Ischemia-reperfusion Model Research Articles

Development of a new cerebral ischemia reperfusion model of Mongolian gerbils and standardized evaluation system.

The Mongolian gerbil is an excellent laboratory animal for preparing the cerebral ischemia model due to its inherent deficiency in the circle of Willis. However, the low incidence and unpredictability of symptoms are caused by numerous complex variant types of the circle. Additionally, the lack of an evaluation system for the cerebral ischemia/reperfusion (I/R) model of gerbils has shackled the application of this model. We created a symptom-oriented principle and detailed neurobehavioral scoring criteria. At different time points of reperfusion, we analyzed the alteration in locomotion by rotarod test and grip force score, infarct volume by triphenyltetrazolium chloride (TTC) staining, neuron loss using Nissl staining, and histological characteristics using hematoxylin-eosin (H&E) straining. With a successful model rate of 56%, 32 of the 57 gerbils operated by our method harbored typical features of cerebral I/R injury, and the mortality rate in the male gerbils was significantly higher than that in the female gerbils. The successfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion; formed obvious infarction; exhibited typical pathological features, such as tissue edema, neuronal atrophy and death, and vacuolated structures; and were partially recovered with the extension of reperfusion time. This study developed a new method for the unilateral common carotid artery ligation I/R model of gerbil and established a standardized evaluation system for this model, which could provide a new cerebral I/R model of gerbils with more practical applications.

Long-Term Protective Effects of Lamotrigine in a Rat Ovarian Ischemia-Reperfusion Model

Ovarium torsion is a gynecological emergency that is common in women of reproductive age and requires early diagnosis and intervention. In this study, we aimed to investigate the long-term anatomical, histological and biochemical protective effects of lamotrigine in ovariums in the ischemia – reperfusion (I–R) model created experimentally in rats. A total of 40 female Sprague-Dawley rats, 14 weeks old, weighing 220-270 grams were used in the study. The subjects were randomly distributed to form 4 groups named SHAM group, I - R group, I - R + Lamotrigine (LTG) group and R + LTG group. Under general anesthesia, the ovaries of the rats were reached and ischemia was created for 3hours with vascular clamps. 20mg / kg LTG was administered intraperitoneally (ip.) to group 3 30minutes before ischemia and to group 4 30minutes before reperfusion. At the third hour of ischemia, the vascular clamps were opened and the abdomen of the rats was closed according to the surgical procedure. The rats were followed up for 28 days postoperatively and the ovarium tissues taken on the 28th day were examined anatomically and histologically. Biochemically, estradiol (E2), follicle stimulating hormone (FSH) and antimullerian hormone (AMH) levels were measured from blood samples taken from their hearts. Granulosa cells with diffuse vaculations were observed in degenerative follicles in group I-R. Again in this group, severe hemorrhage, fibrosis and edematous areas were observed in the ovarium stroma (Ovarian Histopathological Scoring (OHS): 7). In the I - R + LTG group, OHS was statistically significantly lower than the I - R group (OHS: 2; p <0.000). In the R + LTG group, although the OHS score was calculated to be lower than the I - R group, there was no statistically significant difference (OHS: 6; p> 0.05). The protective effect of LTG against experimentally created ischemia and reperfusion damage was determined anatomically and histologically. No protective effect of LTG was observed in terms of FSH, E2 and AMH values measured from the blood sera of rats. These findings may provide a basis for future studies using LTG to treat ovarian ischemia-reperfusion injury.

Piceatannol protects against myocardial ischemia/reperfusion injury by inhibiting ferroptosis via Nrf-2 signaling-mediated iron metabolism

