Metabolomic analysis of swainsonine poisoning in renal tubular epithelial cells.

Locoweed is a poisonous plant widely present in grasslands around the world. Swainsonine (SW), an indole alkaloid that, is the main toxic component of the locoweed. To understand the mechanism of SW-induced toxicity and to delineate the metabolic profile of locoweed poisoning we performed the LC-MS/MS untargeted metabolomic study to analyze metabolites in SW-treated renal tubular epithelial cells (0.8 mg/mL, 12 h) and in order to identify the SW-induced metabolomic changes. The analysis identified 2,563 metabolites in positive ion mode and 1,990 metabolites in negative ion mode. Our results showed that the metabolites were mainly benzenoids, lipids and lipid-like molecules, nucleosides, nucleotides, and analogs, organic acids, and derivatives. The differential metabolites were primarily enriched in pathways involving bile secretion, primary bile acid biosynthesis, riboflavin metabolism, ferroptosis, drug metabolism-cytochrome P450, and primidine metabolism. We have screened out substances such as swainsonine, 3alpha,7alpha-Dihydroxy-5beta-cholestanate, 2-Hydroxyiminostilbene, and glycochenodeoxycholate, which may have the potential to serve as biomarkers for swainsonine poisoning. This study provides insights into the types of metabolomic alteration in renal tubular epithelial cells induced by swainsonine.

Regulatory role of oxidative stress in retrorsine - Induced apoptosis and autophagy in primary rat hepatocytes.

Pyrrolizidine alkaloids (PAs) are a group of naturally occurring alkaloids widely present in plants. PAs are highly hepatotoxic and have been documented to cause many incidents of human and animal poisoning. Retrorsine (RTS) is a pyrrolizidine alkaloid (PA) derived from the Compositae Senecio, which has been shown to cause hepatotoxicity. Human liver poisoning occurs through the consumption of RTS-contaminated food, and animals can also be poisoned by ingesting RTS-containing toxic plants. The mechanism of RTS-induced liver toxicity is not fully understood. In this study, we demonstrated that RTS-induced oxidative stress plays a pivotal role in RTS-induced liver toxicity involving apoptosis and autophagy. The results showed that RTS treatment in the cultured Primary rat hepatocytes caused cytotoxicity and release of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a time- and dose-dependent manner. Our study showed that treatment of RTS induced ROS and MDA (malondialdehyde, a lipid peroxidation marker) in the hepatocytes, and reduced antioxidant capacity (GSH content, SOD activity), suggesting RTS treatment caused oxidative stress response in the hepatocytes. Furthermore, we found that RTS induced apoptosis and autophagy in the hepatocytes, and RTS-induced apoptosis and autophagy could be alleviated by ROS scavenger N-acetylcysteine (NAC) and the MAPK pathway inhibitors suggesting ROS/MAPK signaling pathway plays a role in RTS induced apoptosis and autophagy. Collectively, this study reveals the regulatory mechanism of oxidative stress in RTS-induced apoptosis and autophagy in the hepatocytes, providing important insights of molecular mechanisms of hepatotoxicity induced by RTS and related pyrrolizidine alkaloids in liver. This mechanism provides a basis for the prevention and treatment of PA poisoning in humans and animals.

Autophagic-lysosomal damage induced by swainsonine is protected by trehalose through activation of TFEB-regulated pathway in renal tubular epithelial cells.

