabolism and reproductive activities via binding to its specific receptor. Leptin can inhibit cardiomyocyte apoptosis caused by I/R, but its effect on I/R kidney injury and the underlying mechanisms are still unclear. This study aims to investigate the effect and mechanisms of leptin on renal function, renal histopathology, apoptosis, and autophagy during acute I/R kidney injury. METHODS: Healthy adult male mice were randomly divided into 4 groups: a sham+wild-type mice (ob/+) group, a sham+leptin gene-deficient mice (ob/ob) group, an I/R+ob/+ group, and an I/R+ob/ob group (n=8 per group). For sham operation, a longitudinal incision was made on the back of the mice to expose and separate the bilateral kidneys and renal arteries, and no subsequent treatment was performed. I/R treatment was ischemia for 30 min and reperfusion for 48 h. The levels of BUN and SCr were detected to evaluate renal function; HE staining was used to observe the pathological changes of renal tissue; TUNEL staining was used to observe cell apoptosis, and apoptosis-positive cells were counted; Western blotting was used to detect levels of apoptosis-related proteins (caspase 3, caspase 9), autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3 I, LC3 II], mTOR-dependent signaling pathway proteins [phosphate and tension homology (PTEN), adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (AKT), extracellular regulated protein kinase (ERK), phosphorylated PTEN (p-PTEN), phosphorylated AMPK (p-AMPK), phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK)]. RESULTS: There was no significant difference in the levels of BUN and SCr between the sham+ob/+ group and the sham+ob/ob group (both P>0.05). The levels of BUN and SCr in the I/R+ob/+ group were significantly higher than those in the sham+ob/+ group (both P<0.05). Compared with the mice in the sham+ob/ob group or the I/R+ob/+ group, the levels of BUN and SCr in the I/R+ob/ob group were significantly increased (all P<0.05). There was no obvious damage to the renal tubules in the sham+ob/+ group and the sham+ob/ob group. Compared with sham+ob/+ group and sham+ob/ob group, both the I/R+ob/+ group and the I/R+ob/ob group had cell damage such as brush border shedding, vacuolar degeneration, and cast formation. Compared with the I/R+ob/+ group, the renal tubules of the mice in the I/R+ob/ob group were more severely damaged. The pathological score of renal tubular injury showed that the renal tubular injury was the most serious in the I/R+ob/ob group (P<0.05). Compared with the sham+ob/+ group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, the ratio of LC3 II to LC3 I was significantly increased, and the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/+ group (all P<0.05). Compared with the sham+ob/ob group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, and the ratio of LC3 II to LC3 I was significantly increased, while the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/ob group (all P<0.05). Compared with the I/R+ob/+ group, the levels of p-mTOR, p-PTEN, p-AMPK, p-AKT were more significantly down-regulated, while the levels of caspase 3, caspase 9, PTEN, and LC3 II were more significantly up-regulated, and the ratio of LC3 II to LC3 I was more significantly increase in the I/R+ob/ob group (all P<0.05). CONCLUSIONS Renal function and tubular damage, and elevated levels of apoptosis and autophagy are observed in mice kidneys after acute I/R. Leptin might relieve I/R induced AKI by inhibiting apoptosis and autophagy that through a complex network of interactions between mTOR-dependent signaling pathways.
