Xiang-Hu He

Transduction of PEP-1-heme oxygenase-1 fusion protein reduces myocardial ischemia/reperfusion injury in rats.

Abstract: Recent studies have uncovered that overexpression of heme oxygenase-1 (HO-1) by induction or gene transfer provides myocardial protection. In the present study, we investigated whether HO-1 protein mediated by cell-penetrating peptide PEP-1 could confer cardioprotection in a rat model of myocardial ischemia/reperfusion (I/R) injury. Male Sprague-Dawley rats were subjected to 30 minutes of ischemia by occluding the left anterior descending coronary artery and to 120 minutes of reperfusion to prepare the model of I/R. Animals were randomized to receive PEP-1–HO-1 fusion protein or saline 30 minutes before a 30-minute occlusion. I/R increased myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and reduced myocardial superoxide dismutase activity. Administration of PEP-1–HO-1 reduced myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and increased myocardial superoxide dismutase and HO-1 activities. His-probe protein was only detected in PEP-1-HO-1–transduced hearts. In addition, transduction of PEP-1–HO-1 markedly reduced elevated myocardial tissue nuclear factor-&kgr;B induced by I/R. The results suggested that transduction of PEP-1–HO-1 fusion protein decreased myocardial reperfusion injury, probably by attenuating the production of oxidants and proinflammatory cytokines regulated by nuclear factor-&kgr;B.

HO-1 Protects against Hypoxia/Reoxygenation-Induced Mitochondrial Dysfunction in H9c2 Cardiomyocytes

Background Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial’s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism. Methods and Results The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed. Conclusions During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect.

Protective effect of PNU-120596, a selective alpha7 nicotinic acetylcholine receptor-positive allosteric modulator, on myocardial ischemia-reperfusion injury in rats.

Abstract: The cholinergic anti-inflammatory pathway has been found to exert a protective role in myocardial ischemia–reperfusion injury (MIRI). Alpha7 nicotinic acetylcholine receptor (&agr;7nAChR) is a regulator of cholinergic anti-inflammatory pathway; however, little information is available on the effect of &agr;7nAChR on MIRI. In the present study, we hypothesized that 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a potent positive allosteric modulator of &agr;7nAChR, could play a protective role on MIRI. Fifty-five rats were randomly assigned into 4 groups: Sham group, ischemia–reperfusion group, PNU-120596 group, &agr;-bungarotoxin group. Compared with ischemia–reperfusion group, PNU-120596 treatment markedly decreased infarct size, ultrastructural damage, serum creatine kinase, and lactate dehydrogenase. Serum proinflammatory cytokine production, myocardium endothelial activation and neutrophil infiltration, myocardium malondialdehyde were also significantly decreased, accompanied by increased myocardium superoxide dismutase production, in the PNU-120596 group compared with the ischemia–reperfusion group. Meanwhile, we observed a significant inhibition of nuclear factor kappa B activation in PNU-120596 group compared with ischemia–reperfusion group. Pretreatment of &agr;7nAChR-selective antagonist, &agr;-bungarotoxin, abolished all the protective effects of PNU-120596 on MIRI. In conclusion, PNU might have a protective effect against MIRI. Its action mechanisms might be involved in the inhibition of inflammatory responses, attenuation of lipid peroxidation, and suppression of nuclear factor kappa B activity.

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Reduces Remote Organ Injury Induced by Intestinal Ischemia/Reperfusion

Background A fusion protein composed of heme oxygenase-1 (HO-1) and cell-penetrating peptide PEP-1 has been shown to reduce local intestinal injury after intestinal ischemia/reperfusion (I/R). In this study, we investigated the effects of PEP-1-HO-1 fusion protein on remote organ injury induced by intestinal I/R in rats. Material/methods We randomly assigned 24 male Sprague-Dawley rats to 3 groups: Sham, I/R, and I/R plus PEP-1-HO-1 treatment (HO). The model of intestinal I/R was established by occluding the superior mesenteric artery for 45 min followed by 120-min reperfusion. In HO group, PEP-1-HO-1 was administered intravenously 30 min before ischemia, while animals in the Sham and I/R groups received the equal volume of physiological saline. At the end of the experiment, lung, liver, and blood samples were collected and analyzed. Results Malondialdehyde levels and histological injury scores were increased, and superoxide dismutase activities were decreased in the lung and liver tissues in the I/R group compared with the Sham group (P<0.05). Serum levels of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, interleukin-6, and lung tissue wet weight to dry weight ratio were increased in the I/R group compared with the Sham group (P<0.05). NF-κB expression in intestinal tissues was significantly higher in the I/R group than in the Sham group. These changes were significantly reversed by treatment with PEP-1-HO-1. Conclusions This study demonstrates that administration of PEP-1-HO-1 has a protective role against lung and liver injury after intestinal I/R, attributable to the reduction of released proinflammatory cytokines regulated by NF-κB.

Protection against intestinal injury from hemorrhagic shock by direct peritoneal resuscitation with pyruvate in rats.

Abstract Objective: We explored the effects of direct peritoneal resuscitation with pyruvate-peritoneal dialysis solution (PDS) following intravenous resuscitation (VR) on intestinal ischemia-reperfusion injury in rats with hemorrhagic shock (HS). Methods: Fifty rats were randomly assigned equally to five groups. In group sham, a surgical operation was performed on rats without shock or resuscitation. In group VR, rats were subjected only to VR. In groups NS, LA, and PY, direct peritoneal resuscitation was performed with normal saline (NS), lactate-based PDS (Lac-PDS), and pyruvate-based PDS (Pyr-PDS), respectively, after VR. Mean arterial pressure was monitored in the right common carotid artery. Two hours after resuscitation, the lactate level in arterial blood and the wet weight/dry weight ratio of the intestine were determined. The intestinal mucosal damage index was estimated, and ultrastructural changes in the intestinal mucosa were observed. Malondialdehyde, myeloperoxidase, nitric oxide, and tumor necrosis factor &agr; levels were also measured. Results: Two hours after HS and resuscitation, the increase in arterial blood lactate and intestinal wet weight/dry weight ratio declined significantly in rats from Groups LA and PY compared with groups VR and NS, whereas group PY was more advantageous in the changes of these parameters. The intestinal mucosal damage index and ultrastructural changes were also improved in groups LA and PY when compared with groups VR and NS. Protection was more apparent with Pyr-PDS than Lac-PDS. Hemorrhagic shock resulted in a significant increase in malondialdehyde levels and myeloperoxidase activity and was accompanied by overexpression of tumor necrosis factor &agr; and a reduction in nitric oxide levels. These changes were significantly attenuated by Lac-PDS and Pyr-PDS at 2 h after resuscitation, and Pyr-PDS showed more effective protection for the intestine than Lac-PDS. Conclusions: Direct peritoneal resuscitation with Lac-PDS and Pyr-PDS after VR alleviated intestinal injury from HS in rats, and Pyr-PDS was superior to Lac-PDS in its protective effect. Mechanisms of action might include the elimination of free oxygen radicals, reduction of neutrophil infiltration, inhibition of the inflammatory response, and regulation of intestinal mucosal blood flow and barrier function.

Inhibition of miR-200b/miR-429 contributes to neuropathic pain development through targeting zinc finger E box binding protein-1

Many studies have reported that microRNAs participate in neuropathic pain development. Previously, miR‐200b and miR‐429 are reported to be involved in various diseases. In our current study, we focused on their roles in neuropathic pain and we found that miR‐200b and miR‐429 were significantly decreased in chronic constriction injury (CCI) rat spinal cords and isolated microglials. miR‐200b and miR‐429 overexpression were able to relieve neuropathic pain through modulating PWT and PWL in CCI rats. Meanwhile, we observed that both miR‐200b and miR‐429 upregulation could repress neuroinflammation via inhibiting inflammatory cytokines such as IL‐6, IL‐1β, and TNF‐α in CCI rats. By carry out bioinformatics technology, Zinc finger E box binding protein‐1 (ZEB1) was predicted as target of miR‐200b, and miR‐429 and dual‐luciferase reporter assays confirmed the correlation between them. ZEB1 has been reported to regulate a lot of diseases. Here, we found that ZEB1 was greatly increased in CCI rats and miR‐200b and miR‐429 overexpression markedly suppressed ZEB1 mRNA expression in rat microglial cells. In addition, knockdown of ZEB1 can reduce neuropathic pain development and co‐transfection of LV‐anti‐miR‐200b/miR‐429 reversed this phenomenon in vivo. Taken these together, our results suggested that miR‐200b/miR‐429 can serve as an important regulator of neuropathic pain development by targeting ZEB1.

XIST accelerates neuropathic pain progression through regulation of miR-150 and ZEB1 in CCI rat models

LncRNAs are reported to participate in neuropathic pain development. LncRNA X‐inactive specific transcript (XIST) is involved in the progression of various cancers. However, the role of XIST in neuropathic pain remains unclear. In our present study, we established a chronic constriction injury (CCI) rat model and XIST was found to be greatly upregulated both in the spinal cord tissues and in the isolated microglias of CCI rats. Inhibition of XIST inhibited neuropathic pain behaviors including mechanical and thermal hyperalgesia. Moreover, decrease of XIST repressed neuroinflammation through inhibiting COX‐2, tumor necrosis factor (TNF)‐α and IL‐6 and in CCI rats. Previously, miR‐150 has been reported to restrain neuropathic pain by targeting TLR5. Currently, miR‐150 was predicted to be a microRNA target of XIST, which indicated a negative correlation between miR‐150 and XIST. miR‐150 was remarkably decreased in CCI rats and overexpression of miR‐150 can significantly suppress neuroinflammation‐related cytokines. Furthermore, ZEB1 was exhibited to be a direct target of miR‐150 and we found it was overexpressed in CCI rats. Silencing ZEB1 was able to inhibit neuropathic pain in vivo and downreguation of XIST decreased ZEB1, which can be reversed by miR‐150 inhibitors. Taken these together, we indicated that XIST can induce neuropathic pain development in CCI rats via upregulating ZEB1 by acting as a sponge of miR‐150. It was revealed that XIST/miR‐150/ZEB1 axis can be provided as a therapeutic target in neuropathic pain.

Dexmedetomidine alleviates lipopolysaccharide-induced lung injury in Wistar rats

This study aimed to investigate the protective effects of dexmedetomidine on lipopolysaccharide (LPS)-induced lung injury in Wistar rats. 24 female Wistar rats were randomly assigned into 3 groups (n = 8): a control group, a LPS-challenged group, and a LPS plus dexmedetomidine group. Inflammation, oxidative stress, Nrf2/Keap1, and Akt signal were determined. The results showed that LPS caused inflammation and oxidative stress via increasing pro-inflammatory cytokines and oxidative products. Dexmedetomidine treatment alleviated inflammation and oxidative stress in LPS-challenged rats. Nrf2/Keap1 was inhibited and Akt signal was activated in the lung after exposure to LPS, while dexmedetomidine activated Nrf2/Keap1, which further mediated expressions of antioxidant genes. In conclusion, dexmedetomidine alleviated inflammatory response and oxidative stress in LPS-induced lung injury in rats via influencing Nrf2/Keap1 signal.

EFFECT OF INTRAPERITONEAL RESUSCITATION WITH DIFFERENT CONCENTRATIONS OF SODIUM PYRUVATE ON INTESTINAL ISCHEMIA REPERFUSION INJURY IN HEMORRHAGIC SHOCK RAT.

Objective: To determine the effects of intraperitoneal resuscitation (PR) with different concentrations of sodium pyruvate (PY) on intestinal ischemia reperfusion injury in rats hemorrhagic shock (HS). Methods: Sixty rats were randomly assigned to six groups. These included: group SHAM, intravenous resuscitation only (VR) group, and four PR groups based on resuscitation fluid: glucose-lactate-based peritoneal dialysis solution (LA), and PY-1.1%, PY-1.6%, and PY-2.2% (concentrations in grams/dL). Mean arterial pressure (MAP) was monitored continuously. Blood pH, base excess (BE), lactate, intestinal myeloperoxidase (MPO), malondialdehyde (MDA), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), activated caspase-3, and zonula occludens-1 (ZO-1) were measured; intestinal mucosal damage index (IMDI) and subcellular changes were observed; apoptotic index (AI) was calculated. Results: Three hours after resuscitation, in PY groups, MPO, MDA, IMDI, AI, TNF-alpha, and IL-6 were significantly lower than VR and LA groups, while pH and BE were higher. PY groups showed less expression of activated caspase-3 but elevated ZO-1. Among PY groups, group PY-1.1% had the lowest MPO, MDA and TNF-alpha, and had less pathological damage and subcellular changes than other experimental groups. Conclusions: PR using PY solution combined with VR provided protection against intestinal ischemia-reperfusion injury following HS and resuscitation. Under the same hypertonic condition, 1.1% PY solution showed significant advantages compared with 2.2% and 1.6% solutions. The underlying mechanisms may include the maintenance of hemodynamic stability, regulation of homeostasis, inhibition of oxidative stress and inflammation, and protection of intestinal epithelial tight junction barrier function.

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Attenuates Lung Injury in Septic Shock Rats

Oxidative stress and inflammation have been identified to play a vital role in the pathogenesis of lung injury induced by septic shock. Heme oxygenase-1 (HO-1), an effective antioxidant and anti-inflammatory and antiapoptotic substance, has been used for the treatment of heart, lung, and liver diseases. Thus, we postulated that administration of exogenous HO-1 protein transduced by cell-penetrating peptide PEP-1 has a protective role against septic shock-induced lung injury. Septic shock produced by cecal ligation and puncture caused severe lung damage, manifested in the increase in the lung wet/dry ratio, oxidative stress, inflammation, and apoptosis. However, these changes were reversed by treatment with the PEP-1-HO-1 fusion protein, whereas lung injury in septic shock rats was alleviated. Furthermore, the septic shock upregulated the expression of Toll-like receptor 4 (TLR4) and transcription factor NF-κB, accompanied by the increase of lung injury. Administration of PEP-1-HO-1 fusion protein reversed septic shock-induced lung injury by downregulating the expression of TLR4 and NF-κB. Our study indicates that treatment with HO-1 protein transduced by PEP-1 confers protection against septic shock-induced lung injury by its antioxidant, anti-inflammatory, and antiapoptotic effects.