Swainsonine (SW) is the main toxic component of locoweed. Previous studies have shown that kidney damage is an early pathologic change in locoweed poisoning in animals. Trehalose induces autophagy and alleviates lysosomal damage, while its protective effect and mechanism against the toxic injury induced by SW is not clear. Based on the published literature, we hypothesize that transcription factor EB(TFEB) -regulated is targeted by SW and activating TFEB by trehalose would reverse the toxic effects. In this study, we investigate the mechanism of protective effects of trehalose using renal tubular epithelial cells. The results showed that SW induced an increase in the expression level of microtubule-associated protein light chain 3-II and p62 proteins and a decrease in the expression level of ATPase H+ transporting V1 Subunit A, Cathepsin B, Cathepsin D, lysosome-associated membrane protein 2 and TFEB proteins in renal tubular epithelial cells in a time and dose-dependent manner suggesting TFEB-regulated lysosomal pathway is adversely affected by SW. Conversely, treatment with trehalose, a known activator of TFEB promote TFEB nuclear translocation suggesting that TFEB plays an important role in protection against SW toxicity. We demonstrated in lysosome staining that SW reduced the number of lysosomes and increased the luminal pH, while trehalose could counteract these SW-induced effects. In summary, our results demonstrated for the first time that trehalose could alleviate the autophagy degradation disorder and lysosomal damage induced by SW. Our results provide an interesting method for reversion of SW-induced toxicity in farm animals and furthermore, activation of TFEB by trehalose suggesting novel mechanism of treating lysosomal storage diseases.

Intranasal administration of recombinant prosaposin attenuates neuronal apoptosis through GPR37/PI3K/Akt/ASK1 pathway in MCAO rats.

Studies have reported that Prosaposin (PSAP) is neuroprotective in cerebrovascular diseases. We hypothesized that PSAP would reduce infarct volume by attenuating neuronal apoptosis and promoting cell survival through G protein-coupled receptor 37(GPR37)/PI3K/Akt/ASK1 pathway in middle cerebral artery occlusion (MCAO) rats. Two hundred and thirty-five male and eighteen female Sprague-Dawley rats were used. Recombinant human PSAP (rPSAP) was administered intranasally 1 h (h) after reperfusion. PSAP small interfering ribonucleic acid (siRNA), GPR37 siRNA, and PI3K specific inhibitor LY294002 were administered intracerebroventricularly 48 h before MCAO. Infarct volume, neurological score, immunofluorescence staining, Western blot, Fluoro-Jade C (FJC) and TUNEL staining were examined. The expression of endogenous PSAP and GPR37 were increased after MCAO. Intranasal administration of rPSAP reduced brain infarction, neuronal apoptosis, and improved both short- and long-term neurological function. Knockdown of endogenous PSAP aggravated neurological deficits. Treatment with exogenous rPSAP increased PI3K expression, Akt and ASK1 phosphorylation, and Bcl-2 expression; phosphorylated-JNK and Bax levels were reduced along with the number of FJC and TUNEL positive neurons. GPR37 siRNA and LY294002 abolished the anti-apoptotic effect of rPSAP at 24 h after MCAO. In conclusion, rPSAP attenuated neuronal apoptosis and improved neurological function through GPR37/PI3K/Akt/ASK1 pathway after MCAO in rats. Therefore, further exploration of PSAP as a potential treatment option in ischemic stroke is warranted.

Swainsonine-induced vacuolar degeneration is regulated by mTOR-mediated autophagy in HT22 cells.

The indolizidine alkaloid, swainsonine (SW), is the main toxic component of locoweed, which can cause locoism in animals with characteristic neurological dysfunction. Pathological manifestations at cellular level include extensive vacuolar degeneration. Studies have shown that SW can induces autophagy, but the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear. In this study, we analyzed the role of autophagy in SW-induced cell injury in mouse hippocampal neurons cell line (HT22) using western blotting, qRT-PCR, transmission electron microscopy and immunofluorescence microscopy. The results showed that the expressions of LC3-II, ATG5, Beclin1 and p62 proteins and their mRNAs in HT22 cells were induced by SW treatment. The SW treatment increased the number of autophagosomes with enhanced fluorescence intensity of monodansylcadaverine (MDC) and LC3-II in a time-dose dependent manner. The results of lysosome staining showed that SW could increase the number of lysosomes, increase the intraluminal pH. Transmission electron microscopy results indicate that SW induced autophagosomes, and Baf A1 could effectively alleviate SW-induced vacuolar degeneration. At the molecular level, SW treatment inhibited the expression of p-PI3K, p-AKT, p-ERK, p-AMPK, p-mTOR, p-p70S6K and p-4EBP1 and promoted the expression of p53. Our results collectively suggest, PI3K/AKT/mTOR, ERK/mTOR and p53/mTOR signaling pathways are involved in the regulation of SW-induced autophagy in HT22 cells, while the AMPK/mTOR signaling pathway is not involved in this regulation. Inhibition of autophagic degradation can effectively alleviate SW-induced vacuolar degeneration.

