ZnPP Group Research Articles

A Mechanism Study on the Antioxidant Pathway of Keap1-Nrf2-ARE Inhibiting Ferroptosis in Dopaminergic Neurons.

The pathology of Parkinson's disease (PD) indicates that iron deposition exists in dopaminergic neurons, which may be related to the death of cellular lipid iron peroxide. The extracellular autophagy adaptor SQSTM1(p62) of dopamine (DA) neurons can activate the intracellular Keap1-Nrf2-ARE signaling pathway to inhibit ferroptosis, which has a protective effect on DA neurons. The objective of this study was to investigate the protective mechanism of the Keap1- Nrf2-ARE antioxidant pathway against iron death in dopaminergic neurons. The experiment was divided into a control group (Control group), 1-methyl-4- phenylpyridiniumion control group (MPP+ Control group), p62 overexpression group (MPP+OVp62), and p62 overexpression no-load group (MPP+ OV-P62-NC). The inhibitors brusatol and ZnPP inhibited the activation of NF-E2-related factor 2(Nrf2) and Heme oxygenase-1(HO-1), respectively, and were divided into brusatol group (MPP+OV-p62+brusatol) and ZnPP group (MPP+OV-p62+ZnPP). RT-qPCR was used to detect transfection efficiency, and Cell Counting Kit-8 (CCK8) was used to detect cell activity. FerroOrange, 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Liperfluo probes were used to detect intracellular iron, reactive oxygen species (ROS), and lipid peroxidation (LPO) levels. Western Blotting detected the levels of Nrf2, HO-1, Kelch-like ECH-associated protein1 (Keap1), and their downstream Glutathione peroxidase 4(GPX4) and Acyl-CoA synthetase long-chain family member 4(ACSL4). The levels of LGlutathione (GSH) and Malondialdehyde (MDA) were detected by GSH and MDA kits, and the activation of Keap1-Nrf2-ARE pathway was verified at the cellular level to have an antioxidant protective effect on iron death in dopaminergic neurons. (1) The results of RT-qPCR showed that compared with the control group, the expression of the p62 gene was significantly increased in the MPP+OV-p62 groups (P = 0.039), and the p62 gene was significantly increased in the brusatol and ZnPP groups, indicating successful transfection (P =0.002; P=0.008). (2) The immunofluorescence probe flow results showed that compared to the normal control group, the contents of three kinds of probes in MPP+ model group were significantly increased (P =0.001; P ＜0.001; P＜0.001), and the contents of three kinds of probes in MPP+OV-p62 group were decreased compared to the MPP+ model group (P =0.004). The results indicated that the levels of iron, ROS, and LPO were increased in the MPP+ group and decreased in the MPP+OV-p62 group. (3) Compared with the control group, the expressions of Nrf2, HO-1, and GPX4 in the MPP+OV-p62 group were increased (P =0.007; P =0.004; P=0.010), and the expressions of Keap1 and ACSL4 in MPP+p62 overexpression group were decreased (P =0.017; P =0.005). Compared with the MPP+ control group, Nrf2 and GPX4 were increased in the MPP+OV-p62 group, and ACSL4 was decreased in the MPP+OVp62 group (P =0.041; P ＜0.001; P ＜0.001). The results of the GSH and MDA kit showed that compared with the normal control group, the content of GSH in the MPP+ control group was decreased (P < 0.01), and the content of MDA was increased (P < 0.01). Compared with the MPP+ model group, GSH content was increased (P = 0.003), and MDA content was decreased in the MPP+OV-p62 group (P < 0.001). Nrf2, HO-1, and GPX4 increased in the MPP+p62 overexpression group but decreased in the brusatol group and ZnPP group (P < 0.001). Based on the transfection of P62 plasmid, it was found that P62 plasmid can inhibit the lipid peroxidation of iron death in dopaminergic nerve cells by activating the Nrf2 signaling pathway, thus playing a protective role in dopaminergic nerve cells.

Elevation of HO-1 expression protects the intestinal mucosal barrier in severe acute pancreatitis via inhibition of the MLCK/p-MLC signaling pathway

