Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis:nicotiflorin and JAK2/STAT3 pathway

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius.Previous studies have shown its cerebral protective effect,but the mechanism is undefined.In this study,we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway.The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion.Nicotiflorin（10 mg/kg） was administered by tail vein injection.Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay.Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining.Additionally,p-JAK2,p-STAT3,Bcl-2,Bax,and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay.Nicotiflorin altered the shape and structure of injured neurons,decreased the number of apoptotic cells,down-regulates expression of p-JAK2,p-STAT3,caspase-3,and Bax,decreased Bax immunoredactivity,and increased Bcl-2 protein expression and immunoreactivity.These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury：mechanisms of brain tissue repair

Buyanghuanwu decoction has been shown to protect against cerebral ischemia/reperfusion injury,but the underlying mechanisms remain unclear.In this study,rats were intragastrically given Buyanghuanwu decoction,15 m L/kg,for 3 days.A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion.In rats administered Buyanghuanwu decoction,infarct volume was reduced,serum vascular endothelial growth factor and integrin αvβ3 levels were increased,and brain tissue vascular endothelial growth factor and CD34 expression levels were increased compared with untreated animals.These effects of Buyanghuanwu decoction were partially suppressed by an angiogenesis inhibitor（administered through the lateral ventricle for 7 consecutive days）.These data suggest that Buyanghuanwu decoction promotes angiogenesis,improves cerebral circulation,and enhances brain tissue repair after cerebral ischemia/reperfusion injury.

The role of glycogen synthase kinase 3 beta in brain injury induced by myocardial ischemia/reperfusion injury in a rat model of diabetes mellitus

Myocardial ischemia/reperfusion injury can lead to severe brain injury.Glycogen synthase kinase 3 beta is known to be involved in myocardial ischemia/reperfusion injury and diabetes mellitus.However,the precise role of glycogen synthase kinase 3 beta in myocardial ischemia/reperfusion injury-induced brain injury is unclear.In this study,we observed the effects of glycogen synthase kinase 3 beta on brain injury induced by myocardial ischemia/reperfusion injury in diabetic rats.Rat models of diabetes mellitus were generated via intraperitoneal injection of streptozotocin.Models of myocardial ischemia/reperfusion injury were generated by occluding the anterior descending branch of the left coronary artery.Post-conditioning comprised three cycles of ischemia/reperfusion.Immunohistochemical staining and western blot assays demonstrated that after 48 hours of reperfusion,the structure of the brain was seriously damaged in the experimental rats compared with normal controls.Expression of Bax,interleukin-6,interleukin-8,terminal deoxynucleotidyl transferase d UTP nick end labeling,and cleaved caspase-3 in the brain was significantly increased,while expression of Bcl-2,interleukin-10,and phospho-glycogen synthase kinase 3 beta was decreased.Diabetes mellitus can aggravate inflammatory reactions and apoptosis.Ischemic post-conditioning with glycogen synthase kinase 3 beta inhibitor lithium chloride can effectively reverse these changes.Our results showed that myocardial ischemic post-conditioning attenuated myocardial ischemia/reperfusion injury-induced brain injury by activating glycogen synthase kinase 3 beta.According to these results,glycogen synthase kinase 3 beta appears to be an important factor in brain injury induced by myocardial ischemia/reperfusion injury.

miR-21-3p alleviates neuronal apoptosis during cerebral ischemiareperfusion injury by targeting SMAD2

Cerebral ischemia is due to the formation of blood clots or embolisms in the brain arteries,which leads to local brain tissue necrosis and neural cell apoptosis.Recent studies have shown that microRNA(miRNA)plays an important regulatory role in the pathological process of ischemic injury.The aim of this study is to investigate the role and the mechanism of miR-21-3p and drosophila mothers against decapentaplegic 2(SMAD2)in cerebral ischemic reperfusion injured(CIRI)neural cells.The CIRI model was established by oxygen-glucose deprivation and recovery process for N2a cells.The cell viability and the apoptotic was evaluated by MTT assay and the Flow Cytometer,respectively.The expression of miR-21-3p and SMAD2 mRNA was detected by real-time fluorescence quantitative PCR(qRT-PCR),and the expression of SMAD2 and apoptotic-related proteins were detected by Western Blotting.Our results showed that miR-21-3p is down-regulated,and SMAD2 is up-regulated in CIRI.Overexpression of miR-21-3p inhibits the apoptosis of neural cells in CIRI.miR-21-3p targets SMAD2 and inhibits SMAD2 expression.Overexpression of SMAD2 eliminates the protective effect of over-expression of miR-21-3p on neural cells in CIRI.Token together,this study provides a theoretical basis for the mechanism of ischemic reperfusion injury in neural cells and a new molecular target for ischemic stroke therapy.