Sevoflurane preconditioning promotes mesenchymal stem cells to relieve myocardial ischemia/reperfusion injury via TRPC6-induced angiogenesis.

BACKGROUND: Ischemic heart diseases is one of the leading causes of death worldwide. Although revascularization timely is an effective therapeutic intervention to salvage the ischemic myocardium, reperfusion itself causes additional myocardial injury called ischemia/reperfusion (I/R) injury. Bone marrow-derived mesenchymal stem cells (MSCs) is one of the promising cells to alleviate ischemic myocardial injury. However, this cell therapy is limited by poor MSCs survival after transplantation. Here, we investigated whether sevoflurane preconditioning could promote MSCs to attenuate myocardial I/R injury via transient receptor potential canonical channel 6 (TRPC6)-induced angiogenesis. METHODS: The anti-apoptotic effect of sevoflurane preconditioning on MSCs was determined by Annexin V-FITC/propidium iodide staining. TRPC6, hypoxia-inducible factor-1α (HIF-1α), Chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) protein expressions and VEGF release from MSCs were determined after hypoxia and reoxygenation (H/R). Small interfering RNA (siRNA) was used to knock down TRPC6 gene expression in MSCs. The angiogenesis of human umbilical vein endothelial cells (HUVECs) co-cultured with MSCs was determined by Matrigel tube formation. Myocardial I/R mouse model was induced by occluding left anterior descending coronary artery for 30 min and then reperfusion. MSCs or sevoflurane preconditioned MSCs were injected around the ligature border zone 5 min before reperfusion. Left ventricle systolic function, infarction size, serum LDH, cTnI and inflammatory cytokines were determined after reperfusion. RESULTS: Sevoflurane preconditioning up-regulated TRPC6, HIF-1α, CXCR4 and VEGF expressions in MSCs and VEGF release from MSCs under H/R, which were reversed by knockdown of TRPC6 gene using siRNA in MSCs. Furthermore, sevoflurane preconditioning promoted the angiogenic and anti-inflammatory effect of HUVECs co-cultured with MSCs. Sevoflurane preconditioned MSCs improved left ventricle systolic function and alleviated myocardial infarction and inflammation in mice subjected to I/R insult. CONCLUSION: The current findings reveal that sevoflurane preconditioned MSCs boost angiogenesis in HUVECs subjected to H/R insult and attenuate myocardial I/R injury, which may be mediated by TRPC6 up-regulated HIF-1α, CXCR4 and VEGF.

Inhibition of Aryl Hydrocarbon Receptor Attenuates Hyperglycemia-Induced Hematoma Expansion in an Intracerebral Hemorrhage Mouse Model.

Background Hyperglycemia is associated with greater hematoma expansion (HE) and worse clinical prognosis after intracerebral hemorrhage (ICH). However, the clinical benefits of intensive glucose normalization remain controversial, and there are no approved therapies for reducing HE. The aryl hydrocarbon receptor (AHR) has been shown to participate in hyperglycemia-induced blood-brain barrier (BBB) dysfunction and brain injury after stroke. Herein, we investigated the role of AHR in hyperglycemia-induced HE in a male mouse model of ICH. Methods and Results CD1 mice (n=387) were used in this study. Mice were subjected to ICH by collagenase injection. Fifty percent dextrose was injected intraperitoneally 3 hours after ICH. AHR knockout clustered regularly interspaced short palindromic repeat was administered intracerebroventricularly to evaluate the role of AHR after ICH. A selective AHR inhibitor, 6,2',4'-trimethoxyflavone, was administered intraperitoneally 2 hours or 6 hours after ICH for outcome study. To evaluate the effect of AHR on HE, 3-methylcholanthrene, an AHR agonist, was injected intraperitoneally 2 hours after ICH. The results showed hyperglycemic ICH upregulated AHR accompanied by greater HE. AHR inhibition provided neurological benefits by restricting HE and preserving BBB function after hyperglycemic ICH. In vivo knockdown of AHR further limited HE and enhanced the BBB integrity. Hyperglycemia directly activated AHR as a physiological stimulus in vivo. The thrombospondin-1/transforming growth factor-ß/vascular endothelial growth factor axis partly participated in AHR signaling after ICH, which inhibited the expressions of BBB-related proteins, ZO-1 and Claudin-5. Conclusions AHR may serve as a potential therapeutic target to attenuate hyperglycemia-induced hematoma expansion and to preserve the BBB in patients with ICH.