Myocardial tissue ischemia damages myocardial cells. Although reperfusion is an effective technique to rescue myocardial cell damage, it may also exacerbate myocardial cell damage. Ferroptosis, an iron-dependent cell death, occurs following myocardial ischemia-reperfusion (I/R). Piceatannol (PCT) is a natural stilbene compound with excellent antioxidant properties that protect against I/R injury and exerts protective effects against ferroptosis-induced cardiomyocytes following I/R injury; however, the exact mechanism remains to be elucidated. PurposeThis study aims to investigate the protective effect and mechanism of PCT on myocardial ischemia-reperfusion injury. MethodsAn ischemia-reperfusion model was established via ligation of the left anterior descending branch of mice's hearts and hypoxia-reoxygenation (H/R) of cardiomyocytes. ResultsDuring ischemia-reperfusion, Nuclear factor E2-related factor 2 (Nrf-2) expression was downregulated, the left ventricular function was impaired, intracellular iron and lipid peroxidation product levels were elevated, and cardiomyocytes underwent ferroptosis. Furthermore, ferroptosis was enhanced following treatment with an Nrf-2 inhibitor. After PCT treatment, Nrf-2 expression significantly increased, intracellular ferrous ions and lipid peroxidation products significantly reduced, Ferroportin1 (FPN1) expression increased, and transferrin receptor-1 (TfR-1) expression was inhibited. ConclusionsPCT regulates iron metabolism through Nrf-2 to protect against myocardial cell ferroptosis induced by myocardial I/R injury.

Comprehensive bile acid pool analysis during ex-vivo liver perfusion in a porcine model of ischemia-reperfusion injury

Bile acids (BA) are key for liver regeneration and injury. This study aims at analyzing the changes in the BA pool induced by ischemia-reperfusion (IRI) and investigates the impact of hypothermic oxygenated perfusion (HOPE) on the BA pool compared to static cold storage (SCS). In a porcine model of IRI, liver grafts underwent 30 min of asystolic warm ischemia followed by 6 h of SCS (n = 6) ± 2 h of HOPE (n = 6) and 2 h of ex-situ warm reperfusion. The BA pool in bile samples was analyzed with liquid chromatography coupled with tandem mass spectrometry. We identified 16 BA and observed significant changes in response to ischemia-reperfusion, which were associated with both protective and injury mechanisms. Second, HOPE-treated liver grafts exhibited a more protective BA phenotype, characterized by a more hydrophilic BA pool compared to SCS. Key BA, such as GlycoCholic Acid, were identified and were associated with a decreased transaminase release and improved lactate clearance during reperfusion. Partial Least Square-Discriminant Analysis revealed a distinct injury profile for the HOPE group. In conclusion, the BA pool changes with liver graft IRI, and preservation with HOPE results in a protective BA phenotype compared to SCS.

SNX10 promoted liver IR injury by facilitating macrophage M1 polarization via NLRP3 inflammasome activation

BackgroundLiver ischemia reperfusion (IR) injury is a common cause of liver dysfunction in patients post liver partial resection and liver transplantation. However, the cellular defense mechanisms underlying IR are not well understood. Macrophage mediated sterile inflammation plays critical roles in liver IR injury. Sorting nexin (SNX) 10, a member of the SNX family which functions in regulation of endosomal sorting. This study aimed to explore the role of sorting nexin 10 (SNX10) during liver IR injury with a focus on regulating macrophage function. MethodsBoth the gene and protein expression levels of SNX10 were analyzed in human specimens from 10 patients undergoing liver partial resection with ischemic insult and in a mouse model of liver IR. The in vivo effects of SNX10 in liver IR injury and sterile inflammation in mice were investigated. Bone marrow derived macrophages (BMDMs) were used to determine the role of SNX10 in modulating macrophage function in vitro. ResultsIncreased expression of SNX10 was observed both in human specimens and mice livers post IR. SNX10 knockdown alleviated IR induced sterile inflammation and liver damage in mice. SNX10 promoted M1 polarization of macrophage treated with LPS and facilitated inflammatory response by activating NLRP3 inflammasome. ConclusionsWe report for the first time that SNX10 is upregulated in IR-stressed livers. SNX10 activation aggravates liver IR injury and sterile inflammation by facilitating macrophage M1 polarization and inflammatory response suggesting SNX10 as a potential therapeutic target for liver IR injury.

Amplified P2X3 pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia-reperfusion.

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,β-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.

Retracted: Effect of Dihuang Yinzi on Inflammatory Response in Cerebral Ischemia-Reperfusion Model Rats by Regulating Gut Microbiota.

[This retracts the article DOI: 10.1155/2022/3768880.].

Function and mechanism of the human SOD2 gene in mice cerebral ischemia/ reperfusion injury.

To investigate the neuroprotective effects of the SOD2 gene in cerebral ischemia reperfusion injury function and the underlying mechanisms in a mice model of middle cerebral artery ischemia reperfusion. SOD2 transgenic mice were engineered using transcription activator-like effector nucleases, and the genotype was identified using PCR after every three generations. Transgenic and C57BL/6J wild type mice were simultaneously subjected to the middle cerebral artery occlusion model. SOD2 expression in the brain, heart, kidney, and skeletal muscle of transgenic mice was significantly higher than that in the wild type. Following ischemia reperfusion, the infarct volume of wild type mice decreased after treatment with fenofibrate compared to the CMC group. Infarction volume in SOD2 transgenic mice after CMC and fenofibrate treatment was significantly reduced. The recovery of cerebral blood flow in wild type mice treated with fenofibrate was significantly enhanced compared with that in the CMC group. The expression of SOD2 in transgenic mice was significantly higher than that in wild type mice, the neuroprotective role of fenofibrate depends on an increase in SOD2 expression.

Effect of sugammadex, rocuronium and sevoflurane on oxidative stress and apoptosis in cerebral ischemia reperfusion model in rats.

Cerebral ischemia-reperfusion (I/R) injury causes neurological dysfunction and cell death. Sugammadex, as a large molecule, is normally difficult to pass through the blood-brain barrier (BBB). In ischemia, molecules can pass into the brain tissue. In this study, we aimed to evaluate the effect of sugammadex in the presence of cerebral I/R damage in rats with a general anesthesia model with sevoflurane and rocuronium. Rats were divided into 7 groups; Group 1 (Control), Group 2 (Sham), Group 3 (Sevoflurane), Group 4 (Sugammadex), Group 5 (Sevoflurane + Rocuronium), Group 6 (Sevoflurane + Sugammadex), Group 7 (Sevoflurane + Rocuronium + Sugammadex). Brain tissues of rats with cerebral I/R damage with bilateral carotid occlusion were removed. Tissue Malondialdehyde (MDA), Myeloperoxidase (MPO), and Superoxide dismutase (SOD) levels were examined with ELISA and apoptosis was examined by Caspase-3. The number of caspase-3 positive cells decreased the most in Group 4 compared to the other groups. Group 4's mean MDA and MPO levels were lower than Group 2. There was no significant difference in terms of SOD levels. The apoptotic effect of sugammadex was lowest compared to other agent groups, and it did not increase oxidative damage as much as the other groups.

Is there any effect of lidocaine on ischemia/reperfusion injury in testicular torsion? An experimental study.

This experimental study aimed to evaluate the potential protective effects of lidocaine on ischemia-reperfusion injury resulting from testicular torsion/detorsion in rats. A total of 18 male rats were randomized into three groups. Group 1 served as the control group. Group 2 was designed to evaluate testicular ischemia-reperfusion injury using a torsion/detorsion model. In Group 3, the treatment group, a similar ischemia-reperfusion model was used as in Group 2. Additionally, lidocaine at a dose of 15 mg/kg was administered intraperitoneally five minutes before reperfusion. Blood biochemical analyses and testicular histopathological evaluations were conducted. Blood biochemical analysis showed that malondialdehyde (MDA) and protein carbonyl (PC) levels were significantly higher in Group 2 compared to the other groups (p<0.001 and p=0.008, respectively). Proinflammatory cytokine levels, including interleu-kin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), were lower in Group 3 than in Group 2 (p<0.001, p=0.007, and p=0.026, respectively). Antioxidant enzyme activities, including glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), were higher in Group 3 compared to Group 2 (p=0.005 and p=0.025, respectively). Histopathological evaluations revealed significant improvements in all testicular damage scores, including hemorrhage, edema, vasocongestion, and inflammation in Group 3 compared to Group 2 (p=0.015, p=0.035, p=0.015, and p=0.034, respectively). Additionally, there was a notable improvement in the Johnsen score in Group 3 compared to Group 2 (p=0.034). Lidocaine, an effective local anesthetic, significantly alleviates the effects of testicular ischemia-reperfusion injury.

