Hemin Group Research Articles

Hemin attenuates bleomycin-induced lung fibrosis in mice by regulating the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways.

The objective of this study was to investigate the protective effect and potential mechanism of action of hemin on bleomycin-induced pulmonary fibrosis in mice. Male C57BL/6 mice were randomly divided into control, bleomycin and bleomycin + hemin groups. Mice in the bleomycin and bleomycin + hemin groups were injected intratracheally with bleomycin to establish the pulmonary fibrosis model. The bleomycin + hemin group mice were injected intraperitoneally with hemin starting 7 days before modeling until the end of Day 21 after modeling. Pathological changes in lung tissue were assessed by HE and Masson staining. Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined in lung tissue. Immunohistochemistry was performed to assess the expression of α-SMA and collagen I. The serum levels of IL-6 and TNF-α were measured via ELISA. Western blotting was used to determine the expression of TGF-β1, SIRT1, PGC-1α and HO-1 and the phosphorylation levels of p38, ERK1/2, JNK, AMPK and NF-κB p65 in lung tissue. Hemin significantly reduced lung indices, increased terminal body weight. It also significantly increased SOD and CAT activities; decreased MDA, IL-6 and TNF-α levels; reduced the levels of α-SMA and collagen I-positive cells; upregulated SIRT1, PGC-1α and HO-1 expression; promoted AMPK phosphorylation; and downregulated TGF-β1 expression and p38, ERK1/2, JNK and NF-κB p65 phosphorylation. Hemin might attenuate oxidative damage and inflammatory responses and reduces extracellular matrix deposition by regulating the expression and phosphorylation of proteins associated with the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways, thereby alleviating bleomycin-induced pulmonary fibrosis.

Dietary inclusion of cyanobacteria Arthrospira (Spirulina platensis) spp. decreases the aggravating effect of hemin from red meat in a rat colorectal carcinogenesis model

Colorectal cancer (CRC) risk, associated with a high intake of red/processed meat, may be related to hemin. A potential chemopreventive agent, Arthrospira (Spirulina platensis) spp. (AP) is a cyanobacteria employed as a functional food with the potential to reduce malnutrition in developing countries. Thus, we assessed the effects of dietary AP against hemin-promoting effects in a dimethylhydrazine (DMH)-induced colorectal carcinogenesis model. Male Sprague-Dawley rats were allocated into five groups. G1–G4 received four subcutaneous DMH injections (40 mg/kg body weight, twice a week for 2 weeks) and G5 received DMH vehicle. At weeks 4 to 16, groups were fed a balanced diet (BD, G1 and G5), BD containing 2 % AP (20 g powder/kg chow, G2), BD containing hemin (0.32 g/kg chow, G3) or BD containing hemin+2 % AP (G4), respectively. Hemin group (G3) increased aberrant crypt foci (ACF) development, malondialdehyde (MDA), and Nox-1 levels and reduced glutathione (GSH) and GSH-Px levels in colonic mucosa when compared to DMH group (G1). In contrast, the dietary hemin+2 % AP regimen (G4), reduced both preneoplastic lesion development and MDA levels while increased GSH levels, compared to the DMH+hemin group (G3). Dietary AP may reduce hemin-promoting effects by modulating oxidative stress and ACF lesions.

Intravenous Hemin, a potential heme oxygenase-1 activator, does not protect from post-ERCP acute pancreatitis in humans: Results of a randomized multicentric multinational placebo-controlled trial