Activation of GPR40 attenuates neuroinflammation and improves neurological function via PAK4/CREB/KDM6B pathway in an experimental GMH rat model.

BACKGROUND: Germinal matrix hemorrhage (GMH) is defined by the rupture of immature blood vessels in the germinal matrix, where subsequent hemorrhage enters the subependymal zone and the cerebral lateral ventricles. The consequent blood clot has been identified as the causative factor of secondary brain injury, which triggers a series of complex parallel and sequential harmful mechanisms, including neuroinflammation. The orphan G-protein-coupled receptor 40 (GPR40), a free fatty acid (FFA) receptor 1, has been shown to exert anti-inflammatory effects when activated and improved outcomes in animal models of stroke. We aimed to investigate the anti-inflammatory effects of GPR40 and its underlying mechanisms after GMH. METHODS: GMH model was induced in 7-day-old rat pups by an intraparenchymal injection of bacterial collagenase. GPR40 agonist, GW9508, was administered intranasally 1 h, 25 h, and 49 h after GMH induction. CRISPR targeting GPR40, PAK4, and KDM6B were administered through intracerebroventricular injection 48 h before GMH induction. Neurologic scores, microglia polarization, and brain morphology were evaluated by negative geotaxis, right reflex, rotarod test, foot fault test, Morris water maze, immunofluorescence staining, Western blots, and nissl staining respectfully. RESULTS: The results demonstrated that GW9508 improved neurological and morphological outcomes after GMH in the short (24 h, 48 h, 72h) and long-term (days 21-27). However, the neuroprotective effects of treatment were abolished by GW1100, a selective GPR40 antagonist. GW9508 treatment increased populations of M2 microglia and decreased M1 microglia in periventricular areas 24 h after GMH induction. GW9508 upregulated the phosphorylation of PAK4, CREB, and protein level of KDM6B, CD206, IL-10, which was also met with the downregulation of inflammatory markers IL-1ß and TNF-α. The mechanism study demonstrated that the knockdown of GPR40, PAK4, and KDM6B reversed the neuroprotective effects brought on by GW9508. This evidence suggests that GPR40/PAK4/CREB/KDM6B signaling pathway in microglia plays a role in the attenuation of neuroinflammation after GMH. CONCLUSIONS: In conclusion, the present study demonstrates that the activation of GPR40 attenuated GMH-induced neuroinflammation through the activation of the PAK4/CREB/KDM6B signaling pathway, and M2 microglia may be a major mediator of this effect. Thus, GPR40 may serve as a potential target in the reduction of the inflammatory response following GMH, thereby improving neurological outcomes in the short- and long-term.

TREM (Triggering Receptor Expressed on Myeloid Cells)-1 Inhibition Attenuates Neuroinflammation via PKC (Protein Kinase C) δ/CARD9 (Caspase Recruitment Domain Family Member 9) Signaling Pathway After Intracerebral Hemorrhage in Mice.