In severe acute pancreatitis (SAP), intestinal mucosal barrier damage can cause intestinal bacterial translocation and induce or aggravate systemic infections. Heme oxygenase-1 (HO-1) is a validated antioxidant and cytoprotective agent. This research aimed to investigate the effect and mechanism of HO-1 on SAP-induced intestinal barrier damage in SAP rats. Healthy adult male Sprague-Dawley rats were randomly separated into the sham-operated group, SAP group, SAP + Hemin group, and SAP + Znpp group. The rat model of SAP was established by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Hemin (a potent HO-1 activator) and Znpp (a competitive inhibitor of HO-1) were injected intraperitoneally in the selected groups 24 h before SAP. Serum and intestinal tissue samples were collected for analysis after 24 h in each group. Hemin pretreatment significantly reduced systemic inflammation, intestinal oxidative stress, and intestinal epithelial apoptosis in SAP by increasing HO-1 expression. Meanwhile, pretreatment with Hemin abolished the inhibitory effect on the expression of the tight junction proteins and significantly inhibited the activation of the MLCK/P-MLC signaling pathway. Conversely, ZnPP completely reversed these effects. Our study indicates that upregulation of HO-1 expression attenuates the intestinal mucosal barrier damage in SAP. The protective effect of HO-1 on the intestine is attributed to MLCK/p-MLC signaling pathway inhibition.

Abdominal puncture drainage alleviates severe acute pancreatitis in rats by activating Nrf-2/HO-1 pathway and promoting autophagy

To assess the effect of early abdominal puncture drainage (APD) on autophagy and Nrf-2/HO-1 pathway in rats with severe acute pancreatitis (SAP) and explore the possibile mechanism. Thirty-two male SD rats were randomly divided into sham-operated (SO) group, SAP group with retrograde injection of 4% sodium taurocholate, APD group with insertion of a drainage tube into the lower right abdomen after SAP induction, and APD + ZnPP group with intraperitoneal injection of 30 mg/kg ZnPP 12 h before APD modeling. Blood samples were collected from the rats 12 h after modeling for analysis of amylase and lipase levels and serum inflammatory factors. The pathological changes of the pancreatic tissue were observed with HE staining. Oxidative stress in the pancreatic tissue was detected with colorimetry, and sub-organelle structure and autophagy in pancreatic acinar cells were observed by transmission electron microscopy. The expressions of autophagy-related proteins and Nrf-2/HO-1 pathway were detected using RT-PCR and Western blotting. Compared with those in SAP group, the rats with APD treatment showed significantly alleviated pathologies in the pancreas, reduced serum levels of lipase, amylase and inflammatory factors, lowered levels of oxidative stress, and activated expressions of Nrf-2/HO-1 pathway in the pancreas. The ameliorating effect of ADP was significantly inhibited by ZnPP treatment before modeling. APD obviously reversed mitochondrial and endoplasmic reticulum damages and p62 accumulation induced by SAP. APD treatment can suppress oxidative stress and repair impaired autophagy in rats with SAP by activating the Nrf-2/HO-1 pathway, thereby reducing the severity of SAP.

Heme Oxygenase-1 Protects Bone Marrow Mesenchymal Stem Cells Against Iron Overload through Decreasing ROS and Promoting IL-10 Generation