ObjectiveHemin, a heme oxygenase 1 activator has shown efficacy in the prevention and treatment of acute pancreatitis in mouse models. We conducted a randomized controlled trial (RCT) to assess the protective effect of Hemin administration to prevent post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) in patients at risk. MethodsIn this multicenter, multinational, placebo-controlled, double-blind RCT, we assigned patients at risk for PEP to receive a single intravenous dose of Hemin (4 mg/kg) or placebo immediately after ERCP. Patients were considered to be at risk on the basis of validated patient- and/or procedure-related risk factors. Neither rectal NSAIDs nor pancreatic stent insertion were allowed in randomized patients. The primary outcome was the incidence of PEP. Secondary outcomes included lipase elevation, mortality, safety, and length of stay. ResultsA total of 282 of the 294 randomized patients had complete follow-up. Groups were similar in terms of clinical, laboratory, and technical risk factors for PEP. PEP occurred in 16 of 142 patients (11.3%) in the Hemin group and in 20 of 140 patients (14.3%) in the placebo group (p = 0.48). Incidence of severe PEP reached 0.7% and 4.3% in the Hemin and placebo groups, respectively (p = 0.07). Significant lipase elevation after ERCP did not differ between groups. Length of hospital stay, mortality and severe adverse events rates were similar between groups. ConclusionWe failed to detect large improvements in PEP rate among participants at risk for PEP who received IV hemin immediately after the procedure compared to placebo. Trial registration numberClinicalTrials.gov number, NCT01855841).

Study on the TGF-β Ligand Trap Enhances Erythroid Differentiation in HEL Cell By Inhibiting Smad3 Phosphorylation

Objective: Anemia is the most common clinical manifestation of MF, and its pathogenesis may be closely related to the over activation of TGF-β signaling pathway. This study aims to establish an erythroid differentiation model by inducing HEL cells with hemin and investigate the mechanism of the TGF-β ligand trap luspatercept enhances erythroid differentiation in HEL cell. Methods: 1.ELISA was used to detect the levels of GDF11 in peripheral blood plasma in patients with MF and healthy people. 2.The erythroid differentiation model was established by inducing HEL cells with hemin. The differentiation and proliferation levels of cells were detected by benzidine staining and CCK8. The expression levels of mRNA and protein in the cells were detected by RNA-seq and TMT proteomic analysis, and verified by flow cytometry, RT-PCR and WB. 3.HEL cells were induced with 40 μM hemin as a model of erythroid differentiation. 100ng/mL GDF11 interfered with the over activation of TGF-β signal pathway in differentiated HEL cells. 5μg/mL luspatercept blocked the activation of TGF-β signaling pathway by GDF11. Divided them into three groups, named: hemin group , GDF11 group, and luspatercept group. WB was used to detect the protein levels of pSmad3 and Smad4 in nuclear. Benzidine staining and flow cytometry were used to detect the levels of cell differentiation. RT-PCR and WB were used to detect the expression levels of GATA-1 and KLF1. The changes of RNA expression were detected by RNA-seq and verified by WB. 4.All experimental data were analyzed by SPSS 22.0 statistical software. Results: 1.The GDF11 levels of plasma of MF patients were higher than those of normal people ( P=0.0411). 2.The erythroid differentiation model of HEL cells induced with 40 μM hemin was successfully constructed. The results of RNA-seq and TMT proteome analysis showed that compared with the control group, the expression of hematopoietic related genes such as GATA-1, KLF1 and EPOR in hemin group were up-regulated, and the intracellular proteins related to erythroid differentiation such as HBγ- 2, HB-α and CD235a were up-regulated. Flow cytometry indicated that the expression of CD235a in hemin group was significantly higher than that of the control group (33.83% vs81%, P<0.01). RT-PCR and WB experiment showed that the expression of GATA-1 and KLF1 in hemin group were higher than those in control group. 3.WB showed that compared with hemin group, the hyperphosphorylation of smad3 and Smad4 in nuclear levels were increased in GDF11 group. And luspatercept could inhibit the hyperphosphorylation of Smad3 and reduce Smad4 in nuclear levels of GDF11 group. Luspatercept could inhibit GDF11 mediated activation of TGF-β signaling pathway in HEL cells. 4.Compared with hemin group, the benzidine positivity rate of cells and the expression of CD235a on the cell surface in GDF11 group were significantly decreased (benzidine: 13.0% vs 9%, P<0.01; CD235a: 16.8% vs 33.83%, P<0.01), and the expression of GATA-1 and KLF1 were also decreased. GDF11 could inhibit the erythroid differentiation of HEL cells. 5.Compared with GDF11 group, the benzidine positivity rate of cells and the expression of CD235a on the cell surface in luspatercept group were significantly increased (benzidine: 29.0% vs0%, P<0.01; CD235a: 31.1% vs 16.8%, P<0.01), and the expression of GATA-1 and KLF1 were increased. Luspatercept could reverse the inhibition of GDF11 on the erythroid differentiation of HEL cells. 6.Compared with the hemin group, the expression of GATA-1, KLF1, EPOR and other genes related to hematopoiesis in GDF11 group were down-regulated, and the differentially expressed genes were related to the biological functions of ribosomal metabolism process, chromosome regulation, erythropoiesis, oxygen binding, oxidoreductase activity and so on, mainly involving erythroid differentiation, oxidative phosphorylation, iron metabolism, TGF- β, JAK-STAT and other channels. Luspatercept could restore the expression of GATA-1, KLF1, EPOR and other genes by inhibiting the pathway over-activated by GDF11. Conclusion: Hemin could induce the erythroid differentiation of HEL cells. The TGF-β ligand trap luspatercept could inhibit GDF11-mediated hyperphosphorylation of Smad3 in HEL cells and promote the erythroid differentiation of HEL cells. Luspatercept could be a therapeutic tool for patients with myelofibrosis associated anemia.