A Brain Ischemia-Reperfusion Model for the Study of Tau Phosphorylation and O-GlcNAcylation.

Cerebral ischemia produces a decrease, loss, or instability of the assembly processes in the neuronal cytoskeleton, related to the alteration in the normal processes of phosphorylation of the Tau protein, triggering its hyperphosphorylation and altering the normal processes of formation of neuronal microtubules. Here we describe the methods used to study the impact of middle cerebral artery occlusion (MCAo) on neurological functions and Tau phosphorylation in Wistar rat brain.

Effects of ketamine on penile tissues in an experimental priapism model in rats.

This study aimed to evaluate the histopathological and biochemical effects of ketamine on penile tissues following ischemia-reperfusion injury induced by priapism. Twenty-four male rats were randomized into three groups. Group 1 served as the control group. Group 2 underwent the priapism model to induce ischemia-reperfusion injury. Group 3, the treatment group, experienced a similar ischemia-reperfusion model as Group 2; additionally, 50 mg/kg of ketamine was administered intraperitoneally just before reperfusion. Blood biochemical analyses and penile histopathological evaluations were performed. In Group 3, significant improvements were observed in all histopathological scores, including desquamation, edema, inflammation, and vasocongestion compared to Group 2 (p<0.001). Blood biochemical analyses showed that the malondialdehyde (MDA) levels were recorded as 10 in Group 2, with a significant decrease in Group 3 (p=0.013). Similarly, proinflammatory cytokine levels, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), were found to be suppressed in Group 3 compared to Group 2 (p=0.003, p=0.022, and p=0.028, respectively). Antioxidant enzyme activities, such as glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), were higher in Group 3 compared to Group 2 (p=0.016 and p=0.024, respec-tively). Ketamine is an effective anesthetic agent in alleviating the effects of penile ischemia-reperfusion injury.

The Anti-Apoptotic Effect of Ranolazine on Cerebral Protection during Cardiopulmonary Bypass and Carotid Artery Surgery.

We aimed to determine the usability of ranolazine (Rn) as a neuroprotective during cardiac surgeries and carotid artery interventions where cerebral blood flow is interrupted. Female Wistar albino rats were used. The rats were divided into 4 groups of 8 rats each. The first group (Group 1) was the control group. Group 2 underwent ischemia induction but was not treated with Rn. Group 3 received 25 mg/kg/day and Group 4 50 mg/kg/day Rn intraperitoneally, starting 3 days before ischemia induction. Bilateral carotid arteries were explored and clamped simultaneously. Ischemia was induced for 15 minutes. After 72 hours, the experimental animals were sacrificed. Superoxide dismutase, alkaline phosphatase, and interleukin 6 levels were similar among the 4 groups. Acetylcholine esterase (Group 3: p = 0.007, Group 4: p = 0.002), tumor necrosis factor-alpha (Group 4: p = 0.01), and annexin V (Group 3: p = 0.001) levels were statistically significantly lower in the Rn-treated groups. Malondialdehyde (Group 3: p = 0.003, Group 4: p = 0.009), reduced glutathione (Group 4: p = 0.04), acid phosphatase (Group 3: p = 0.04), noradrenaline (Group 3: p = 0.01), and Bcl-2 (Group 4: p = 0.004) levels were significantly higher in the Rn-treated groups. The results of this study demonstrated the antiapoptotic effect of Rn in a brain ischemia-reperfusion model of rats receiving Rn before the procedure.

Comparison of the protective effects of vanillic and rosmarinic acid on cardiac tissue: Lower limb ischemia-reperfusion model in rats.