AMP-Activated Protein Kinases/metabolism* ; Acute Kidney Injury/pathology* ; Animals ; Apoptosis ; Apoptosis Regulatory Proteins/pharmacology* ; Autophagy ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Female ; Humans ; Ischemia ; Kidney/pathology* ; Leptin/pharmacology* ; Male ; Mammals/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Reperfusion/adverse effects* ; Reperfusion Injury/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

Cerebral ischemia-reperfusion injury(CIRI) is an important factor hindering the recovery of ischemic stroke patients after blood flow recanalization. Mitochondria, serving as the "energy chamber" of cells, have multiple important physiological functions, such as supplying energy, metabolizing reactive oxygen species, storing calcium, and mediating programmed cell death. During CIRI, oxidative stress, calcium overload, inflammatory response, and other factors can easily lead to neuronal mitochondrial dyshomeostasis, which is the key pathological link leading to secondary injury. As reported, the mitochondrial quality control(MQC) system, mainly including mitochondrial biosynthesis, kinetics, autophagy, and derived vesicles, is an important endogenous mechanism to maintain mitochondrial homeostasis and plays an important protective role in the damage of mitochondrial structure and function caused by CIRI. This paper reviewed the mechanism of MQC and the research progress on MQC-targeting therapy of CIRI in recent 10 years to provide theoretical references for exploring new strategies for the prevention and treatment of ischemic stroke with traditional Chinese medicine.
Brain Ischemia/prevention & control* ; Calcium/metabolism* ; Humans ; Ischemic Stroke ; Mitochondria/pathology* ; Reactive Oxygen Species/metabolism* ; Reperfusion Injury/prevention & control*

This study established the EA.hy926 cell myocardial ischemia model to compare the effects of two Kaixin Powder prescriptions, Buxin Decoction(BXD) and Dingzhi Pills(DZP), at three dosages(500, 200, and 100 μg·mL~(-1)) on the cell viability. Further, the public databases(TCMSP, TCMID, SYMMAP, and STRING) and the network pharmacology methods such as KEGG pathway enrichment were employed to decipher the possible molecular mechanism of BXD in exerting the cardioprotective effect. The pharmacological effect of BXD was evaluated with the rat model of isoprenaline(ISO)-induced myocardial ischemia. The expression levels of proteins involved in the phosphatidylinositol-3-kinase/protein kinase B(PI3 K/AKT) signaling pathway were measured by Western blot. BXD significantly increased the viability of EA.hy926 cells, showing the performance superior to DZP. The network pharmacology analysis predicted that BXD might exert cardiac protection through the PI3 K/AKT signaling pathway. The in vivo experiment on rats showed that BXD treatment significantly increased the cardiac ejection fraction(EF), fractional shortening(FS), diastolic left ventricular anterior wall(LVAWd), systolic left ventricular anterior wall(LVAWs), and diastolic left ventricular posterior wall(LVPWd), significantly decreased the beat per minute(BPM) and diastolic left ventricular internal diameter(LVIDd), and significantly improved the ST segment in the electrocardiogram. The pathological results(Masson staining) showed that BXD restored the myocardial thickness, decreased the collagen fiber, increased the muscle fiber, and reduced the infarct area to alleviate myocardial ischemia. Furthermore, BXD lowered the serum levels of inflammatory cytokines [tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6)] and myocardial enzymes [creatine kinase(CK) and lactate dehydrogenase(LDH)], increased the p-AKT/AKT ratio, up-regulated the protein levels of PI3 K, NF-κB, IKK-α, and Bcl-xl, and down-regulated that of the apoptotic protein Bax. In conclusion, BXD may exert cardiac protection effect by regulating the PI3 K/AKT signaling pathway.
Rats ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Powders ; Network Pharmacology ; Signal Transduction ; Myocardial Ischemia ; Myocardium/pathology* ; Creatine Kinase ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Prescriptions

OBJECTIVE: To examine the effect of Shenmai Injection (SMJ) on ferroptosis during myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism. METHODS: A total of 120 SPF-grade adult male SD rats, weighing 220-250 g were randomly divided into different groups according to a random number table. Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of 120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration of Fe²⁺ in heart tissues and the levels of creatine kinase-MB (CK-MB), troponin I (cTnl), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured using assay kits, and the expressions of Nrf2, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were examined by Western blot. RESULTS: Compared with the sham group, I/R significantly injured heart tissues, as evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum CK-MB, cTnI and MDA levels, and myocardial Fe²⁺ concentrations; and the decreases in SOD activity (P<0.05). These results were accompanied by ultrastructural alterations to the mitochondria, increased expression of ACSL4 and inhibited the activation of Nrf2/GPX4 signalling (P<0.05). Compared with I/R group, pretreatment with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury, Fe²⁺ concentrations and ACSL4 expression and attenuated mitochondrial impairment, while 14 mg/kg erastin exacerbated myocardial I/R injury (P<0.05). In addition, cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385, as evidenced by the increased myocardial infarct size, CK-MB, cTnI, MDA and Fe²⁺ concentrations, and the decreased SOD activity (P<0.05). CONCLUSIONS Ferroptosis is involved in myocardial I/R injury. Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a strategy for the prevention and treatment of ischemic heart diseases.
Animals ; Male ; Rats ; Coenzyme A ; Creatine Kinase ; Ferroptosis ; Ligases ; Malondialdehyde ; Myocardial Infarction/drug therapy* ; Myocardial Ischemia/drug therapy* ; Myocardial Reperfusion Injury/pathology* ; Myocytes, Cardiac/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Rats, Sprague-Dawley ; Superoxide Dismutase/metabolism* ; Troponin I

OBJECTIVES: Sufentanil has a good protective effect on myocardial and liver injury caused by ischemia reperfusion (IR), but its protective effect on kidney is still unclear. This study aims to investigate whether sufentanil can prevent IR-induced acute kidney injury (AKI) and to determine whether its efficacy is related to miR-145-mediated autophagy. METHODS: A total of 40 rats were randomly divided into 5 groups (n=8 in each group): A sham group, an IR group, a sufentanil group, a sufentanil+miR-145 inhibitor control group (an anti-NC group) and a sufentanil+miR-145 inhibitor group (an anti-miR-145 group). Except for the sham group, the other groups established a rat AKI model induced by IR. The sufentanil group, the sufentanil+anti-NC group, and the sufentanil+anti-miR-145 were injected with sufentanil (1 μg/kg) through femoral vein 30 min before ischemia. The sufentanil+anti-NC group and the sufentanil+anti-miR-145 group were injected with miR-145 inhibitor control or anti-miR-145 (80 mg/kg) through the tail vein before sufentanil pretreatment. The structure and function of kidneys harvested from the rats were evaluated, and the protein levels of autophagy-related proteins, oxidative stress levels, and apoptosis levels were measured. RESULTS: Compared with the IR group, the renal structure and function were improved in the sufentanil group. The levels of blood urea nitrogen (BUN), creatinine (Cr), urinary kidney injury molecule 1 (KIM-1), neutrophil gelatinase related lipid transporter (NGAL), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and ROS were significantly decreased (all P<0.05). In addition, compared with the IR group, the levels of Beclin-1 and LC3 in renal tissues in the sufentanil group were significantly increased (both P<0.05), and the apoptosis in renal tissues was significantly reduced (P<0.05). Compared with the sufentanil+anti-NC group, the levels of BUN, Cr, KIM-1, NGAL, TNF-α, IL-1β, IL-6 and ROS in the sufentanil+anti-miR-145 group were significantly increased (all P<0.05), the levels of Beclin-1 and LC3 in renal tissues were significantly decreased (both P<0.05), and the apoptosis in renal tissues was significantly increased (P<0.05). CONCLUSIONS Sufentanil can prevent the AKI induced by IR, which is related to the up-regulation of miR-145-mediated autophagy.