Background and Purpose: Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high mortality and disability. Inflammatory response promotes secondary brain injury after ICH. TREM (triggering receptor expressed on myeloid cells)-1 is a key regulator of inflammation. The aim of this study was to evaluate the role of TREM-1 in neuroinflammatory response after ICH in mice. Methods: CD1 mice (n=275) were used in this study. Mice were subjected to ICH by autologous blood injection. TREM-1 knockout CRISPR was administered intracerebroventricularly to evaluate the role of TREM-1 after ICH. A selective TREM-1 inhibitor, LP17, was administered intranasally 2 hours after ICH. To elucidate TREM-1 signaling pathway, CARD9 (caspase recruitment domain family member 9) activation CRISPR was administered with LP17 and TREM-1 activating anti-mouse TREM-1 monoclonal antibody (mAb) was administered with Rottlerin, a specific PKC (protein kinase C) δ inhibitor. Lastly, to evaluate the role of HMGB1 (high-mobility group box 1) in TREM-1 mediated microglia activation, glycyrrhizin, an inhibitor of HMBG1 was administered with TREM-1 activating mAb. Neurobehavioral test, brain water content, Western blot, immunofluorescence staining, and coimmunoprecipitation was performed. Results: TREM-1 knockout reduced ICH-induced neurobehavioral deficits and neuroinflammatory response. The temporal expression of HMGB1, TREM-1, PKC δ, and CARD9 increased after ICH. TREM-1 was expressed on microglia. Intranasal administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 and 72 hours after ICH. LP17 promoted M2 microglia polarization and reduced proinflammatory cytokines after ICH, which was reversed with CARD9 activation CRISPR. TREM-1 mAb increased neurobehavior deficits, proinflammatory cytokines, and reduced M2 microglia after ICH, which was reversed with Rottlerin. HMBG1 interaction with TREM-1 increased after ICH, and glycyrrhizin reduced neuroinflammation and promoted M2 microglia which was reversed with TREM-1 mAb. Conclusions: This study demonstrated that TREM-1 enhanced neuroinflammation by modulating microglia polarization after ICH, and this regulation was partly mediated via PKC δ/CARD9 signaling pathway and increased HMGB1 activation of TREM-1.

Kisspeptin-54 attenuates oxidative stress and neuronal apoptosis in early brain injury after subarachnoid hemorrhage in rats via GPR54/ARRB2/AKT/GSK3ß signaling pathway.

Oxidative stress-induced neuron apoptosis plays a crucial role in the early brain injury (EBI) after subarachnoid hemorrhage (SAH). Kisspeptin has been reported as antioxidant to reduce oxidative stress-induced neuronal cell death through G protein-coupled receptor 54 (GPR54). The goal of this study was to determine the neuroprotection of the Kisspeptin/GRP54 signaling pathway against EBI after SAH. Two hundred and ninety-two Sprague Dawley male rats were used and SAH was induced by the endovascular perforation. Exogenous Kisspeptin 54 (KP54) was delivered intranasally. Small interfering ribonucleic acid (siRNA) for endogenous KISS1, a selective GPR54 antagonist kisspeptin 234, or ß-arrestin 2 siRNA for ARRB2 (a functional adaptor of GPR54) were administered intracerebroventricularly. Post-SAH evaluations included neurobehavioral tests, SAH grade, Western blot, immunofluorescence, Fluoro-Jade C, TUNEL, and Nissl staining. The results showed that endogenous KISS1 knockdown aggravated but exogenous KP54 (1.0 nmol/kg) treatment attenuated neurological deficits, brain oxidative stress, and neuronal apoptosis at 24 h after SAH. The benefits of KP54 persisted to 28 days after SAH, which significantly improved cognitive function in SAH rats. The GPR54 blockade or the ARRB2 knockout offset the neuroprotective effects of KP54 in SAH rats. In conclusion, our results suggested that administration of KP54 attenuated oxidative stress, neuronal apoptosis and neurobehavioral impairments through GPR54/ARRB2/AKT/GSK3ß signaling pathway after SAH in rat. Thus, KP54 may provide an effective treatment strategy for SAH patients.

Sestrin2 is involved in the Nrf2-regulated antioxidative signaling pathway in luteolin-induced prevention of the diabetic rat heart from ischemia/reperfusion injury.