Ischemia/reperfusion injury is one of the most challenging postoperative situations in vascular surgery, both in elective procedures with prolonged clamping time and in delayed emergency cases with vascular occlusion. The inflammatory response that develops during ischemia and the oxygen-free radicals that proliferate during reperfusion have detrimental effects on the brain, heart, and kidneys. In this study, we aimed to compare the effects of vanillic and rosmarinic acid in preventing ischemia/reperfusion injury in a lower limb ischemia-reperfusion model in rats. Thirty-two female Sprague-Dawley rats weighing 185-240 g were randomly divided into four groups of eight animals each. Group 1 was designated as the control, Group 2 as ischemia/reperfusion (I/R), Group 3 as ischemia/reperfusion + vanillic acid (I/R + VA), and Group 4 as ischemia/reperfusion + rosmarinic acid (I/R + RA). In all groups except the control, the infrarenal abdominal aorta was clamped, and 60 minutes of ischemia followed by 120 minutes of reperfusion was performed. Vanillic acid was administered intra-abdominally 15 minutes before the start of reperfusion in Group 3, and rosmarinic acid in Group 4. At the end of the reperfusion phase, blood samples and hearts were collected, and the rats were euthanized. Histopathologically, myofibrillar edema, myocytolysis, focal hemorrhages, and infiltration of polymorphonuclear leukocytes (PMNL) in cardiac tissue were examined. Total antioxidant capacity (TAC), total oxidative status (TOS), oxidative stress index (OSI), 8-OH-deoxyguanosine, lactonase, and arylesterase activity were measured in blood samples. Myofibrillar edema was most pronounced in the I/R group and less pronounced in the I/R + VA and I/R + RA groups (p=0.005 and p=0.066, respectively). There was no difference between the ischemia/reperfusion groups regarding myocytolysis, focal hemorrhage, and PMNL infiltration (p>0.99). Among all groups, TOS and OSI were lowest in the control group, while TAC was highest. TAC was similar in the I/R + VA and I/R + RA groups but was significantly higher in these two groups than in the I/R group. The lactonase activity in the I/R + VA group was similar to that in the control group but was significantly higher compared to the I/R and I/R + RA groups. Our study shows that vanillic and rosmarinic acids reduce myofibrillar edema in the heart after lower limb ischemia and increase TAC. However, vanillic acid increases the activity of lactonase, an enzyme known for its antioxidant effect, more than rosmarinic acid.

C/EBPα alleviates hepatic ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress via HDAC1-mediated deacetylation of ATF4.

Hepatic ischemia-reperfusion (IR) injury is a complex systemic process causing a series clinical problem. C/EBPα is a key transcription factor for hepatocyte function, but its role and mechanism in regulating hepatic IR injury are largely unknown. Occluding portal vein and hepatic artery was used to establish a mouse model of hepatic IR injury. C/EBPα expression was decreased in IR-injured liver compared with the sham, accompanied by increased contents of serum alanine transaminase(ALT), aspartate transaminase(AST),high mobility group box-1, and proportion of hepatic cells. Oxygen and glucose deprivation/recovery (OGD/R) was used to establish a cellular hepatic IR model in WRL-68 hepatocytes in vitro, and C/EBPα was overexpressed in the hepatocytes to evaluate its effect on hepatic IR injury. OGD/R promoted oxidative stress, cell apoptosis and endoplasmic reticulum (ER) stress in hepatocytes, which was reversed by C/EBPα overexpression. Then, we found that C/EBPα promoted histone deacetylase 1 (HDAC1) transcription through binding to HDAC1 promoter. Moreover, HDAC1 deacetylated the activating transcription factor 4 (ATF4), a key positive regulator of ERstress. Trichostatin-A(an HDAC inhibitor) or ATF4 overexpression reversed the improvement of C/EBPα on OGD/R-induced ER stress and hepatocyte dysfunction. 4-Phenylbutyric acid(an endoplasmic reticulum stress inhibitor) also reversed the hepatic IR injury induced by ATF4 overexpression. Finally, lentivirus-mediated C/EBPα overexpression vector was applied to administrate hepatic IR mice, and the results showed that C/EBPα overexpression ameliorated IR-inducedhepatic injury, manifesting with reduced ALT/AST, oxidative stress and ER stress. Altogether, our findings suggested that C/EBPα ameliorated hepatic IR injury by inhibiting ER stress via HDAC1-mediated deacetylation of ATF4 promoter.

Increased A1 astrocyte activation-driven hippocampal neural network abnormality mediates delirium-like behavior in aged mice undergoing cardiac surgery.

Delirium is the most common neurological complication after cardiac surgery with adverse impacts on surgical outcomes. Advanced age is an independent risk factor for delirium occurrence but its underlying mechanisms are not fully understood. Although increased A1 astrocytes and abnormal hippocampal networks are involved in neurodegenerative diseases, whether A1 astrocytes and hippocampal network changes are involved in the delirium-like behavior of aged mice remains unknown. In the present study, a mice model of myocardial ischemia-reperfusion mimicking cardiac surgery and various assessments were used to investigate the different susceptibility of the occurrence of delirium-like behavior between young and aged mice and the underlying mechanisms. The results showed that surgery significantly increased hippocampal A1 astrocyte activation in aged compared to young mice. The high neuroinflammatory state induced by surgery resulted in glutamate accumulation in the extrasynaptic space, which subsequently decreased the excitability of pyramidal neurons and increased the PV interneurons inhibition through enhancing N-methyl-D-aspartate receptors' tonic currents in the hippocampus. These further induced the abnormal activities of the hippocampal neural networks and consequently contributed to delirium-like behavior in aged mice. Notably, the intraperitoneal administration of exendin-4, a glucagon-like peptide-1 receptor agonist, downregulated A1 astrocyte activation and alleviated delirium-like behavior in aged mice, while IL-1α, TNF-α, and C1q in combination administered intracerebroventricularly upregulated A1 astrocyte activation and induced delirium-like behavior in young mice. Therefore, our study suggested that cardiac surgery increased A1 astrocyte activation which subsequently impaired the hippocampal neural networks and triggered delirium development.

Enhanced the treatment of ischemic stroke through intranasal temperature-sensitive hydrogels of edaravone and borneol inclusion complex

Since ischemic stroke occurs by a combination of multiple mechanisms, therapies that modulate multiple mechanisms are required for its treatment. The combination of edaravone (EDA) and borneol can significantly ameliorate the symptoms of neurological deficits in cerebral ischemia–reperfusion model in rats. In this study, the solubility of borneol and edaravone was improved by hydroxypropyl-β-cyclodextrin and PEG400. Furthermore, a nasal temperature-sensitive hydrogel containing both edaravone and borneol inclusion complex (EDA-BP TSGS) was developed to overcome the obstacles of ischemic stroke treatment including the obstruction of the blood–brain barrier (BBB) and the unavailability and untimely of intravenous injection. The effectiveness of the thermosensitive hydrogel was investigated in transient middle cerebral artery occlusion/reperfusion model rats (MCAO/R). The results showed that EDA-BP TSGS could significantly alleviate the symptoms of neurological deficits and decrease the cerebral infarct area and the degree of brain damage. In summary, nasal EDA-BP TSGS is a secure and effective brain-targeting formulation that may provide a viable option for the clinical prophylaxis and treatment of ischemic stroke.

M6A methylase WTAP participates in renal ischemia-reperfusion injury by regulating FOXO1 expression

To investigate the expression of WTAP, a m6A methylase, in a mouse model of renal ischemia-reperfusion (I/R) injury and the effect of WTAP knockdown on biological behavior of renal tubular epithelial cells exposed to I/R injury. Sixteen C57BL/6 mice with renal I/R injury or sham operation (n=8) were examined for blood urea nitrogen (BUN) and creatinine (Scr) levels to assess renal function, and renal pathologies were observed with HE staining. The expressions of WTAP and FOXO1 proteins in the kidneys of the mice were detected using immunohistochemistry. Human renal tubular epithelial cells (HK-2) were transfected with si-WTAP or si-NC followed by hypoxia-reoxygenation (H/R) exposure, Protein and mRNA expression were assessed by Western blot and qRT-PCR, and changes and changes in cell viability and apoptosis were assessed using CCK8 assay and TUNEL staining, respectively; LDH release level and caspase-3 activity of the cells were measured using commercial assay kits. FOXO1 m6A modification sites were predicted using SRAMP website (http://www.cuilab.cn/sramp/), and the interaction between WTAP and FOXO1 mRNA was analyzed with RIP experiment; the level of FOXO1 modified by m6A was detected by MeRIP-qPCR. Compared with sham-operated mice, the mice with renal I/R injury showed significantly increased Scr and BUN levels (P < 0.001) and renal expressions of WTAP mRNA and protein (P < 0.001). In cultured HK-2 cells, H/R exposure significantly decreased the cell viability (P < 0.001) and increased cellular LDH release (P < 0.001) and expressions of WTAP mRNA and protein (P < 0.001). WTAP knockdown obviously reduced the cell damage induced by I/R injury and significantly decreased the mRNA and protein levels of FOXO1 in the cells (P < 0.001). RIP experiment confirmed WTAP binding to FOXO1 mRNA, and inhibition of WTAP expression significantly reduced FOXO1 m6A level in HK-2 cells (P < 0.001). WTAP expression is up-regulated in the kidneys of mice with renal I/R injury and in HK-2 cells with H/R exposure. Inhibition of WTAP alleviates H/R-induced apoptotic damage in HK-2 cells possibly by inhibiting FOXO1 expression.

APOBEC-1 deletion enhances cisplatin-induced acute kidney injury

Cisplatin (CP) induces acute kidney injury (AKI) whereby proximal tubules undergo regulated necrosis. Repair is almost complete after a single dose. We now demonstrate a role for Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec-1) that is prominently expressed at the interface between acute and chronic kidney injury (CKD), in the recovery from AKI. Apobec-1 knockout (KO) mice exhibited greater mortality than in wild type (WT) and more severe AKI in both CP- and unilateral ischemia reperfusion (IR) with nephrectomy. Specifically, plasma creatinine (pCr) 2.6 ± 0.70 mg/dL for KO, n = 10 and 0.16 ± 0.02 for WT, n = 6, p < 0.0001 in CP model and 1.34 ± 0.22 mg/dL vs 0.75 ± 0.06, n = 5, p < 0.05 in IR model. The kidneys of Apobec-1 KO mice showed increased necrosis, increased expression of KIM-1, NGAL, RIPK1, ASCL4 and increased lipid accumulation compared to WT kidneys (p < 0.01). Neutrophils and activated T cells were both increased, while macrophages were reduced in kidneys of Apobec-1 KO animals. Overexpression of Apobec-1 in mouse proximal tubule cells protected against CP-induced cytotoxicity. These findings suggest that Apobec-1 mediates critical pro-survival responses to renal injury and increasing Apobec-1 expression could be an effective strategy to mitigate AKI.

Analysis of lipid metabolites derived from gut microbiota in ischemia-reperfusion model.

Disruption of intestinal barrier caused by intestinal ischemia due to hemorrhagic shock is associated with the pathogenesis of multiple organ dysfunction (MOD) after severe trauma. Mesenteric lymph (ML) plays an important role as a route for transporting inflammatory mediators, including lipids. Postbiotics, such as 10-hydroxy-cis-12-octadecenoic acid (HYA), have received much attention as a treatment option. However, the relationship between postbiotics and MOD has yet to be clarified. The aim of the present study was to analyze lipid metabolites derived from gut microbiota in the intestinal ischemia-reperfusion (IR) rat model. Male Sprague-Dawley rats underwent laparotomy, and their ML duct and superior mesenteric artery were exposed. The superior mesenteric artery was clamped for 60 minutes, followed by 120 minutes of reperfusion. The ML and the plasma were collected before and after intestinal IR. Lipids were extracted from plasma and ML, and liquid chromatography-tandem mass spectrometry was performed. The concentration of linoleic acid in plasma samples was not different before and after IR; however, the linoleic acid concentration in the ML samples increased after intestinal IR. Eicosapentaenoic acids and docosahexaenoic related to linoleic acids showed similar changes with IR-induced increase in the ML. The concentration of HYA, a linoleic acid-derived bioactive metabolite produced by gut bacteria, was high in ML samples, while that in plasma samples was low. The relative increase rate of HYA in ML samples after IR was higher than that of the plasma samples (the ML samples: relative increase, 3.23 ± 1.36; the plasma samples: relative increase, 0.95 ± 0.35; n = 3, p = 0.048). The present study demonstrated increased linoleic acids and high concentrations of HYA, lipid metabolites derived from gut bacteria in the ML after intestinal IR. These findings may contribute to clarifying the relation between gut microbiota and MOD after severe trauma.