MRI Investigation of New Approach to Improve the Recovery of Myocardial Ischemia Reperfusion Injury by Treatment with Intralipid®

Background: Despite advances in revascularization and thrombolytic therapy, the outcome of patients after acute myocardial infarction (MI) could be complicated by ischemia reperfusion injury (IRI) and subsequent ventricular remodeling. Inflammation plays a central role in IRI. Intralipid® has been shown to reduce infarct size after IRI, but its effects on myocardial inflammation have not been addressed. The goal of this study is to investigate the effects of Intralipid® on in-situ myocardial inflammation and to better characterize its cardio-protective effects. Methods and Results: Cellular MRI was used to evaluate myocardial inflammation of iron-oxide-labeled macrophage infiltration. Cardiac MRI was used to evaluate global and regional ventricular wall motion, as well as myocardial perfusion and infarction in a rat model. Our results show that the Intralipid® treatment following IRI can preserve global and regional ventricular wall motion, and reduce the infarct size. The In-tralipid®-treated rats exhibit reduced myocardial macrophage infiltration, indicating reduced in-situ myocardial inflammation. Conclusions: Our results indicate that the Intralipid® treatment can protect the heart against IRI and can specifically reduce in-situ myocardial inflammation. Additional study is needed to assess if treatment using Intralipid® after LAD occlusion could improve the recovery of patients suffering from a heart attack and also reduce future development of heart failure.

Changes of cTnI in myocardial ischemic and reperfusion injury during correction of cardiac defects in children

Objective The purpose of this study is to investgate changes of cTnI in myocardial ischemic and reperfusion injury during correction of cardiac defects in children. Methods From June, 1999 to May,2000,45 children (30 male, 15 female) undergoing correction of cardiac defects were divided into three groups randomly: group Ⅰ no myocardial ischemia,group Ⅱ myocardial ischemia less than 60 minutes, group Ⅲmyocardial ischemia 】 60 minutes. There were no significant differences in the three groups in age, sex ratio, C/T ratio, or left ventricular function. Blood samples for analysis were collected before skin incision and at time intervals up to 6 days postoperatively. Analysis of creatine kinase MB.LDH and cardiac-specific troponin I was used for the detection of myocardial damage. Meantime, the ECG was checked for myocardial infarction. After the reperfusion, myocardial tissue was obtained from the free wall of right ventricle myocardial structure studies. Results The level of cTnI was increased

By regulating IP3/PKC/TRPV4 pathway hyperoside induces endothelium-dependent vasodilatation in rat basilar artery following four vessel occlusion ischemia reperfusion

OBJECTIVE To investigate regulatory effects of hyperoside(Hyp)on IP3/PKC/TRPV4 pathway in rat cerebral basilar artery(CBA)subjected to global cerebral ischemia-reperfusion(I/R).METHODS The model of global cerebral I/R in rats was established by four-vessel occlusions methods.The treated rats were administrated with Hyp(50 mg·kg^-1)group,Hyp(50 mg·kg^-1)+HC-067047(10 mg·kg^-1),Hyp(50 mg·kg^-1)+2APB(2 mg·kg^-1),Hyp(50 mg·kg^-1)+BisI(2.5 mg·kg^-1),Hyp(50 mg·kg^-1)+2APB(2 mg·kg^-1)+BisI(2.5 mg·kg^-1).Hematoxylin-eosin(HE)and Nissl staining were performed and the contents of methane dicarboxylic aldehyde(MDA),neuron-specific enolase(NSE),S100β and the activity of lactic dehydrogenase(LDH)in serum were measured by enzyme-linked immunosorbnent assay(ELISA).The specific blocker N-nitro-L-arginine-methyl-ester(L-NAME)and indomethacin(Indo)were used to delete the prostacyclin(PGI2)and nitric oxide(NO)dependent relaxation.The protein expression level of TRPV4 was detected by Western blotting.Ca2+intensity in vascular smooth muscle cells was measured by confocal laser scanning microscope and flow cytometry was performed to observe the apoptosis of CBA endothelial cells after in vivo administration.RESULTS Hyp induced a dose-dependent relaxation of CBA in IR rats via a PGI2 and NO independent manner,as evidenced by alleviated patho⁃logical changes and up-regulated expression of TRPV4 protein in the endothelial cells from cerebral vessels.Hyp signifi⁃cantly reduced the contents of MDA,NSE,S100βand the activity of LDH in serum and decreased the fluorescence intensity of Ca2+in cerebral vascular smooth muscle cells by in vivo administration.The apoptotic rate of endothelial cells in Hyp treated group was significantly less than that in IR group.CONCLUSION Hyp does in fact ameliorate I/R injury by regulatingIP3/PKC/TRPV4 pathway.