Luteolin attenuates myocardial ischemia/reperfusion (I/R) injury in diabetes through activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidative response. Though sestrin2, a highly conserved stress-inducible protein, is regarded as a modulator of Nrf2 and reduces I/R injury, the effect of sestrin2 on luteolin-induced prevention of the diabetic heart from I/R injury remains unclear. We hypothesized that luteolin could relieve myocardial I/R injury in diabetes by activating the sestrin2-modulated Nrf2 antioxidative response. Diabetes was induced in rats using a single dose of streptozotocin (65 mg kg-1, i.p.) for 6 weeks, and then luteolin (100 mg kg-1 d-1, i.g.), Nrf2 inhibitor brusatol, or sestrin2 blocker leucine was administered for 2 consecutive weeks. After that, the hearts were isolated and exposed to global I/R (30 min/120 min). Luteolin markedly improved cardiac function, myocardial viability and expressions of Nrf2-regulated antioxidative genes, and reduced lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R hearts. Ca2+-induced mitochondrial permeability transition and membrane potential disruption were markedly inhibited in luteolin-treated diabetic ventricular myocytes. All these effects of luteolin were significantly reversed by Nrf2 inhibitor brusatol or sestrin2 inhibitor leucine. Luteolin-induced diminished Keap1 and augmented nuclear translocation and ARE binding activity of Nrf2 were hampered by leucine in the diabetic I/R heart. In addition, luteolin-induced augmented transcription of sestrin2 was markedly blocked by brusatol in the diabetic I/R heart. These data suggest that sestrin2 and Nrf2 positively interact to promote antioxidative actions and attenuate mitochondrial damage, by which luteolin relieves diabetic myocardial I/R injury.

Pituitary Adenylate Cyclase-Activating Polypeptide: A Promising Neuroprotective Peptide in Stroke.

The search for viable, effective treatments for acute stroke continues to be a global priority due to the high mortality and morbidity. Current therapeutic treatments have limited effects, making the search for new treatments imperative. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a well-established cytoprotective neuropeptide that participates in diverse neural physiological and pathological activities, such as neuronal proliferation, differentiation, and migration, as well as neuroprotection. It is considered a promising treatment in numerous neurological diseases. Thus, PACAP bears potential as a new therapeutic strategy for stroke treatment. Herein, we provide an overview pertaining to the current knowledge of PACAP, its receptors, and its potential neuroprotective role in the setting of stroke, as well as various mechanisms of neuroprotection involving ionic homeostasis, excitotoxicity, cell edema, oxidative stress, inflammation, and cell death, as well as the route of PACAP administration.

Pituitary Adenylate Cyclase-Activating Polypeptide Attenuates Brain Edema by Protecting Blood-Brain Barrier and Glymphatic System After Subarachnoid Hemorrhage in Rats.

Brain edema is a vital contributor to early brain injury after subarachnoid hemorrhage (SAH), which is responsible for prolonged hospitalization and poor outcomes. Pharmacological therapeutic targets on edema formation have been the focus of research for decades. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to participate in neural development and brain injury. Here, we used PACAP knockout CRISPR to demonstrate that endogenous PACAP plays an endogenous neuroprotective role against brain edema formation after SAH in rats. The exogenous PACAP treatment provided both short- and long-term neurological benefits by preserving the function of the blood-brain barrier and glymphatic system after SAH. Pretreatment of inhibitors of PACAP receptors showed that the PACAP-involved anti-edema effect and neuroprotection after SAH was facilitated by the selective PACAP receptor (PAC1). Further administration of adenylyl cyclase (AC) inhibitor and sulfonylurea receptor 1 (SUR1) CRISPR activator suggested that the AC-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) axis participated in PACAP signaling after SAH, which inhibited the expression of edema-related proteins, SUR1 and aquaporin-4 (AQP4), through SUR1 phosphorylation. Thus, PACAP may serve as a potential clinical treatment to alleviate brain edema in patients with SAH.

Luteolin Attenuates Cardiac Ischemia/Reperfusion Injury in Diabetic Rats by Modulating Nrf2 Antioxidative Function.