Background:Iron overload caused by frequent transfusions of suspended red blood cells which is of major concern in hematological disease. Heme oxygenase-1(HO-1) is well known as a crucial factor to cells against oxidative stress. In this study, significant upregulation of HO-1 was observed in bone marrow mesenchymal stem cells (BMMSCs) in the early phase of IO and predicted good prognosis in mice.Aim: Given that the role of HO-1 in protecting (BMMSCs) against damage from IO was still unknown. In the study, we investigated the effects and mechanisms of iron overload-related ROS by inducing IL-10 by regulating HO-1.METHODS:Male C57BL/6Ly5.2 (Ly45.2) mice were housed under specific pathogen-free conditions and allowed to acclimate for 2 weeks before experiments. The mice were housed (5 individuals per cage) and used at a weight of approximately 20.0 g. Thirty male mice were recipient mice, and another five were competitive mice. The thirty-five mice were randomly divided into four groups: (a) a control group; (b) an IO group (25 mg/ml); (c) an IO + hemin group and (d) an IO + ZnPP group. The depositions of iron in the liver, spleen and BM were assessed using HE staining and BM iron staining. Then, the expression of HO-1 and IL-10 were examined by Western blotting and q-PCR. Cell proliferation was measured by CCK-8 assay. The quantity of Ferritin, Serum iron, Hepcidin and IL-10 in the blood serum from iron overload cell were detected by ELISA in mouse model and iron overload patients. Finally, the levels of apoptotic rate, intracellular ROS and labile iron pool (LIP) were analyzed by flow cytometer.Result :We found that HO-1 was upregulated in the excess iron culture of BMMSCs in the early phase. Meanwhile, the apoptosis rate of IO BMMSCs was increased by 3.0 times compared with that of CON group. On the contrary, the apoptotic rate of BMMSCs down-regulated by transcription of si-HO-1 virus increased by 7.12 times compared to the CON group. In addition, the apoptosis rate of HO-1 overexpressing BMMSCs was significantly decreased in BM-1 virus transfected with HO-1 virus. Next, we also observed LIP in IO BMMSCs transfected with HO-1 virus was significantly lower than IO BMMSCs in un-transfected group. It suggested that HO-1 up-regulated the BMMSCs was protected from IO damage by decreasing intracellular iron content in vitro.To get an insight into the mechanism involved in cells protection from IO by HO-1 upregulation. We further explore production of ROS and secrection of IL10. As HO-1 was reported to have ability to reduce ROS, and IL-10 could promote excretion of intracellular iron. The results showed the ROS level in IO BMMSC was improved by 5.94 times compared with that of CON group, and the level of ROS in IO BMMSCs transcribed with si-HO-1 and HO-1 virus increased by 6.18 and 4.53 times, respectively. And IL-10 was increased significantly in the HO-1 up-regulated group.Next, we used U0126 (an ERK pathway inhibitor) to co-culture with IO BMMSCs that transcribed HO-1 virus. We observed the secretion of ferritin and the expression of IL-10 were consistent with the BMMSCs concentration changed. In vivo, we found that the serum iron and ferritin levels in the peripheral blood of IO mice were reduced by 1.92 and 1.3 times through ELISA analysis. The levels of IL-10 and ferritin were detected by ELISA. The results of deposition detection in bone marrow were consistent with the in vitro model. Finally, from November 2015 to December 2016, 12 patients diagnosed iron overload were enrolled. Including 6 males and 6 females, aged 44 to 77 years old. By q-PCR, we also found that iron overload in BMMSCs with high expression of HO-1 and IL-10 was reduced in serum ferritin after deferoxamine treatment. Therefore, we hypothesized that HO-1 overexpression of iron overload patients with better prognosis.Conclusions: we hypothesized that HO-1 overexpression of iron overload patients with better prognosis. The results of this study allow for further study of mechanisms of IO-related disease and provide new therapeutic targets for the treatment of IO patients. DisclosuresNo relevant conflicts of interest to declare.

κ‑opioid receptor agonist U50488H attenuates postoperative cognitive dysfunction of cardiopulmonary bypass rats through the PI3K/AKT/Nrf2/HO‑1 pathway.

Postoperative cognitive dysfunction (POCD) is a common complication following cardiopulmonary bypass (CPB). U50488H, a κ-opioid receptor (KOR) agonist, can specifically activate KORs on hippocampal nerve cells, resulting in neuroprotective effects. The present study established a CPB rat model, observed the protective effect of U50488H on CPB-induced POCD and brain damage and explored the regulatory mechanism of the PI3K/AKT/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 pathway. Sprague-Dawley rats were divided into the following groups: Sham operation (Sham group), CPB (CPB group), KOR agonist (U50488H) + CPB (U50488H group), CPB + U50488H + HO-1 antagonist (ZnPP-IX; ZnPP group) and CPB + U50488H + PI3K antagonist (LY294002; LY294002 group), with 10 rats in each group. Neurological scores and the Morris water maze test were used to evaluate cognitive function; hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed to observe hippocampal neuron damage in rats. Immunofluorescence was used to detect reactive oxygen species, glial fibrillary acidic protein and Nrf2 expression in the hippocampus. Enzyme-linked immunosorbent assays were used to detect inflammatory and oxidative stress factors. Western blotting was used to examine the expression of PI3K/AKT/Nrf2/HO-1-related proteins. It was demonstrated that U50488H significantly reduced the neural function score of rats with POCD induced by CPB, relieved cognitive dysfunction, reduced hippocampal neuron damage, inhibited the rate of apoptosis, repaired oxidative stress injury and protected against brain damage caused by CPB. In addition, U50488H could promote Nrf2 entry into the nucleus and upregulate HO-1 and thioredoxin 1 (Trx1) expression. In CPB rats treated with PI3K inhibitors, less Nrf2 was detected in the nucleus and HO-1 and Trx-1 expression levels were reduced in the nucleus. Therefore, U50488H, a KOR agonist, can activate Nrf2/HO-1 via the PI3K/AKT pathway to improve cognitive function and reduce brain damage in CPB rats.

Effects of curcumin on liver fibrosis induced by cholestasis in mice

To investigate the protective effects of curcumin on bile duct ligation(BDL)-induced liver cholestasis in mice, so as to provide a new treatment strategy for liver fibrosis. Forty-two healthy adult male BALB/c mice were randomly divided into sham group (n =6), sham+curcumin group (n=6), BDL treatment group (n=10), BDL+curcumin group(n=10), BDL+curcumin+ZnPP group (n=10). Seven days after BDL operation, the sham operation + curcumin group and the BDL+ curcumin group were treated with curcumin at the dose of 30 mg/kg by intraperitoneal injection once a day for 7 days.The mice in BDL+ curcumin +ZnPP group were treated with curcumin (30 mg/kg) and ZnPP (50 μmol/kg) by intraperitoneal injection once a day for 7 days. For the sham group and the BDL group, mice were treated with equal-volume saline daily by intraperitoneal injection. After 14 days of BDL, the plasma and liver tissues were collected, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. The pathological changes of liver tissue and liver fibrosis were observed, and the protein expression of HO-1 in liver tissue was detected. Compared with the sham group, mice in the BDL group had enlarged liver gallbladder and the serum levels of ALT and AST were increased significantly (P＜0.05). Meanwhile, the results of Sirius red staining and qRT-PCR of pro-fibrosis related genes showed collagen deposition in the liver, and immunohistochemistry of macrophages and neutrophils showed inflammatory cell infiltration in the liver. Compared with the BDL group, the serum levels of ALT and AST in the curcumin treatment group were decreased significantly (P＜0.05), collagen deposition and inflammatory cell infiltration were improved, and HO-1 expression was increased (P＜0.05) after curcumin treatement. In the curcumin treatment group, the protective effect of curcumin on liver injury could be reversed by HO-1 active inhibitor ZnPP. Curcumin can improve liver inflammation and fibrosis caused by BDL, and this protective effect is related to the regulation of HO-1 activity by curcumin.

HO-1 protects the nerves of rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.

Objective: This study aimed to investigate the neuroprotective effect of heme oxygenase-1 (HO-1) on the PI3K/AKT signaling pathway in rats with cerebral hemorrhage.Materials and methods: Adult male Sprague-Dawley rats were randomly divided into: a sham group, a model group and an HO-1 inhibitor group (ZnPP group). Functional defects after surgery were scored according to the Longa5 standard. Hemotoxylin and eosin staining was used to detect whether the model was constructed successfully. Superoxide dismutase (SOD) vitality and malondialdehyde (MDA) content were calculated by the xanthine oxidase method and thiobarbituric acid method, respectively. Blood-brain barrier permeability was measured by Evans Blue. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The expression of Bcl-2 and BAX was evaluated by immunohistochemistry and the expression of PI3K, p-PI3K, AKT and p-AKT was tested by Western blotting.Results: The rat intracerebral hemorrhage model was successfully constructed. Compared with the model group, the bleeding in the ZnPP group was more serious, the cell edema and deformation were aggravated, and the neurological deficit score in the rat was significantly increased. In addition, the content of Evans blue, MDA, the number of apoptotic cells, the water content of brain tissue and the expression of BAX were significantly increased, while the SOD activity and the expressions of Bcl-2, p-PI3K and p-AKT protein were decreased.Conclusion: HO-1 could protect the nerves of rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.

Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α

To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group ( P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased ( P<0.05), while in ZnPP group it increased ( P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group ( P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced ( P<0.05); the expression of HO-1 protein in ZnPP group decreased ( P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased ( P<0.05), and the expression of p-P65 protein was not significantly changed ( P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced ( t=3.076, P=0.031). HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.

Biliary tract external drainage protects against multiple organs injuries of severe acute pancreatitis rats via heme oxygenase-1 upregulation

ObjectiveTo investigate the effect of biliary tract external drainage (BTED) on severe acute pancreatitis (SAP) in rats and the relationship with heme oxygenase-1 (HO-1) pathway. MethodsThirty SD rats weighing 250–300 g were randomly assigned into five groups (n = 6): sham surgery (SS) group, SAP group, SAP + BTED group, SAP + zinc protoporphyrin IX (ZnPP) group, SAP + BTED + ZnPP group. The SAP model was induced via retrograde injection of 4% sodium taurocholate (1 mL/kg) into biliopancreatic duct through duodenal wall. BTED was performed by inserting a cannula into the bile duct of SAP rats. Tissue and blood samples were collected 24 h after surgery. Pathological changes in organs were scored. The level of amylase, alanine transaminase (ALT), aspartate aminotransferase (AST), diamine oxidase (DAO), lipopolysaccharide (LPS), myeloperoxidase (MPO) and ability to inhibit hydroxyl radical(·OH) in serum were measured. The expression of hemeoxygenase-1 (HO-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in tissues were analyzed by RT- PCR and western-blot. ResultsOrgans damage in SAP rats was significantly alleviated by BTED (p < 0.05). Compared to the SAP group, the serum level of amylase, ALT, AST, DAO, MPO, and LPS were significantly lower in the SAP + BTED group, and the ability to inhibit ·OH was significantly higher (p < 0.05). The BETD treatment led to a significant reduction of TNF-α, IL-6 level and a significant increase of HO-1 level in tissues than in SAP rats (p < 0.05). ZnPP significantly inhibited all above mentioned changes. ConclusionsBTED protected multiple organs against SAP related injuries via HO-1 upregulation.

Hydrogen-rich water protects against inflammatory bowel disease in mice by inhibiting endoplasmic reticulum stress and promoting heme oxygenase-1 expression.

AIMTo investigate the therapeutic effect of hydrogen-rich water (HRW) on inflammatory bowel disease (IBD) and to explore the potential mechanisms involved.METHODSMale mice were randomly divided into the following four groups: control group, in which the mice received equivalent volumes of normal saline (NS) intraperitoneally (ip); dextran sulfate sodium (DSS) group, in which the mice received NS ip (5 mL/kg body weight, twice per day at 8 am and 5 pm) for 7 consecutive days after IBD modeling; DSS + HRW group, in which the mice received HRW (in the same volume as the NS treatment) for 7 consecutive days after IBD modeling; and DSS + HRW + ZnPP group, in which the mice received HRW (in the same volume as the NS treatment) and ZnPP [a heme oxygenase-1 (HO-1) inhibitor, 25 mg/kg] for 7 consecutive days after IBD modeling. IBD was induced by feeding DSS to the mice, and blood and colon tissues were collected on the 7th d after IBD modeling to determine clinical symptoms, colonic inflammation and the potential mechanisms involved.RESULTSThe DSS + HRW group exhibited significantly attenuated weight loss and a lower extent of disease activity index compared with the DSS group on the 7th d (P < 0.05). HRW exerted protective effects against colon shortening and colonic wall thickening in contrast to the DSS group (P < 0.05). The histological study demonstrated milder inflammation in the DSS + HRW group, which was similar to normal inflammatory levels, and the macroscopic and microcosmic damage scores were lower in this group than in the DSS group (P < 0.05). The oxidative stress parameters, including MDA and MPO in the colon, were significantly decreased in the DSS + HRW group compared with the DSS group (P < 0.05). Simultaneously, the protective indicators, superoxide dismutase and glutathione, were markedly increased with the use of HRW. Inflammatory factors were assessed, and the results showed that the DSS + HRW group exhibited significantly reduced levels of TNF-α, IL-6 and IL-1β compared with the DSS group (P < 0.05). In addition, the pivotal proteins involved in endoplasmic reticulum (ER) stress, including p-eIF2α, ATF4, XBP1s and CHOP, were dramatically reduced after HRW treatment in contrast to the control group (P < 0.05). Furthermore, HRW treatment markedly up-regulated HO-1 expression, and the use of ZnPP obviously reversed the protective role of HRW. In the DSS + HRW + ZnPP group, colon shortening and colonic wall thickening were significantly aggravated, and the macroscopic damage scores were similar to those of the DSS + HRW group (P < 0.05). The histological study also showed more serious colonic damage that was similar to the DSS group.CONCLUSIONHRW has a significant therapeutic potential in IBD by inhibiting inflammatory factors, oxidative stress and ER stress and by up-regulating HO-1 expression.

Endogenous carbon monoxide downregulates hepatic cystathionine-γ-lyase in rats with liver cirrhosis.

The aim of the present study was to investigate the effect of endogenous carbon monoxide (CO) on the hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) pathway in cirrhotic rat livers. The rats were allocated at random into four groups: Sham, cirrhosis, cobalt protoporphyrin (CoPP) and zinc protoporphyrin IX (ZnPP). The expression of hepatic CSE mRNA was evaluated using a quantitative polymerase chain reaction, while CSE protein expression was determined using immunohistochemical analysis. Hematoxylin and eosin staining was performed for the histological evaluation of liver fibrosis. The levels of H2S, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and carboxyhemoglobin (COHb) in the arterial blood were determined, in addition to the portal vein pressure. The mRNA and protein expression levels of hepatic CSE and the serum levels of H2S were significantly decreased in the cirrhosis group compared with those in the sham group (P<0.05). Compared with the cirrhosis group, rats in the ZnPP group had significantly lower levels of serum ALT, AST and TBIL, arterial COHb and hepatic fibrosis, while hepatic CSE expression and the production of H2S were significantly increased (P<0.05). The CoPP group exhibited decreased hepatic CSE expression and H2S production, but aggravated hepatic function and fibrosis (P<0.05). In conclusion, the H2S/CSE pathway is involved in the formation of liver cirrhosis and serves a crucial function in protecting liver cells against the progression of liver fibrosis. Endogenous CO downregulates hepatic CSE mRNA and protein expression and the production of H2S in rats with liver cirrhosis.

Inflammation and Edema in the Lung and Kidney of Hemorrhagic Shock Rats Are Alleviated by Biliary Tract External Drainage via the Heme Oxygenase-1 Pathway.

The lung and kidney are two organs that are easily affected by hemorrhagic shock (HS). We investigated roles of biliary tract external drainage (BTED) in inflammation and edema of the lung and kidney in HS and its relationship with the heme oxygenase-1 (HO-1) pathway. Rat models of HS were induced by drawing blood from the femoral artery until a mean arterial pressure (MAP) of 40 ± 5mmHg was achieved. A MAP of 40 ± 5mmHg was maintained for 60min. Thirty-six Sprague-Dawley rats were randomized to the following groups: sham group; HS group; HS + zinc protoporphyrin IX (ZnPP), a specific HO-1 inhibitor, group; HS + BTED group; HS + BTED + ZnPP group; and HS + BTED + bile infusion (BI) group. HO-1 levels, aquaporin-1 levels, and ratios of dry/wet in the lung and kidney increased markedly after BTED, but tumor necrosis factor-α and myeloperoxidase levels in the lung and kidney decreased significantly after BTED under HS conditions. Under the condition that HO-1 was inhibited by ZnPP, all these effects induced by BTED disappeared in the lung and kidney. These results demonstrated that inflammation and edema of the lung and kidney of HS rats are alleviated by BTED via the HO-1 pathway.

Biliary tract external drainage alleviates kidney injury in shock

BackgroundKidney injury is common in hemorrhagic shock (HS). Kidney injury leads to a systemic increase in serum chemokines and cytokines and causes injuries to other vital organs. Our previous studies showed that vitamin C led to organ protection and inflammation inhibitory effects in rat models of HS via induction heme oxygenase-1 (HO-1). We also found that biliary tract external drainage (BTED) increased the expression levels of HO-1 in rat livers. We investigated roles of BTED in kidney injury and its relationship with the HO-1 pathway in HS in this research. MethodsRat models of HS were induced by drawing blood from the femoral artery. BTED was performed by inserting a catheter into the bile duct. Thirty-six Sprague–Dawley rats were randomized to sham group; HS group; zinc protoporphyrin IX (Znpp) group; BTED group; BTED + Znpp group, and BTED + bile infusion group. The expression levels of HO-1 in the kidney were analyzed by Western blotting. The expression levels of occludin messenger RNA in the kidney were analyzed by real-time reverse transcription-polymerase chain reaction. The expression levels of occludin in the kidney were analyzed by immunohistochemistry. Histology of renal was performed by hematoxylin and eosin staining. ResultsOccludin messenger RNA and protein levels in the kidney increased markedly after BTED under HS conditions. Renal histopathologic scores decreased significantly after BTED under HS conditions. Znpp significantly inhibited all mentioned effects. ConclusionsBTED alleviates kidney injury in rats of HS via the HO-1 pathway.

Hemin inhibits NLRP3 inflammasome activation in sepsis-induced acute lung injury, involving heme oxygenase-1

NLRP3 inflammasome activation contributes to acute lung injury (ALI), accelerating caspase-1 maturation, and resulting in IL-1β and IL-18 over-production. Heme oxygenase-1 (HO-1) plays a protective role in ALI. This study investigated the effect of hemin (a potent HO-1 inducer) on NLRP3 inflammasome in sepsis-induced ALI. The sepsis model of cecal ligation and puncture (CLP) was used in C57BL6 mice. In vivo induction and suppression of HO-1 were performed by pretreatment with hemin and zinc protoporphyrin IX (ZnPP, a HO-1 competitive inhibitor) respectively. CLP triggered significant pulmonary damage, neutrophil infiltration, increased levels of IL-1β and IL-18, and edema formation in the lung. Hemin pretreatment exerted inhibitory effect on lung injury and attenuated IL-1β and IL-18 secretion in serum and lung tissue. In lung tissues, hemin down-regulated mRNA and protein levels of NLRP3, ASC and caspase-1. Moreover, hemin reduced malondialdehyde and reactive oxygen species production, and inhibited NF-κB and NLRP3 inflammasome activity. Meanwhile, hemin significantly increased HO-1 mRNA and protein expression and HO-1 enzymatic activity. In contrast, no significant differences were observed between the CLP and ZnPP groups. Our study suggests that hemin-inhibited NLRP3 inflammasome activation involved HO-1, reducing IL-1β and IL-18 secretion and limiting the inflammatory response.

Isoflurane post-treatment improves pulmonary vascular permeability via upregulation of heme oxygenase-1

ABSTRACTIsoflurane (ISO) has been shown to attenuate acute lung injury (ALI). Induction of heme oxygenase-1 (HO-1) and suppression of inducible nitric oxide synthase (iNOS) expression provide cytoprotection in lung and vascular injury. The aim of this study was to investigate the effect of post-treatment with isoflurane on lung vascular permeability and the role of HO-1 in an ALI rat model induced by cecal ligation and puncture (CLP). Male Sprague–Dawley rats were randomly assigned to one of four groups: sham group, sham rats post-treated with vehicle (Sham); CLP group, CLP rats post-treated with vehicle (CLP); ISO group, CLP rats post-treated with isoflurane (ISO); and ZnPP group, CLP rats injected with zinc protoporphyrin IX (ZnPP), a competitive inhibitor of HO-1, 1 hour before the operation, and post-treated with isoflurane (ZnPP). Isoflurane (1.4%) was administered 2 hour after CLP. At 24 hour after CLP, the extent of ALI was evaluated by lung wet/dry ratio, Evans blue dye (EBD) extravasation, lung permeability index (LPI), as well as histological and immunohistochemical examinations. We also determined pulmonary iNOS and HO-1 expression. Compared with the CLP group, the isoflurane post-treatment group showed improved pulmonary microvascular permeability as detected by EBD extravasation, LPI, as well as histological and immunohistochemical examinations. Furthermore, isoflurane decreased iNOS and increased HO-1 expression in lung tissue. Pretreatment with ZnPP prevented the protective effects of isoflurane in rats. These findings indicate that the protective role of isoflurane post-conditioning against CLP-induced lung injury may be associated with its role in upregulating HO-1 in ALI.

Heme oxygenase/carbon monoxide pathway inhibition plays a role in ameliorating fibrosis following splenectomy

Splenectomy is a recognized therapy for liver cirrhosis with splenomegaly, since it decreases free iron concentration that accompanies the destruction of red blood cells. Heme oxygenase (HO)-1 and its by-products, iron and carbon monoxide (CO), play crucial roles in hepatic fibrosis. The aim of the present study was to determine whether splenectomy in cirrhotic rats induced by bile duct ligation (BDL), through the HO/CO pathway, could slow down the development of liver fibrosis. Male Sprague-Dawley rats were divided randomly into the sham, BDL, splenectomy, Fe, zinc protoporphyrin (Znpp) and cobalt protoporphyrin (Copp) treatment groups, for inhibiting and inducing HO-1 expression. The level of HO-1 was detected by western blot analysis and reverse transcription-polymerase chain reaction. Serum carboxyhemoglobin (COHb), iron and portal vein pressure (PVP) were also quantified. Liver iron was measured by atomic absorption spectrometry with acetylene-air flame atomization. HO-1 and α-smooth muscle actin (α-SMA) were localized by immunohistochemistry. Liver and spleen iron were visualized by Perls' Prussian blue staining. Hepatic fibrosis was assessed using hematoxylin and eosin (H&E) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum transforming growth factor-β1 (TGF-β1). The results showed that liver, spleen and serum levels of HO-1, COHb and iron were greatly enhanced in the BDL group compared with the sham group; they were reduced following splenectomy and Znpp treatment, but were elevated in the Copp and Fe groups. Hydroxyproline, TGF-β1, α-SMA, PVP and malonaldehyde levels were lower in the splenectomy and Znpp groups compared to BDL, while higher levels were observed in the Copp and Fe-treated groups. Our study shows that splenectomy reduces iron and CO levels in part by reducing HO-1 expression, and it decreases portal pressure and slightly decreases hepatic fibroproliferation.

The effects of Zinc Protoporphyrin and Heme on the expression of HO-1 in cochlear and the change of auditory brainstem response on diffuse traumatic brain injury model of rats

Objective To explore the effects of Zinc Protoporphyrin and Heme on the expression of HO-1 in cochlear and the change of auditory brainstem response on diffuse traumatic brain injury model of rats.Methods Diffuse traumatic brain injury model of rats were established and randomly divided into thirteen groups.Then auditory brainstem response examination,light microscope,immunohistochemistry technique were used to evaluate the change of auditory brainstem response and the expression of HO-1 in cochlear.Results The differences of auditory brainstem response threshold and latency of wave between the experimental and the normal control group were obvious(P＜0.05).The expression of HO-1 in the control group was normal,whereas there were obvious changes of inner ear HO-1 expression in the traumatic groups.The grey value of HO-1 expression in trauma group,Znpp group and heme group was significantly associated with auditory function change(P＜0.05).Conclusion There were influence of Zinc Protoporphyrin and Hemeon the inner ear HO-1 expression and the change of auditory brainstem response with diffuse traumatic brain injury model of rats.The protective effect of heme on auditory function may be associated with the increased expression of HO-1. Key words: Diffuse traumatic brain injury; Zinc Protoporphyrin; Heme; Cochlear; Heme oxygenase; Auditory Brainstem Response

A NOVEL VWF A3-GPI MODIFIED EPCS TO ENHANCE ITS ADHESION ABILITY TO DAMAGED VESSEL SEGMENT-COLLEGEN

ObjectivesThis study was designed to investigate the relationship between hame oxygenase (HO)/carbon monoxide (CO) system and nitric oxide synthase (NOS)/nitric monoxide (NO) system in diet-induced atherosclerosis.MethodsThe rabbits serving as animal...

Effects and mechanisms of heme oxygenase-1 on rats with postresuscitation myocardial dysfunction

To explore the effects and mechanisms of heme oxygenase-1 on rats with postresuscitation myocardial dysfunction. Male Sprague-Dawley rats were asphyxiated for 9 minutes and resuscitated. They were randomly divided into 4 groups: sham-operated, cardiopulmonary resuscitation (CPR), hemin and hemin + ZnPP (zinc protoporphyrin IX). Resuscitated groups had 2 observation points: 6 and 24 hours post-CPR (n = 8 for each time point). And the sham-operated group of 12 rats were divided in two observation points, according to 6 or 24 hours post-operation (n = 6 each). Hemodynamic was observed. The expression of heme oxygenase-1 (HO-1) in cardiac tissue was detected by Western blot. And the activity of cardiac homogenate superoxide dismutase (SOD) was determined by xanthine oxidase method and the level of malondialdehyde (MDA) measured by the thiobarbituric acid method. Nitrotyrosine protein expression in cardiac tissue was analyzed by immunohistochemistry. (1) The mean blood pressure (MAP) significantly decreased in resuscitated groups after resuscitation (all P < 0.05). No difference existed between the subgroups. The scores of dP/dt40 and -dP/dt significantly decreased in CPR and hemin + ZnPP groups after resuscitation (all P < 0.05). But dP/dt40 in hemin group did not differ significantly after resuscitation and -dP/dt decreased only 0.5 hour and 1 hour post-resuscitation (3341.60 ± 524.85 and 3711.40 ± 502.39 vs 4284.20 ± 800.87, all P < 0.05). The scores of dP/dt40 and -dP/dt in hemin group at all time points post-resuscitation were significantly higher than those in CPR and hemin + ZnPP groups (all P < 0.05). (2) Compared with the sham-operated group, the HO-1 expression, MDA level and nitrotyrosine protein expression significantly increased while the activities of SOD decreased after resuscitation in the CPR, hemin and hemin + ZnPP groups (all P < 0.05). Compared with the CPR and hemin + ZnPP groups, the expression of HO-1 and the activity of SOD increased, while MDA level and nitrotyrosine protein expression were decreased in group hemin (all P < 0.05). No difference existed in the above indices between the CPR and hemin + ZnPP groups. HO-1 can reduce myocardial oxidative stress injury after cardiopulmonary resuscitation and effectively improve post-resuscitation myocardial function in rats.

Effect of heme oxygenase-1 on renal function in rats with liver cirrhosis

To investigate the role of heme oxygenase-1 (HO-1) in pathogenesis of experimental hepatorenal syndrome (HRS). Rats were divided into liver cirrhotic group, zinc protoporphyrin IX (ZnPP) treatment group, cobalt protoporphyrin (CoPP) treatment group and sham group. Biliary cirrhosis was established by bile duct ligation in the first three groups. Rats in the ZnPP and CoPP treatment groups received intraperitoneal injection of ZnPP and CoPP, respectively, 24 h before sample collection. Expression of HO-1 mRNA in kidney was detected by reverse-transcription polymerase chain reaction, while protein expression was determined by immunohistochemical analysis. Hematoxylin and eosin staining was performed to observe liver cirrhosis and renal structure. Renal artery blood flow, mean arterial pressure and portal vein pressure, 24 h total urinary volume, serum and urine sodium concentrations, and creatinine clearance rate (Ccr) were also measured. The HO-1 mRNA and protein expression levels in kidney, 24 h total urinary volume, renal artery blood flow, serum and urine sodium concentration and Ccr were lower in cirrhotic group than in sham group (P < 0.05). However, they were significantly lower in ZnPP treatment group than in cirrhotic group and significantly higher in CoPP treatment group than in cirrhotic group (P < 0.05). Low HO-1 expression level in kidney is an important factor for experimental HRS.