Establishment of an animal model of ischemic type intrahepatic biliary lesion in rabbits

AIM:To explore a method to establish an animal model of ischemic type intrahepatic biliary lesion in rabbits. METHODS:Forty Japanese white rabbits of clean grade were divided randomly into four groups(10 rabbits per group)including sham operation(SO) group,and artery-bile obstruction(ABO)-1 h group, ABO-2 h group and ABO-3 h group.All the rabbits in this study underwent the same initial surgical procedure in which the liver was prepared as for graft removal during liver transplantation.Subsequently in the SO group,no additional vascular intervention was performed,while in groups ABO-1 h,ABO-2 h and ABO-3 h,the animals underwent combined clamping of the hepatic artery and common bile duct with microvascular clips for 1,2 and 3 h,respectively.After the scheduled occlusion time,the clip was removed to recover blood supply.The animals were killed 4 wk after operation.The survival rate,liver function, cholangiography and histopathological manifestation of the rabbits in each group were observed. RESULTS:The survival rate was 100%in groups SO,ABO-1 h and ABO-2 h,while it was 60%in group ABO-3 h.At each observation time,the change degree of the indexes of liver function was proportional to the clamping time(ABO-3 h>ABO-2 h>ABO-1 h> SO,P<0.05).Cholangiographical and histopathologic manifestations both showed that intrahepatic biliary lesion aggravated proportionally with the increase of the clamping time. CONCLUSION:An animal model of ischemic type intrahepatic biliary lesion in rabbits is successfully established,which may provide a reliable technique for basic and clinical research into the etiology, development and prophylaxis of ischemic type intrahepatic biliary lesion after liver transplantation.

Effects of Compound Shenhua Tablet(复方肾华片) on Renal Tubular Na^+-K^+-ATPase in Rats with Acute Ischemic Reperfusion Injury

Objective： To observe the effect of Compound Shenhua Tablet （复方肾华片, SHT） on the sodium- potassium-exchanging adenosinetriphosphatase （Na＋-K＋-ATPase） in the renal tubular epithelial cells of rats with acute ischemic reperfusion and to investigate the mechanisms underlying the effects of SHT on renal ischemic reperfusion injury （RIRI）. Methods： Fifty male Wistar rats were randomly divided into the sham surgery group, model group, astragaloside group [150 mg/（kg.d）], SHT low-dose group [1.5 g/（kg,d）] and SHT high-dose group [3.0 g/（kg.d）], with 10 rats in each group. After 1 week of continuous intragastric drug administration, surgery was performed to establish the model. At either 24 or 72 h after the surgery, 5 rats in each group were sacrificed, blood biochemistry, renal pathology, immunoblot and immunohistochemical examinations were performed, and double immunofluorescence staining was observed under a laser confocal microscope. Results： Compared with the sham surgery group, the serum creatinine （SCr） and blood urea nitrogen （BUN） levels were significantly increased, Na＋-K＋-ATPase protein level was decreased, and kidney injury molecule-1 （KIM-1） protein level was increased in the model group after the surgery （P〈0.01 or P〈0.05）. Compared with the model group, the SCr, BUN, pathological scores, Na＋-K＋-ATPase, and the KIM-1 protein level of the three treatment groups were significantly improved at 72 h after the surgery （P〈0.05 or P〈0.01）. And the SCr, BUN of the SHT low- and high-dose groups, and the pathological scores of the SHT high-dose group were significantly lower than those of the astragaloside group （P〈0.05）. The Iocalizations of Na＋-K＋-ATPase and megalin of the model group were disrupted, with the distribution areas overlapping with each other and alternately arranged. The severity of the disruption was slightly milder in three treatment groups compared with that of the model group. The results of immunofluorescence staining showed that the SHT high-dose group had a superior effect as compared with the astragaloside group and the SHT low-dose group. Conclusions： The SHT effectively alleviated RIRI caused by ischemic reperfusion, promoted the recovery of the polarity of renal tubular epithelial cells, and protected the renal tubules. The therapeutic effects of SHT were superior to those of astragaloside as a single agent.