Luteolin has been reported to attenuate ischemia/reperfusion (I/R) injury in the diabetic heart through endothelial nitric oxide synthase- (eNOS-) related antioxidative response. Though the nuclear factor erythroid 2-related factor 2 (Nrf2) is regarded as a key endogenous factor to reduce diabetic oxidative stress, whether luteolin reduces cardiac I/R injury in the diabetic heart via enhancing Nrf2 function needs to be clarified. We hypothesized that pretreatment with luteolin could alleviate cardiac I/R injury in the diabetic heart by affecting the eNOS/Nrf2 signaling pathway. The diabetic rat was produced by a single injection of streptozotocin (65 mg/kg, i.p.) for 6 weeks, and then, luteolin (100 mg/kg/day, i.g.), eNOS inhibitor L-NAME, or Nrf2 inhibitor brusatol was administered for the succedent 2 weeks. After that, the isolated rat heart was exposed to 30 min of global ischemia and 120 min of reperfusion to establish I/R injury. Luteolin markedly ameliorated cardiac function and myocardial viability; upregulated expressions of heme oxygenase-1, superoxide dismutase, glutathione peroxidase, and catalase; and reduced myocardial lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R heart. All these ameliorating effects of luteolin were significantly reversed by L-NAME or brusatol. Luteolin also markedly reduced S-nitrosylation of Kelch-like ECH-associated protein 1 (Keap1) and upregulated Nrf2 and its transcriptional activity. This effect of luteolin on Keap1/Nrf2 signaling was attenuated by L-NAME. These data reveal that luteolin protects the diabetic heart against I/R injury by enhancing eNOS-mediated S-nitrosylation of Keap1, with subsequent upregulation of Nrf2 and the Nrf2-related antioxidative signaling pathway.

Luteolin alleviates cardiac ischemia/reperfusion injury in the hypercholesterolemic rat via activating Akt/Nrf2 signaling.

Myocardial ischemia/reperfusion (I/R) injury in hypercholesterolemia is associated with oxidative stress, while luteolin is known to reduce oxidative stress by activating Akt/nuclear factor erythroid-2-related factor 2 (Nrf2) signaling and alleviate cardiac I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in hypercholesterolemic rats by activating Akt/Nrf2 signaling. Hypercholesterolemic rats were produced by 2% cholesterol diet for 8 weeks. Luteolin (100 mg/kg/day, i.g.) or LY294002 was administered for the last 2 weeks. The hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. Pretreatment with luteolin significantly improved left ventricular function throughout reperfusion, increased cardiac tissue viability, reduced coronary lactate dehydrogenase release and the myocardial malondialdehyde level, upregulated p-Akt and p-GSK3ß expressions, inhibited nuclear translocation of Fyn, and activated Nrf2 function in hypercholesterolemic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by LY294002. Ca2+-induced mitochondrial permeability transition pore (mPTP) opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated hypercholesterolemic rats, which were attenuated by LY294002. These results indicate that luteolin protects the hypercholesterolemic heart against I/R injury due to upregulation of Akt-mediated Nrf2 antioxidative function and inhibition of mPTP.

Systematic evaluation of a targeted gene capture sequencing panel for molecular diagnosis of retinitis pigmentosa.

BACKGROUND: Inherited eye diseases are major causes of vision loss in both children and adults. Inherited eye diseases are characterized by clinical variability and pronounced genetic heterogeneity. Genetic testing may provide an accurate diagnosis for ophthalmic genetic disorders and allow gene therapy for specific diseases. METHODS: A targeted gene capture panel was designed to capture exons of 283 inherited eye disease genes including 58 known causative retinitis pigmentosa (RP) genes. 180 samples were tested with this panel, 68 were previously tested by Sanger sequencing. Systematic evaluation of our method and comprehensive molecular diagnosis were carried on 99 RP patients. RESULTS: 96.85% targeted regions were covered by at least 20 folds, the accuracy of variants detection was 99.994%. In 4 of the 68 samples previously tested by Sanger sequencing, mutations of other diseases not consisting with the clinical diagnosis were detected by next-generation sequencing (NGS) not Sanger. Among the 99 RP patients, 64 (64.6%) were detected with pathogenic mutations, while in 3 patients, it was inconsistent between molecular diagnosis and their initial clinical diagnosis. After revisiting, one patient's clinical diagnosis was reclassified. In addition, 3 patients were found carrying large deletions. CONCLUSIONS: We have systematically evaluated our method and compared it with Sanger sequencing, and have identified a large number of novel mutations in a cohort of 99 RP patients. The results showed a sufficient accuracy of our method and suggested the importance of molecular diagnosis in clinical diagnosis.