Animals ; Rats ; Acute Kidney Injury/pathology* ; Antagomirs ; Autophagy ; Beclin-1/metabolism* ; Creatinine ; Interleukin-6/metabolism* ; Ischemia ; Kidney/pathology* ; Lipocalin-2 ; MicroRNAs/metabolism* ; Reactive Oxygen Species ; Reperfusion ; Reperfusion Injury/metabolism* ; Sufentanil/therapeutic use* ; Tumor Necrosis Factor-alpha ; Up-Regulation

OBJECTIVE: The purpose of this study was to determine whether xenon post-conditioning affects mTOR signaling as well as endoplasmic reticulum stress (ERS)-apoptosis pathway in rats with spinal cord ischemia/reperfusion injury. METHODS: Fifty male rats were randomized equally into sham-operated group (Sham group), I/R model group (I/R group), I/R model+ xenon post-conditioning group (Xe group), I/R model+rapamycin (a mTOR signaling pathway inhibitor) treatment group (I/R+ Rapa group), and I/R model + xenon post- conditioning with rapamycin treatment group (Xe + Rapa group).. In the latter 4 groups, SCIRI was induced by clamping the abdominal aorta for 85 min followed by reperfusion for 4 h. Rapamycin (or vehicle) was administered by daily intraperitoneal injection (4 mg/kg) for 3 days before SCIRI, and xenon post-conditioning by inhalation of 1∶1 mixture of xenon and oxygen for 1 h at 1 h after initiation of reperfusion; the rats without xenon post-conditioning were given inhalation of nitrogen and oxygen (1∶ 1). After the reperfusion, motor function and histopathologic changes in the rats were examined. Western blotting and real-time PCR were used to detect the protein and mRNA expressions of GRP78, ATF6, IRE1α, PERK, mTOR, p-mTOR, Bax, Bcl-2 and caspase-3 in the spinal cord. RESULTS: The rats showed significantly lowered hind limb motor function following SCIRI (P < 0.01) with a decreased count of normal neurons, increased mRNA and protein expressions of GRP78, ATF6, IRE1α, PERK, and caspase-3, and elevated p-mTOR/mTOR ratio and Bax/Bcl-2 ratio (P < 0.01). Xenon post-conditioning significantly decreased the mRNA and protein levels of GRP78, ATF6, IRE1α, PERK and caspase-3 (P < 0.05 or 0.01) and reduced p-mTOR/mTOR and Bax/Bcl-2 ratios (P < 0.01) in rats with SCIRI; the mRNA contents and protein levels of GRP78 and ATF6 were significantly decreased in I/R+Rapa group (P < 0.01). Compared with those in Xe group, the rats in I/R+Rapa group and Xe+Rapa had significantly lowered BBB and Tarlov scores of the hind legs (P < 0.01), and caspase-3 protein level and Bax/Bcl-2 ratio were significantly lowered in Xe+Rapa group (P < 0.05 or 0.01). CONCLUSION By inhibiting ERS and neuronal apoptosis, xenon post- conditioning may have protective effects against SCIRI in rats. The mTOR signaling pathway is partially involved in this process.
Animals ; Apoptosis ; Caspase 3/metabolism* ; Endoplasmic Reticulum Stress ; Endoribonucleases/pharmacology* ; Injections, Intraperitoneal ; Male ; Neurons/pathology* ; Nitrogen/metabolism* ; Oxygen/metabolism* ; Protein Serine-Threonine Kinases ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Sirolimus/pharmacology* ; Spinal Cord Ischemia/pathology* ; TOR Serine-Threonine Kinases/metabolism* ; Xenon/therapeutic use* ; bcl-2-Associated X Protein/metabolism*

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.
Animals ; Autophagy ; physiology ; Beclin-1 ; metabolism ; Brain ; metabolism ; pathology ; Brain Ischemia ; metabolism ; pathology ; therapy ; Cystatin C ; genetics ; metabolism ; Disease Models, Animal ; Gene Expression ; Gene Knockdown Techniques ; Hyperbaric Oxygenation ; Lysosomes ; metabolism ; pathology ; Male ; Microtubule-Associated Proteins ; metabolism ; Neurons ; metabolism ; pathology ; Neuroprotection ; physiology ; Oxygen ; therapeutic use ; Random Allocation ; Rats, Sprague-Dawley ; Rats, Transgenic ; Reperfusion Injury ; metabolism ; pathology ; therapy