Protective Mechanism of Cordyceps sinensis Treatment on Acute Kidney Injury-Induced Acute Lung Injury through AMPK/mTOR Signaling Pathway

Objective: To investigate protective effect of Cordyceps sinensis(CS) through autophagyassociated adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling pathway in acute kidney injury(AKI)-induced acute lung injury(ALI). Methods: Forty-eight male Sprague-Dawley rats were divided into 4 groups according to a random number table, including the normal saline(NS)-treated sham group(sham group), NS-treated ischemia reperfusion injury(IRI) group(IRI group), and low-(5 g/kg·d) and high-dose(10 g/kg·d) CS-treated IRI groups(CS1 and CS2 groups), 12 rats in each group. Nephrectomy of the right kidney was performed on the IRI rat model that was subjected to 60 min of left renal pedicle occlusion followed by 12, 24, 48, and 72 h of reperfusion. The wet-to-dry(W/D) ratio of lung, levels of serum creatinine(Scr), blood urea nitrogen(BUN), inflammatory cytokines such as interleukin-βand tumor necrosis factor-α, and biomarkers of oxidative stress such as superoxide dismutase, malonaldehyde(MDA) and myeloperoxidase(MPO), were assayed. Histological examinations were conducted to determine damage of tissues in the kidney and lung. The protein expressions of light chain 3 Ⅱ/light chain 3 Ⅰ(LC3-Ⅱ/LC3-Ⅰ), uncoordinated-51-like kinase 1(ULK1), P62, AMPK and mTOR were measured by Western blot and immunohistochemistry, respectively. Results: The renal IRI induced pulmonary injury following AKI,resulting in significant increases in W/D ratio of lung, and the levels of Scr, BUN, inflammatory cytokines, MDA and MPO(P<0.01);all of these were reduced in the CS groups(P<0.05 or P<0.01). Compared with the IRI groups, the expression levels of P62 and mTOR were significantly lower(P<0.05 or P<0.01), while those of LC3-Ⅱ/LC3-Ⅰ, ULK1, and AMPK were significantly higher in the CS2 group(P<0.05 or P<0.01). Conclusion:CS had a potential in treating lung injury following renal IRI through activation of the autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI.

CARDIOPULMONARY BYPASS WITH AUTOLOGOUS LUNG AS SUBSTITUTE FOR ARTIFICIAL OXYGENATOR ATTENUATES INFLAMMATORY RESPONSIVE INSPIRATORY DYSFUNCTION

Objective To study if using autologous lung as a substitute of oxygenator in cardiopulmonary bypass is better than the conventional cardiopulmonary bypass with artificial oxygenator in pulmonary preservation. Methods Twelve piglets were randomly divided into two groups （ n = 6）. The isolated lung perfusion model was established. The experimental animals underwent continuous lung perfusion for about 120 rain. While the control animals underwent 90 rain lung ischemia followed by 30 rain reperfusion. Another 12 piglets were randomly divided into two groups （ n =6）. The experimental animals underwent bi-ventricular bypass with autologous lung perfusion. While control animals underwent conventional cardiopulmonary bypass with artificial oxygenator. The bypass time and aortic cross clamping time were 135 rain and 60 rain respectively for each animal. The lung static compliance （Cstat）, alveolus-artery oxygen difference （PA-aO2 ） , TNF-α, IL-6 and wet to dry lung weight ratio （W/D） were measured. Histological and ultra-structural changes of the lung were also observed after bypass. Results After either isolated lung perfusion or cardiopulmonary bypass, the Cstat decreased, the PA-aO2 increased and the content of TNF-α increased for both groups, but the changes of experimental group were much less than those of control group. The lower W/D ratio and mild pathological changes in experimental group than those in control group were also demonstrated. Conclusion Autologous lung is able to tolerate the nonpulsatile perfusion. It can be used as a substitute to artificial ogygenator in cardiopulmonary bypass to minimize the inflammatory pulmonary injury caused mainly by ischemic reperfusion and interaction of the blood to the non-physiological surface of artificial oxygenator.