Effect of X-ray irradiation on hepatocarcinoma cells and erythrocytes in salvaged blood.

The broad clinical acceptance of intraoperative blood salvage and its applications in cancer surgery remain controversial. Until now, a method that can safely eliminate cancer cells while preserving erythrocytes does not exist. Here, we investigated whether X-ray generated from linear accelerator irradiation at a certain dose can kill hepatocarcinoma cells while preserving erythrocytes. HepG2, SK-Hep1 or Huh7 cells were mixed into the aliquots of erythrocytes obtained from healthy volunteers. After the mixed cells were exposed to 30 Gy and 50 Gy X-rays irradiation, the viability, clonogenicity, DNA synthesis and tumorigenicity of the tumor cells were determined by the MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine incorporation, and subcutaneous xenograft implantation into immunocompromised mice. The ATP, 2,3-DPG, free Hb, osmotic fragility, blood gas variables in erythrocytes and morphology of erythrocytes at 0 h, 12 h, 24 h, 48 h, 72 h after irradiation were analyzed. X-ray irradiation at 30 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SK-Hep1 and Huh7 cells without noticeably damaging the ability of oxygen-carrying, membrane integrity and morphology of erythrocytes. Theses results suggest that X-ray at 30 Gy irradiation might be safe to eliminate hepatocarcinoma cells while preserving erythrocytes in salvaged blood.

Effects of CYP3A4 polymorphisms on efficiency of general anesthesia combined with epidural block in patients undergoing cardiac valve replacement.

This study aims to investigate the effects of CYP3A4 polymorphisms (*4, *5 and *6) on efficiency of general anesthesia (GA) combined with epidural block (EB) in patients undergoing cardiac valve replacement. From January 2014 to October 2015, a total of 511 patients undergoing cardiac valve replacement (case group) and 503 healthy individuals (control group) were selected for the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for genotyping of CYP3A4 gene. Central venous pressure (CVP), mean arterial pressure (MAP), heart rate (HR), pulse oximetry (SPO2), extubation and duration of intensive care unit (ICU) stay during the surgery were observed and recorded. A nine-month follow-up was conducted. Genotype and allele frequency of CYP3A4*4 were significantly different between the case and control groups (all P < 0.05). Compared with wild-type *1*1 patients with heterozygous *1*4 of CYP3A4*4 showed significant difference in HR, MAP, SPO2 and CVP and in the time of extubation and ICU stay. CYP3A4*4 polymorphism may be associated with the effect of GA combined with EB in cardiac surgery. These results demonstrate that CYP3A4*4 polymorphism is correlated with the effects of GA combined with EB in cardiac surgery. CYP3A4 polymorphisms increase the risk of GA combined with EB among patients undergoing cardiac valve replacement.

A novel mutation in NF1 is associated with diverse intra-familial phenotypic variation and astrocytoma in a Chinese family.

Neurofibromatosis type 1 (NF1) is a dysregulated neurocutaneous disorder, characterized by neurofibromas and café-au-lait spots. NF1 is caused by mutations in the NF1 gene, encoding neurofibromin. Here, we present a clinical molecular study of a three-generation Chinese family with NF1. The proband was a male patient who showed café-au-lait spots and multiple subcutaneous neurofibromas over the whole body, but his siblings only had regional lesions. The man's daughter presented with severe headache and vomiting. Neurological examination revealed an intracranial space occupying lesion. Surgery was undertaken and the histopathological examination showed a grade I-II astrocytoma. Next-Generation sequencing (Illumina HiSeq2500 Analyzers; Illumina, San Diego, CA, USA) and Sanger sequencing (ABI PRISM 3730 automated sequencer; Applied Biosystems, Foster City, CA, USA) identified the c.227delA mutation in the NF1 gene in the man. The mutation is co-segregated with the disease phenotypes among the affected members of this family and was absent in 100 healthy controls. This novel mutation results in a frameshift (p.Asn78IlefsX7) as well as truncation of neurofibromin by formation of a premature stop codon. Our results not only extended the mutational and phenotypic spectra of the gene and the disease, but also highlight the importance of the other genetic or environmental factors in the development and severity of the disease.

Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System.

An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery.