Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

Zhongfeng decoction attenuates cerebral ischemia-reperfusion injury by inhibiting autophagy via regulating the AGE-RAGE signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tackling phlegm and improving blood circulation is vital in the treatment of ischemic stroke (IS), culminating in the development of Zhongfeng Decoction (ZFD), a method grounded in this approach and serving as an effective therapy for IS. Nonetheless, the defensive mechanism of the ZFD in preventing cerebral ischemia-reperfusion damage remains ambiguous. AIM OF THE STUDY: Determine the active ingredients in ZFD that have neuroprotective effects, and identify its mechanism of action against IS. MATERIALS AND METHODS: A cerebral ischemia model in rats was developed, utilizing TTC, Nissl staining, and an oxidative stress kit to evaluate the neuroprotective impact of ZFD on this rat model. Following this, an amalgamation of LC-MS and network pharmacology techniques was employed to pinpoint potential active components, primary targets, and crucial action mechanisms of ZFD in treating IS. Finally, key targets and signaling pathways were detected using qRT-PCR, ELISA, Western blotting, electron microscopy, and other methods. RESULTS: Through LC-MS and network analysis, 15 active ingredients and 6 hub targets were identified from ZFD. Analysis of pathway enrichment revealed that ZFD predominantly engages in the AGE-RAGE signaling route. Kaempferol, quercetin, luteolin, baicalein, and nobiletin in ZFD are the main active ingredients for treating IS. In vivo validation showed that ZFD can improve nerve damage in cerebral ischemic rats, reduce the mRNA expression of IL6, SERPINE1, CCL2, and TGFB1 related to inflammation. Furthermore, we also confirmed that ZFD can inhibit the protein expression of AGEs, RAGE, p-IKBα/IKBα, p-NF-κB p65/NF-κB p65, reduce autophagy levels, and thus decrease neuronal apoptosis. CONCLUSIONS: The mechanism of action of ZFD in treating IS primarily includes inflammation suppression, oxidative stress response alleviation, post-stroke cell autophagy and apoptosis regulation, and potential mediation of the AGE-RAGE signaling pathway. This study elucidates how ZFD functions in treating IS, establishing a theoretical basis for its clinical application.

Melastoma dodecandrum lour. Protects against cerebral ischemia-reperfusion injury by ameliorating oxidative stress and endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Melastoma dodecandrum Lour. (MD), a traditional Chinese medicine used by the She ethnic group, has been used to treat cerebral ischemia-reperfusion (CIR) injury due to its efficacy in promoting blood circulation and removing blood stasiss; however, the therapeutic effects and mechanisms of MD in treating CIR injury remain unclear. AIM: To investigate the protective effects of MD on CIR injury, in addition to its impact on oxidative stress, endoplasmic reticulum (ER) stress, and cell apoptosis. MATERIALS AND METHODS: The research was conducted using both cell experiments and animal experiments. The CCK-8 method, immunofluorescence staining, and flow cytometry were used to analyze the effects of MD-containing serum on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cell viability, reactive oxygen species (ROS) clearance, anti-inflammatory, neuroprotection and inhibition of apoptosis. Furthermore, 2,3,5-Triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, Nissl staining, and immunohistochemistry were used to detect infarct size, pathological changes, Nissl corpuscula and neuronal protein expression in middle cerebral artery occlusion (MCAO) rats. Polymerase chain reaction and Western Blotting were conducted in cell and animal experiments to detect the expression levels of ER stress-related genes and proteins. RESULTS: The MD extract enhanced the viability of PC12 cells under OGD/R modeling, reduced ROS and IL-6 levels, increased MBP levels, and inhibited cell apoptosis. Furthermore, MD improved the infarct area in MCAO rats, increased the number of Nissl bodies, and regulated neuronal protein levels including Microtubule-Associated Protein 2 (MAP-2), Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP), and Neurofilament 200 (NF200). Additionally, MD could regulate the expression levels of oxidative stress proteins malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Both cell and animal experiments demonstrated that MD could inhibit ER stress-related proteins (GRP78, ATF4, ATF6, CHOP) and reduce cell apoptosis. CONCLUSION: This study confirmed that the therapeutic mechanism of the MD extract on CIR injury was via the inhibition of oxidative stress and the ER stress pathway, in addition to the inhibition of apoptosis.

SYK promotes the formation of neutrophil extracellular traps by inducing PKM2 nuclear translocation and promoting STAT3 phosphorylation to exacerbate hepatic ischemia-reperfusion injury and tumor recurrence.

BACKGROUND: At present, hepatic ischemia-reperfusion injury (IRI) is an important complication of partial hepatectomy and liver transplantation, and it is an important cause of poor prognosis. Spleen tyrosine kinase(SYK) plays an important role in a variety of signaling pathways in the liver, but its role in hepatic IRI is still unclear. This study aims to investigate the role and mechanism of SYK in hepatic IRI and tumor recurrence. METHODS: We first observed the activation of SYK in the liver of mice in response to hepatic IRI. Subsequently, Pharmacological inhibitions of SYK were used to evaluated the effect of SYK on neutrophil recruitment and NETosis, and further explored the effect of SYK on IRI and tumor recurrence. RESULTS: Our study shows that SYK is activated in response to hepatic IRI and aggravates liver injury. On the one hand, neutrophils SYK during the early stage of liver reperfusion increases neutrophil extracellular traps (NETs) production by promoting Pyruvate kinase M2(PKM2) nuclear translocation leading to upregulation of phosphorylated STAT3, thereby exacerbating liver inflammation and tumor recurrence. On the other hand, macrophages SYK can promote the recruitment of neutrophils and increase the activation of NLRP3 inflammasome and IL1ß, which further promotes the formation of NETs. CONCLUSIONS: Our study demonstrates that neutrophil and macrophage SYK synergistically promote hepatic IRI and tumor recurrence, and SYK may be a potential target to improve postoperative hepatic IRI and tumor recurrence.

Mild hypothermia therapy alleviates neuronal damage and repairs cerebral ischemia-reperfusion injury through the SIRT1/AMPK pathway.

Cerebrovascular disease, one of the high-risk diseases worldwide, is high in morbidity, disability, mortality, and recurrence rates, which brings many harms to human beings such as physical and mental harm, economic losses, and impairment of social relations. Cerebral ischemia-reperfusion injury (CIRI) is one of the most common pathological manifestations, with mild hypothermia therapy being the most commonly used treatment in clinical practice. In this study, the research team established a CIRI animal model and found that the neuronal apoptosis rate was significantly increased, accompanied by significant ferroptosis, increased inflammation and oxidative stress damage in brain tissue, and obviously inhibited SIRT1/AMPK pathway. However, after mild hypothermia treatment, the pathological changes of CIRI rats were significantly reversed, and the SIRT1/AMPK pathway was reactivated. Therefore, mild hypothermia may achieve the purpose of CIRI repair by activating the SIRT1/AMPK signaling pathway, and targeted regulation of the SIRT1/AMPK signaling pathway may be a research direction for optimizing mild hypothermia therapy or developing new treatment plans for CIRI.

Spatial transcriptomics defines injury specific microenvironments and cellular interactions in kidney regeneration and disease.

Kidney injury disrupts the intricate renal architecture and triggers limited regeneration, together with injury-invoked inflammation and fibrosis. Deciphering the molecular pathways and cellular interactions driving these processes is challenging due to the complex tissue structure. Here, we apply single cell spatial transcriptomics to examine ischemia-reperfusion injury in the mouse kidney. Spatial transcriptomics reveals injury-specific and spatially-dependent gene expression patterns in distinct cellular microenvironments within the kidney and predicts Clcf1-Crfl1 in a molecular interplay between persistently injured proximal tubule cells and their neighboring fibroblasts. Immune cell types play a critical role in organ repair. Spatial analysis identifies cellular microenvironments resembling early tertiary lymphoid structures and associated molecular pathways. Collectively, this study supports a focus on molecular interactions in cellular microenvironments to enhance understanding of injury, repair and disease.

Neutrophil extracellular traps and citrullinated fibrinogen contribute to injury in a porcine model of limb ischemia and reperfusion.

Background: Ischemia/reperfusion injury (IRI) is a complex pathological process, triggered by the restoration of blood flow following an interrupted blood supply. While restoring the blood flow is the only option to salvage the ischemic tissue, reperfusion after a prolonged period of ischemia initiates IRI, triggering a cascade of inflammatory responses ultimately leading to neutrophil recruitment to the inflamed tissue, where they release neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin and neutrophilic proteins, including peptidyl-arginine deiminase 2 and 4 (PAD2, PAD4), that, once outside, can citrullinate plasma proteins, irreversibly changing their conformation and potentially their function. While the involvement of NETs in IRI is known mainly from rodent models, we aimed to determine the effect of NET formation and especially PADs-mediated extracellular protein citrullination in a porcine model of limb IRI. Methods: We conducted our study on amputated pig forelimbs exposed to 1 h or 9 h of ischemia and then reperfused in vivo for 12 h. Limb weight, edema formation, compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Fibrin tissue deposition, complement deposition and NETs were investigated by immunofluorescence. Citrullinated plasma proteins were immunoprecipitated and citrullinated fibrinogen was identified in the plasma by Western blot and in the tissue by immunofluorescence and Western blot. Results: Our data consolidate the involvement of NETs in a porcine model of limb IRI, correlating their contribution to damage extension with the duration of the ischemic time. We found a massive infiltration of NETs in the group subjected to 9 h ischemia compared to the 1 h and citrullinated fibrinogen levels, in plasma and tissue, were higher in 9 h ischemia group. We propose fibrinogen citrullination as one of the mechanisms contributing to the worsening of IRI. NETs and protein citrullination represent a potential therapeutic target, but approaches are still a matter of debate. Here we introduce the idea of therapeutic approaches against citrullination to specifically inhibit PADs extracellularly, avoiding the downstream effects of hypercitrullination and keeping PADs' and NETs' intracellular regulatory functions.

RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1.

One of the most critical axes for cell fate determination is how cells respond to excessive reactive oxygen species (ROS)-oxidative stress. Extensive lipid peroxidation commits cells to death via a distinct cell death paradigm termed ferroptosis. However, the molecular mechanism regulating cellular fates to distinct ROS remains incompletely understood. Through siRNA against human receptor-interacting protein kinase (RIPK) family members, we found that RIPK4 is crucial for oxidative stress and ferroptotic death. Upon ROS induction, RIPK4 is rapidly activated, and the kinase activity of RIPK4 is indispensable to induce cell death. Specific ablation of RIPK4 in kidney proximal tubules protects mice from acute kidney injury induced by cisplatin and renal ischemia/reperfusion. RNA sequencing revealed the dramatically decreased expression of acyl-CoA synthetase medium-chain (ACSM) family members induced by cisplatin treatment which is compromised in RIPK4-deficient mice. Among these ACSM family members, suppression of ACSM1 strongly augments oxidative stress and ferroptotic cell death with induced expression of ACS long-chain family member 4, an important component for ferroptosis execution. Our lipidome analysis revealed that overexpression of ACSM1 leads to the accumulation of monounsaturated fatty acids, attenuation of polyunsaturated fatty acid (PUFAs) production, and thereby cellular resistance to ferroptosis. Hence, knockdown of ACSM1 resensitizes RIPK4 KO cells to oxidative stress and ferroptotic death. In conclusion, RIPK4 is a key player involved in oxidative stress and ferroptotic death, which is potentially important for a broad spectrum of human pathologies. The link between the RIPK4-ASCM1 axis to PUFAs and ferroptosis reveals a unique mechanism to oxidative stress-induced necrosis and ferroptosis.

ANKRD1 aggravates renal ischaemia‒reperfusion injury via promoting TRIM25-mediated ubiquitination of ACSL3.

BACKGROUND: Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. METHODS: The mouse model of IRI-AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation-mass spectrometry was performed to identify potential ANKRD1-interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. RESULTS: First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno-associated virus (rAAV9)-mitigated ischaemia/reperfusion-induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti-ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post-translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63-linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. CONCLUSIONS: Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI-AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. KEY POINTS/HIGHLIGHTS: Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro. ANKRD1 knockdown mitigates kidney damage and preserves renal function. Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI. ANKRD1 promotes acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway. The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1-mediated ubiquitination of ACSL3.

Neuroprotective effects of salvianolic acids combined with Panax notoginseng saponins in cerebral ischemia/reperfusion rats concerning the neurovascular unit and trophic coupling.

BACKGROUND: The neurovascular unit (NVU) and neurovascular trophic coupling (NVTC) play a key regulatory role in brain injury caused by ischemic stroke. Salvianolic acids (SAL) and Panax notoginseng saponins (PNS) are widely used in China to manage ischemic stroke. Neuroprotective effects of SAL and PNS, either taken alone or in combination, were examined in this research. METHODS: Wistar rats were randomly divided into the following groups: Sham group (Sham), cerebral ischemia/reperfusion group (I/R), I/R with SAL group (SAL), I/R with PNS group (PNS), I/R with SAL combined with PNS (SAL + PNS), and I/R with edaravone group (EDA). Treatment was administered once daily for two days after modeling of middle cerebral artery occlusion/reperfusion (MCAO/R). RESULTS: Compared with the I/R group, SAL, PNS, or SAL + PNS treatment reduced infarct size, improved neurological deficit score, reduced Evans blue extravasation, increased expression of CD31 and tight junction proteins (TJs), including zonula occludens-1 (ZO-1), zonula occludens-2 (ZO-2), and junctional adhesion molecule-1 (JAM-1). Furthermore, SAL, PNS, or SAL + PNS suppressed the activations of microglia and astrocyte and led to the amelioration of neuron and pericyte injury. Treatment also inhibited NVU dissociation of GFAP/PDGFRß and Collagen IV/GFAP while upregulated the expression level of BDNF/TrkB and BDNF/NeuN. CONCLUSIONS: SAL and PNS have significantly remedied structural and functional disorders of NVU and NVTC in I/R injury. These effects were more pronounced when SAL and PNS were combined than when used separately.

[Buyang Huanwu Decoction attenuates cerebral ischemia-reperfusion injury in rats via miR-26a-5p mediated PTEN/PI3K/Akt signaling pathway].

This study aims to investigate the mechanism of Buyang Huanwu Decoction in treatment of cerebral ischemia-reperfusion injury in rats. A total of 180 SD rats were randomly divided into 5 different groups: sham group, model group, Buyang Huanwu Decoction group, Buyang Huanwu Decoction + miR-26a-5p agomir(agomir) group, Buyang Huanwu Decoction + miR-26a-5p agomir negative control(agomir NC) group. There were 36 rats in each group. Each group was then subdivided into three subgroups for the duration of reperfusion(3, 7, 14 d). A ligature-induced middle cerebral artery occlusion(MCAO) model was carried out on all groups other than sham group. Reperfusion was performed following ischemia for 90 min. Buyang Huanwu Decoction group, agomir group, and agomir NC group were given Buyang Huanwu Decoction twice daily by gavage 24 h after the formation of the model. Sham group and model group were given an equal amount of physiological saline by gavage until the day before sacrifice. At 24 h after ischemia induction, miR-26a-5p agomir was injected into the lateral ventricle in agomir group, miR-26a-5p NC in agomir NC group, and equal amounts of physiological saline in the other groups. 24 h after ischemia induction, BrdU was intraperitoneally injected once daily until the day before sacrifice. Modified neurological severity score(mNSS) was used to evaluate neurological deficits, 2,3,5-triphenyltetrazolium chloride(TTC) staining was used to determine the cerebral infarct volume, TUNEL staining was used to assess the apoptosis of parenchymal ischemic brain tissue, and double immunofluorescence staining was used to examine BrdU/NeuN double positive neurons in the parenchymal ischemic brain tissue to evaluate the neuronal regeneration. We employed a luciferase reporter assay to identify and validate that the target gene of miR-26a-5p is PTEN. Real-time quantitative polymerase chain reaction(RT-qPCR) was used to assess gene expression levels of PTEN and miR-26a-5p and Western blot to assess the protein levels of PTEN, PI3K, p-PI3K, Akt, and p-Akt. The results revealed that compared with model group, Buyang Huanwu Decoction treatment promoted neural function recovery, reduced the cerebral infarct volume, increased the number of BrdU~+/NeuN~+ neurons, upregulated the expression of miR-26a-5p, regulated the PTEN/PI3K/Akt signaling pathway, and promoted neuronal regeneration in the cerebral ischemia-reperfusion rats. These effects were significantly enhanced after lateral ventricle injection of miR-26a-5p agomir. The findings prove that Buyang Huanwu Decoction treatment can promote neural function recovery, reduce the cerebral infarct volume, and promote neuronal regeneration in a cerebral ischemia-reperfusion rat model, which is likely to be achieved via miR-26a-5p mediated PTEN/PI3K/Akt signaling pathway.

Phillygenin attenuates cell apoptosis and microglia activation in cerebral ischaemia-reperfusion rats through activation of peroxisome proliferator-activated receptor γ.

Ischaemic stroke is a common condition that can lead to cerebral ischaemia-reperfusion injury. Phillygenin (PHI), a natural bioactive compound derived from Forsythia suspensa, has been shown to play a crucial role in regulating inflammation across various diseases. However, its specific regulatory effects in ischaemic stroke progression remain unclear. In this study, we established a middle cerebral artery occlusion (MCAO) rat model. Treatment with PHI (50 or 100 mg/kg) significantly reduced cerebral infarction in MCAO rats. PHI treatment also mitigated the increased inflammatory response observed in these rats. Additionally, PHI suppressed microglial activation by reducing iNOS expression, a marker of M1-type polarization of microglia, and attenuated increased brain tissue apoptosis in MCAO rats. Furthermore, PHI's anti-inflammatory effects in MCAO rats were abrogated upon co-administration with GW9662, a peroxisome proliferator-activated receptor γ (PPARγ) inhibitor. In summary, PHI attenuated microglial activation and apoptosis in cerebral ischaemia-reperfusion injury through PPARγ activation, suggesting its potential as a therapeutic agent for mitigating cerebral ischaemia-reperfusion injury.

Therapeutic and Mechanistic Effects of Granulocyte Colony Stimulating Factor and Neurotrophin Receptor on Cerebral Ischemia-Reperfusion Injury: An Experimental Pilot Study.

OBJECTIVE: To study the therapeutic effect and protective mechanism of granulocyte colony stimulating factor (G-CSF) and neurotrophin receptor (NTR) on cerebral ischemia-reperfusion injury. METHODS: Rat models of permanent focal middle cerebral artery occlusion (MCAO) were constructed by using a modified suture method, and the rats were assigned into three groups such as treatment group (the rats were injected with mixed G-CSF and NTR once), sham operation group and PBS control group. The volume of the cerebral infarction was detected using Triphenyltetrazolium Chloride (TTC) staining method; the motor function in rats was evaluated; and qRT-PCR detection, double immunofluorescence histochemistry and immunohistochemistry were performed to observe various effects. RESULTS: After G-CSF and NTR treatment, the infarct volume induced by MCAO in the treatment group was significantly lower than that in the PBS control group (P<0.05). The motor function in the treatment group was significantly improved on day 7 and day 14 compared to the PBS control group (P<0.05). The levels of MCP-1, TNF-α, TGF-ß and IL-10 mRNA in the treatment group decreased by 22% compared with PBS control group, and the difference was statistically significant (P<0.05). The Bcl-2 protein level in the treatment group was greater than that in the PBS control group, while the Bax level in the treatment group was lower than in the control group; and both the differences were statistically significant (P<0.05). The number of BrdU + cells in the treatment group was significantly greater than that in the PBS control group (P<0.05). CONCLUSION: G-CSF can promote the regeneration of neurons, promote the formation of new blood vessels, promote the reconstruction of neural network in rat MCAO models through anti apoptosis, anti-inflammation and mobilization of bone marrow hematopoietic cells to exert its powerful protective effect on neurons, and contribute to the repair of neural function and improvement of prognosis.

Renal-Targeted Drug Delivery by Chitosan Oligosaccharide Micelles with HSA-Enriched Protein Corona for the Treatment of Ischemia/Reperfusion-Induced Acute Kidney Injury.

Renal-specific nanoparticulate drug delivery systems have shown great potential in reducing systemic side effects and improving the safety and efficacy of treatments for renal diseases. Here, stearic acid-grafted chitosan oligosaccharide (COS-SA) was synthesized as a renal-targeted carrier due to the high affinity of the 2-glucosamine moiety on COS to the megalin receptor expressed on renal proximal tubular epithelial cells. Specifically, COS-SA/CLT micelles were prepared by encapsulating celastrol (CLT) with COS-SA, and different proportions of human serum albumin (HSA) were then adsorbed onto its surface to explore the interaction between the protein corona and cationic polymeric micelles. Our results showed that a multilayered protein corona, consisting of an inner "hard" corona and an outer "soft" corona, was formed on the surface of COS-SA/CLT@HSA8, which was beneficial in preventing its recognition and phagocytosis by macrophages. The formation of HSA protein corona on COS-SA/CLT micelles also increased its accumulation in the renal tubules. Furthermore, the electropositivity of COS-SA/CLT micelles affected the conformation of adsorbed proteins to various degrees. During the adsorption process, the protein corona on the surface of COS-SA/CLT@HSA1 was partially denatured. Overall, COS-SA/CLT and COS-SA/CLT@HSA micelles demonstrated sufficient safety with renal targeting potential, providing a viable strategy for the management of ischemia/reperfusion-induced acute kidney injury.

Unveiling the Renoprotective Mechanisms of Schisandrin B in Ischemia-Reperfusion Injury Through Transcriptomic and Pharmacological Analysis.

Objective: This study investigates the targets, pathways, and mechanisms of Schisandrin B (Sch B) in alleviating renal ischemia-reperfusion injury (RIRI) using RNA sequencing and network pharmacology. Methods: The effects of Sch B on RIRI were assessed using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining, along with measurements of blood creatinine and urea nitrogen (BUN). Differential gene expression in mouse models treated with RIRI and Sch B+RIRI was analyzed through RNA-Seq. Key processes, targets, and pathways were examined using network pharmacology techniques. The antioxidant capacity of Sch B was evaluated using assays for reactive oxygen species (ROS), mitochondrial superoxide, and JC-1 membrane potential. Molecular docking was employed to verify the interactions between key targets and Sch B, and the expression of these targets and pathway was confirmed using qRT-PCR, Western blot, and immunofluorescence. Results: Sch B pre-treatment significantly reduced renal pathological damage, inflammatory response, and apoptosis in a mouse RIRI model. Pathological damage scores dropped from 4.33 ± 0.33 in the I/R group to 2.17 ± 0.17 and 1.5 ± 0.22 in Sch B-treated groups (p < 0.01). Creatinine and BUN levels were also reduced (from 144.6 ± 21.05 µmol/L and 53.51 ± 2.34 mg/dL to 50.44 ± 5.61 µmol/L and 17.18 ± 0.96 mg/dL, p < 0.05). Transcriptomic analysis identified four key targets (AKT1, ALB, ACE, CCL5) and the PI3K/AKT pathway. Experimental validation confirmed Sch B modulated these targets, reducing apoptosis and oxidative stress, and enhancing renal recovery. Conclusion: Sch B reduces oxidative stress, inflammation, and apoptosis by modulating key targets such as AKT1, ALB, ACE, and CCL5, while activating the PI3K/AKT pathway, leading to improved renal recovery in RIRI.

Protective effects of atractylenolide III on oxygen-glucose-deprivation/reperfusion-induced injury in HT22 cells.

BACKGROUND: Atractylenolide III (ATL III) is a natural bioactive compound, that possesses anti-inflammatory, antioxidant, and neuroprotective properties. However, whether ATL III can protect against neuronal injury induced by cerebral ischemia/reperfusion (I/R) have not yet been studied. This study aimed to investigate the protective effects of ATL III on neuronal injury using an oxygen-glucose deprivation/reperfusion (OGD/R) model in HT22 cells. METHODS: Establishment of OGD/R model to induce HT22 cell injury in vitro. Cell viability, live-dead cell staining, oxidative stress levels, and pro-inflammatory cytokine levels were detected using kits. Cell apoptosis was observed by flow cytometry, and the expression of Bax, Bcl-2, and Caspase-3 proteins was detected by western blot. RESULTS: ATL III significantly alleviates OGD/R-induced cell injury, as evidenced by the increased cell viability and reduced apoptosis rate. ATL III increased the levels of superoxide dismutase (SOD) and glutathione (GSH), while reducing malondialdehyde (MDA), reactive oxygen species (ROS), and the levels of TNF-α, IL-1ß, and IL-6. The protein expression of Bax and Caspase-3 was downregulated, while Bcl-2 expression was upregulated by ATL III. CONCLUSION: ATL III as a potential therapeutic agent for reducing neuronal injury by mitigating oxidative stress, apoptosis, and inflammation.

Rehmanniae Radix Praeparata aqueous extract improves hepatic ischemia/reperfusion injury by restoring intracellular iron homeostasis.

Hepatic ischemia/reperfusion injury (HIRI) is a common pathophysiological condition occurring during or after liver resection and transplantation, leading to hepatic viability impairment and functional deterioration. Recently, ferroptosis, a newly recognized form of programmed cell death, has been implicated in IRI. Rehmanniae Radix Praeparata (RRP), extensively used in Chinese herbal medicine for its hepatoprotective, anti-inflammatory, and antioxidant properties, presents a potential therapeutic approach. However, the mechanisms by which RRP mitigates HIRI, particularly through the regulation of ferroptosis, remain unclear. In this study, we developed a HIRI mouse model and monocrotaline (MCT)- and erastin-induced in vitro hepatocyte injury models. We conducted whole-genome transcriptome analysis to elucidate the protective effects and mechanisms of RRP on HIRI. The RRP aqueous extract was characterized by the presence of acteoside, rehmannioside D, and 5-hydroxymethylfurfural. Our results demonstrate that the RRP aqueous extract ameliorated oxidative stress, reduced intracellular iron accumulation, and attenuated HIRI-induced liver damage. Additionally, RRP significantly inhibited hepatocyte death by restoring intracellular iron homeostasis both in vivo and in vitro. Mechanistically, the RRP aqueous extract reduced intrahepatocellular iron accumulation by inhibiting ZIP14-mediated iron uptake, promoting hepcidin- and ferroportin-mediated iron efflux, and ameliorating mitochondrial iron aggregation through upregulation of Cisd1 expression. Moreover, siRNA-mediated inhibition of hamp synergistically enhanced the RRP aqueous extract's inhibitory effect on ferroptosis. In conclusion, our study elucidates the mechanisms by which RRP aqueous extracts alleviate HIRI, highlighting the restoration of iron metabolic balance. These findings position RRP as a promising candidate for clinical intervention in HIRI treatment.

Evaluation of Endoplasmic Reticulum Stress in an Experimental Intestinal Ischemia-Reperfusion Model in Rats: The Role of Ozone Therapy and Trimetazidine.

AIM: The objective of the study was to assess the impact of ozone (O3) and trimetazidine on the intestines following ischemia-reperfusion (I/R) injury through the investigation of endoplasmic reticulum stress. METHODS: Forty Sprague Dawley rats were separated into five groups. The groups were named as follows: control, O3, I/R, I/R + trimetazidine (TMZ), and I/R + O3. The control group had laparotomy and exploration of the superior mesenteric artery (SMA) only. Furthermore, alongside laparotomy and SMA exploration, an intraperitoneal (i.p.) administration of a 0.7 mg/kg ozone-oxygen (O3-O2) combination was given to the O3 group. In the experimental groups, the SMA was blocked with the silk suture ligation technique for a duration of 1 h and then restored to normal blood flow for another hour. In the I/R + O3 group, ozone was delivered i.p. at a dosage of 0.7 mg/kg, 30 min after ischemia. In the I/R + TMZ group, a dose of 20 mg/kg/day of trimetazidine was administered orally via gavage for a duration of 7 days, beginning 1 week prior to the induction of ischemia. Intestinal tissues were taken to assess indicators of intestinal mucosal injury and oxidative stress. RESULTS: The level of the lipid peroxidation marker malondialdehyde (MDA) was significantly reduced in the experimental groups as compared to the I/R group (p < 0.05). The experimental groups had considerably greater levels of glutathione (GSH), which reflects antioxidant capacity, compared to the I/R group (p < 0.05). Nevertheless, the concentration of GSH was observed to be increased in the I/R + O3 group in comparison to the I/R + TMZ group (p < 0.05). The histopathological damage score showed a substantial decrease in the experimental groups as compared to the I/R group (p < 0.05). The I/R + O3 group had the lowest injury score. The experimental groups exhibited significantly reduced positivity of the endoplasmic reticulum (ER) stress markers C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP)-78 compared to the I/R group (p < 0.05). CONCLUSIONS: The findings provide evidence for the potential advantages of utilizing ozone therapy in the treatment of intestinal ischemia-reperfusion injury. Additionally, they propose that ozone should be assessed in more extensive clinical trials in the future as a therapeutic agent that can disrupt endoplasmic reticulum stress.

Role of caspase-11 non-canonical inflammasomes in retinal ischemia/reperfusion injury.

BACKGROUND: Retinal ischemia/reperfusion (IR) injury is a common pathological process in many ophthalmic diseases. Interleukin-1ß (IL-1ß) is an important inflammatory factor involved in the pathology of retinal IR injury, but the mechanism by which IL-1ß is regulated in such injury remains unclear. Caspase-11 non-canonical inflammasomes can regulate the synthesis and secretion of IL-1ß, but its role in retinal IR injury has not been elucidated. This study aimed to evaluate the role of caspase-11 non-canonical inflammasomes in retinal IR injury. METHODS: Retinal IR injury was induced in C57BL/6J mice by increasing the intraocular pressure to 110 mmHg for 60 min. The post-injury changes in retinal morphology and function and in IL-1ß expression were compared between caspase-11 gene knockout (caspase-11-/-) mice and wild-type (WT) mice. Morphological and functional changes were evaluated using hematoxylin-eosin staining and retinal whole mount staining and using electroretinography (ERG), respectively. IL-1ß expression in the retina was measured using enzyme-linked immunosorbent assay (ELISA). The levels of caspase-11-related protein were measured using western blot analysis. The location of caspase-11 in the retina was determined via immunofluorescence staining. Mouse type I astrocytes C8-D1A cells were used to validate the effects of caspase-11 simulation via hypoxia in vitro. Small-interfering RNA targeting caspase-11 was constructed. Cell viability was evaluated using the MTT assay. IL-1ß expression in supernatant and cell lysate was measured using ELISA. The levels of caspase-11-related protein were measured using western blot analysis. RESULTS: Retinal ganglion cell death and retinal edema were more ameliorated, and the ERG b-wave amplitude was better after retinal IR injury in caspase-11-/- mice than in WT mice. Further, caspase-11-/- mice showed lower protein expressions of IL-1ß, cleaved caspase-1, and gasdermin D (GSDMD) in the retina after retinal IR injury. Caspase-11 protein was expressed in retinal glial cells, and caspase-11 knockdown played a protective role against hypoxia in C8-D1A cells. The expression levels of IL-1ß, cleaved caspase-1, and GSDMD were inhibited after hypoxia in the si-caspase-11 constructed cells. CONCLUSIONS: Retinal IR injury activates caspase-11 non-canonical inflammasomes in glial cells of the retina. This results in increased protein levels of GSDMD and IL-1ß and leads to damage in the inner layer of the retina.

Specific mode electroacupuncture stimulation opens the blood-brain barrier of the infarcted border zone in rats during MCAO/R recovery via modulation of tight junction protein expression by VEGFA and NF-κB.

The blood-brain barrier (BBB) strictly limits the entry of most exogenous therapeutic drugs into the brain, which brings great challenges to the drug treatment of refractory central diseases, including the treatment of ischemic stroke. Our previous studies have shown that specific mode electroacupuncture stimulation (SMES) can temporarily open the BBB, but with the mechanisms largely unknown. This study explored whether SMES opens the BBB in the infarcted border zone of rats during middle cerebral artery occlusion/reperfusion recovery, and whether this is related to p65 or vascular endothelial growth factor A (VEGFA) modulation of tight junction protein expression through in vivo and in vitro studies. Evans blue, FITC-dextran, mouse-derived nerve growth factor (NGF), and transendothelial electrical resistance values were used to evaluate the permeability of the BBB. Additionally, microvascular endothelial cells and astrocytes were utilized for in vitro study. Immunofluorescence, immunohistochemistry, western blot, and ELISA were employed to assess related protein expression. SMES significantly increased vascular permeability for Evans blue and NGF in the infarcted border zone, and increased the expression of VEGFA by activating p-p65, thereby reducing the expression of tight junction proteins Occludin and ZO-1. Correspondingly, oxygen glucose deprivation/reoxygenation activated p-p65 in and induced VEGFA secretion from astrocytes in vitro. Their conditioned medium reduced the expression of Occludin in bEnd.3 cells and increased the permeability of FITC-dextran. The mechanism of SMES opening infarcted border zone BBB is partly related to its actions on p65, VEGFA, and tight junction proteins.