Monocarboxylate transporter 4 plays a significant role in the neuroprotective mechanism of ischemic preconditioning in transient cerebral ischemia

Monocarboxylate transporters（MCTs）, which carry monocarboxylates such as lactate across biological membranes, have been associated with cerebral ischemia/reperfusion process. In this study, we studied the effect of ischemic preconditioning（IPC） on MCT4 immunoreactivity after 5 minutes of transient cerebral ischemia in the gerbil. Animals were randomly designated to four groups（sham-operated group, ischemia only group, IPC ＋ sham-operated group and IPC ＋ ischemia group）. A serious loss of neuron was found in the stratum pyramidale of the hippocampal CA1 region（CA1）, not CA2/3, of the ischemia-only group at 5 days post-ischemia; however, in the IPC ＋ ischemia groups, neurons in the stratum pyramidale of the CA1 were well protected. Weak MCT4 immunoreactivity was found in the stratum pyramidale of the CA1 in the sham-operated group. MCT4 immunoreactivity in the stratum pyramidale began to decrease at 2 days post-ischemia and was hardly detected at 5 days post-ischemia; at this time point, MCT4 immunoreactivity was newly expressed in astrocytes. In the IPC ＋ sham-operated group, MCT4 immunoreactivity in the stratum pyramidale of the CA1 was increased compared with the sham-operated group, and, in the IPC ＋ ischemia group, MCT4 immunoreactivity was also increased in the stratum pyramidale compared with the ischemia only group. Briefly, present findings show that IPC apparently protected CA1 pyramidal neurons and increased or maintained MCT4 expression in the stratum pyramidale of the CA1 after transient cerebral ischemia. Our findings suggest that MCT4 appears to play a significant role in the neuroprotective mechanism of IPC in the gerbil with transient cerebral ischemia.

Protective Effects of the Preconditioning with Different Doses of Sodium Aescinate on Tourniquet-induced Ischemic Reperfusion

Objective：To evaluate the protective effects of sodium aescinate（SA）preconditioning on the tourniquet-induced ischemia-reperfusion（l/R）injury after limbs operation.Methods：Seventy-five patients with gradeⅠ-Ⅱissued by American Society of Anesthesiology undergoing lower limb operation were randomly assigned to 3 groups：the control group,low-dose SA-treated group and high-dose SA-treated group;each group enrolled 25 patients.The patients were treated with 5 mg and 10 mg SA 30 min before tourniquet inflation in the two treatment groups separately,while the patients in the control group received normal saline.Venous blood samples were obtained before tourniquet was inflated（T0 baseline）.And 5（T1）,10（T2）,20（T3）min after tourniquet was released.The nitric oxide（NO）,malondialdehyde（MDA）and superoxide dismutase（SOD）levels were determined by commercial kits.Meanwhile,arterial pressure（MAP）and heart rate（HR）were monitored from an automatic invigilator.Results：In the control group,MDA and NO levels were increased,and SOD and MAP were decreased significantly after tourniquet deflation compared to TO baseline（P0.05）.After tourniquet deflation,MDA and NO levels in the two treated groups were significantly decreased;meanwhile,SOD levels and MAP were increased,and the variations of HR were more stable compared with the control group（all P0.05）.There was no significant difference in all of the above between the two treated groups（P0.05）. Conclusion：The protective effects of SA preconditioning on tourniquet-induced limb l/R injury might possibly contribute to the increasing of SOD levels,and MAP and the decreasing of MDA and NO levels.

Remote ischemia conditioning-an endogenous cardioprotective strategy from outside the heart

Objective A general review was made of studies involving： （1） The experimental evidence of remote ischemic preconditioning （RIPC） and relative clinical studies, （2） The experimental and clinical evidences of remote ischemic postconditioning （RIPOC）, （3） The potential mechanistic pathways underlying their protective effects.Data sources The data used in this review were mainly from manuscripts listed in PubMed that were published in English from 1986 to 2010. The search terms were ＂myocardial ischemia reperfusion injury＂, ＂ischemia preconditioning＂,＂ischemia postconditioning＂, ＂remote preconditioning＂ and ＂remote postconditioning＂.Study selection （1） Clinical and experimental evidence that both RIPC and RIPOC produce preservation of ischemia reperfusion injury （IRI） of myocardium and other organs, （2） Studies related to the potential mechanisms, by which remote ischemic conditioning protects myocardium against IRI.Results Both RIPC and RIOPC have been shown to attenuate myocardial IRI in laboratory animals. Also, their cardioprotective effects have appeared in some clinical studies. Except the external, the detailed internal mechanisms of remote ischemic conditioning have been generally described. Through these descriptions better protocols can be developed to provide improved cardioprotective procedures.Conclusions Remote ischemic conditioning is an endogenous cardioprotective mechanism from outside the heart that protects against myocardial IRI and represents a general form of inter-organ protection. Remote ischemic conditioning may have an immense impact on clinical practice in the near future.