Hemin protects against cell stress induced by estrogen and progesterone in rat mammary glands via modulation of Nrf2/HO-1 and NF-κB pathways.

Mammary gland hyperplasia is one of the risk factors for breast cancer. Till date, there is no study that has addressed the effect of hemin in this condition. Thus, this study was designed to evaluate the effect of the heme oxygenase 1 (HO-1) inducer (hemin) and its inhibitor (zinc protoporphyrin-IX) (ZnPP-IX) on mammary gland hyperplasia (MGH) induced by estrogen and progesterone in adult albino rats. Forty adult female albino rats were divided into the control group, MGH group, MGH + Hemin group, and MGH + Hemin + ZnPP-IX group. Serum levels of estradiol and progesterone were measured. Breast tissues were taken for estimation of oxidative, inflammatory, and apoptotic markers. Mammary gland histology was performed, and expression of Ki-67, Beclin, and P53 in breast tissue was also measured. Estrogen and progesterone administration induced hyperplasia of cells lining the ducts of the breast tissues associated with increased diameter and height of the nipples as well as increased oxidative stress markers, inflammatory markers, antiapoptotic markers, and cell autophagy. Hemin administration during induction of MGH can reverse all the affected parameters. Then, these effects were abolished by ZnPP-IX administration. We concluded that hemin administration can antagonize the cell stress induced by estrogen and progesterone and protect against the development of mammary gland hyperplasia via modulation of Nrf2/HO-1 and NF-κB pathways.

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.

Administration of hemin ameliorates ovarian ischemia reperfusion injury via modulation of heme oxygenase-1 and p-JNK/p-NF-κBp65/iNOS signaling pathway

AimsOvarian torsion is the fifth common gynecological emergency that can affect females of all ages particularly during reproductive age and its management by detorsion leads to ovarian ischemia reperfusion (IR) injury. Therefore, prophylactic measures are required to protect the ovarian function after detorsion. So that, our study aimed to assess the effect and underlying mechanisms of heme oxygenase-1 (HO-1) inducer; hemin against ovarian damage induced by IR injury in rats. Main methodsFemale rats were divided into: sham group, hemin group, ovarian IR (OIR) groups with and without hemin treatment. Serum levels of reduced glutathione (GSH) and interleukin 1 β (IL-1β) were measured in addition to ovarian levels of malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD). Ovarian phospho-Janus kinase (p-JNK) levels and gene expressions of HO-1 and inducible nitric oxide synthase (iNOS) were determined. Moreover, histopathological changes and expressions of phospho-nuclear factor kappa B p65 (p-NF-κB p65) and cleaved caspase-3 were done. Key findingsTreatment of OIR rats with hemin led to significant attenuation of ovarian damage through histological examination which was associated with significant increase in ovarian expression of HO-1, ovarian SOD and serum GSH levels with significant decrease in ovarian p-JNK levels, expressions of p-NF-κB p65, iNOS and cleaved caspase-3 in addition to serum IL-1β levels. SignificanceThe protective effect of hemin can be attributed to the increased expression of HO-1 which showed antioxidant, anti-inflammatory and anti-apoptotic effects. Therefore, hemin can be administered to prevent ovarian IR injury which occurs after detorsion.

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.

Superior Proton-Transfer Catalytic Promiscuity of Cytochrome c in Self-Organized Media.

Evolutionarily elderly proteins commonly feature greater catalytic promiscuity. Cytochrome c is among the first set of proteins in evolution to have known prospects in electron transport and peroxidative properties. Here, we report that cyt c is also a proficient proton-transfer catalyst and enhances the Kemp elimination (KE; model reaction to show proton transfer catalytic property) by ∼750-fold on self-organized systems like micelles and vesicles. The self-organized systems mimic the mitochondrial environment in vitro for cyt c. Using an array of biophysical and biochemical mutational assays, both acid-base and redox mechanistic pathways have been explored. The histidine moiety close to hemin group (His18) is mainly responsible for proton abstraction to promote the concerted E2 pathway for KE catalysis when cyt c is in its oxidized form; this has also been confirmed by a H18A mutant of cyt c. However, the redox pathway is predominant under reducing conditions in the presence of dithiothreitol over the pH range 6-7.4. Interestingly, we found almost 750-fold enhanced KE catalysis by cyt c compared to aqueous buffer. Overall, in addition to providing mechanistic insights, the data reveal an unprecedented catalytic property of cyt c that could be of high importance in an evolutionary perspective considering its role in delineating the phylogenic tree and also towards generating programmable designer biocatalysts.

Effect of ascorbic acid and hemin on experimental colon carcinogenesis in mice

Colon cancer is known as the third most widespread cancer in the world. The interaction of heme-iron and ascorbic acid (AA) in colon carcinogenesis is not evident. Hemin (ferric chloride heme) is an iron-containing porphyrin with chlorine that can be formed from a heme group. The purpose of this study was to investigate the protective effect of AA on the formation of pre-neoplastic lesions induced by azoxymethane (AOM)/dextran sodium sulfate (DSS) plus hemin in mice. Forty-five ICR male mice were divided into three experimental groups; AOM/ DSS treatment (control group), hemin (2 g hemin/kg of b.w.), hemin + AA (1.0% in drinking water). The mice had three s.c. injections (0–2nd weeks of the experiment) of AOM (10 mg/kg b.w.) weekly and 2% DSS as drinking water for the next one week and the animals fed on AIN-76A purified rodent diet for 6 weeks. The numbers of aberrant crypt foci (ACF) and aberrant crypts (ACs) in colonic mucosa were counted after methylene blue staining. Lipid peroxidation in feces was measured by the thiobarbituric acid-reactive substances (TBARS) assay. The numbers of ACF and ACs per colon significantly increased in Hemin group compared to the control group. However, the numbers of ACF and ACs per colon notably decreased in hemin + AA group compared to the control group or hemin group (p<0.05). In feces, the TBARS value of hemin group was higher than the control group (p<0.01). The TBARS value of hemin + AA group was slightly decreased compared to Hemin group. These results indicate that hemin can promote the experimental colon carcinogenesis in ICR mice. On the other hand, additional supplement of AA via drinking water has a protective effect against the colon carcinogenesis. The related mechanisms need to be illustrated by further studies in future.

Carbon Monoxide Inhibits the Expression of Proteins Associated with Intestinal Mucosal Pyroptosis in a Rat Model of Sepsis Induced by Cecal Ligation and Puncture.

BackgroundCarbon monoxide (CO) has anti-inflammatory effects and protects the intestinal mucosal barrier in sepsis. Pyroptosis, or cell death associated with sepsis, is mediated by caspase-1 activation. This study aimed to investigate the role of CO on the expression of proteins associated with intestinal mucosal pyroptosis in a rat model of sepsis induced by cecal ligation and puncture (CLP).Material/MethodsThe rat model of sepsis was developed using CLP. Male Sprague-Dawley rats (n=120) were divided into six study groups: the sham group (n=20); the CLP group (n=20); the hemin group (treated with ferric chloride and heme) (n=20); the zinc protoporphyrin IX (ZnPPIX) group (n=20); the CO-releasing molecule 2 (CORM-2) group (n=20); and the inactive CORM-2 (iCORM-2) group (n=20). Hemin and CORM-2 were CO donors, and ZnPPIX was a CO inhibitor. In the six groups, the seven-day survival curves, the fluorescein isothiocyanate (FITC)-labeled dextran 4000 Da (FD-4) permeability assay, levels of intestinal pyroptosis proteins caspase-1, caspase-11, and gasdermin D (GSDMD) were measured by confocal fluorescence microscopy. Proinflammatory cytokines interleukin (IL)-18, IL-1β, and high mobility group box protein 1 (HMGB1) were measured by Western blot and enzyme-linked immunosorbent assay (ELISA).ResultsCO reduced the mortality rate in rats with sepsis and reduced intestinal mucosal permeability and mucosal damage. CO also reduced the expression levels of IL-18, IL-1β, and HMGB1, and reduced pyroptosis by preventing the cleavage of caspase-1 and caspase-11.ConclusionsIn a rat model of sepsis induced by CLP, CO had a protective role by inhibiting intestinal mucosal pyroptosis.

Electroacupuncture ameliorates endotoxin-induced acute lung injury in rabbits by regulating heme oxygenase-1 expression to improve mitochondrial dynamics

BackgroundElectroacupuncture (EA) mitigates endotoxin-induced acute lung injury (ALI) during bacterial sepsis, but the mechanisms are unclear. This study aimed to investigate the potential mechanisms of EA improving endotoxin-induced ALI. MethodsEndotoxin-induced ALI was established by lipopolysaccharide (LPS) administration. Electroacupuncture was started 5 days before LPS administration and non-acupoint EA (punctured at non-acupoints with shallow insertion and no electrical stimulation) was used as the control treatment. In addition, hemin was applied to active HO-1 and ZnPP to suppress HO-1. Briefly, 70 rabbits were divided into seven treatment groups: untreated control (C), LPS, LPS + EA, LPS + non-acupoint EA, LPS + EA + hemin, LPS + EA + ZnPP, and LPS + EA + ZnPP + hemin. Lung samples were collected at 6 h after LPS administration for analysis of tissue injury (edema, inflammation, hemorrhage, necrosis), oxidative stress, mitochondrial membrane potential (MMP), respiratory control ratio (RCR), ATP production, and changes in expression of mitochondrial fusion and fission markers. ResultsElectroacupuncture alleviated LPS-induced lung injury and mitochondria dysfunction as indicated by decreasing wet/dry tissue weight ratio, lung injury score, ROS production, and suppressing expression of mitochondrial fission proteins. Furthermore, EA also increased ATP content, mitochondrial membrane potential, respiratory control ratio, and expressions of mitochondrial fusion proteins (LPS + EA group vs. LPS group). These effects of EA were further enhanced by pretreatment with hemin (LPS + EA + hemin group) but suppressed by Znpp-IX (LPS + EA + ZnPP and LPS + EA + ZnPP + hemin groups). ConclusionsElectroacupuncture protects against endotoxin-induced ALI by upregulating heme oxygenase-1 expression, thereby preserving the balance between mitochondrial fusion and fission.

Amelioration of estrogen-induced endometrial hyperplasia in female rats by hemin via heme-oxygenase-1 expression, suppression of iNOS, p38 MAPK, and Ki67.

Although heme oxygenase-1 (HO-1) is part of an endogenous defense system implicated in the homeostatic response, its role in cell proliferation and tumor progression is still controversial. Endometrial hyperplasia (EH) is associated with high risk of endometrial cancer (EC). Therefore, we aimed to evaluate the effect of hemin, a HO-1 inducer, against EH. Thirty-two female rats (60-70 days old) were divided into 4 groups treated for 1 week: vehicle control group, hemin group (25 mg/kg; i.p. 3 times/week), estradiol valerate (EV) group (2 mg/kg per day, p.o.), and hemin plus EV group. Sera were obtained for reduced glutathione level. Uterine malondialdehyde, superoxide dismutase, total nitrite/nitrate, and interleukin-1β levels were estimated. HO-1 and p38 mitogen-activated protein kinase expressions were obtained in uterine tissue. Uterine histological and immunohistochemical assessment of iNOS and Ki67 were also done. Results demonstrated that upregulation of HO-1 expression in hemin plus EV rats led to amelioration of EH which was confirmed with histological examination. This was associated with significant decrease in oxidative stress parameters, p38 mitogen-activated protein kinase expression, and interleukin-1β level. Also, uterine iNOS and Ki67 expressions were markedly suppressed. In conclusion, upregulation of HO-1 expression via hemin has ameliorative effect against EH through its antioxidant, anti-inflammatory, and antiproliferative actions.

Hemin attenuated oxidative stress and inflammation to improve wound healing in diabetic rats

Oxidative stress and persistent inflammation play crucial role in the progression of diabetic wound complications. Hemeoxgenase-1 (HO-1) by degrading hemin has been shown to display anti-oxidant and anti-inflammatory effects. Further, hemin is a potent HO-1 inducer. Thus, the current study was aimed to evaluate the effect of topical application of hemin on diabetic wound in rats. Four hundred square millimeter open excision wound were created 2weeks after induction of diabetes with single intraperitoneal injection of streptozotocin (60mg/kg), and the diabetic rats were divided into three groups namely diabetic control, hemin, and tin protoporphyrin (SnPPIX). Ointment base, hemin (0.5% in ointment base), and SnPPIX (0.5% in ointment base) were applied topically to wounded area in diabetic control, hemin, and SnPPIX group rats, respectively, twice daily for 19days. Hemin significantly increased the wound contraction in comparison to control and SnPPIX-treated rats. Time-dependent analysis revealed significant increase in anti-oxidants with concomitant decrease in oxidants in hemin-treated rats as compared to diabetic control rats. Further, mRNA expression decreased for inflammatory cytokine and increased for anti-inflammatory cytokine in hemin group as compared to diabetic control rats. Expression of HO-1 also increased in hemin group as compared to diabetic control rats. However, SnPPIX group results were in disagreement with results of hemin which is clearly reflected in histopathology. Results indicate the ability of hemin to accelerate wound healing in diabetic rats by combating inflammation and oxidative stress probably via HO-1.

Adipose-derived mesenchymal stem cells stereotactic transplantation alleviate brain edema from intracerebral hemorrhage.

Adipose-derived mesenchymal stromal cells (ADSCs) exhibited high potential in tissue repair and regeneration, and it has been proved that ADSCs could protect brain cells from apoptosis and maintaining blood-brain barrier stability after cerebral vascular disease. In this study, we evaluated the therapeutic potential and mechanism of ADSCs stereotactic transplantation in intracerebral hemorrhage (ICH) mice model and hemin-treated astrocytes. Mice were divided into three groups: sham group, ICH + PBS group, and ICH + ADSC group. Mice in ICH + ADSC group received ADSCs cell suspension stereotactic transplantation into the area beside the bleeding region. Astrocytes were divided into three groups: control group, hemin group, and hemin + ADSC group. Astrocytes in hemin + ADSC group were cultured in ADSCs-astrocyte no-contact coculture system and treated with 30 μM hemin solution. The results showed that ADSCs stereotactic transplantation improved functional outcomes and reduced cell apoptosis after ICH. Moreover, ADSCs stereotactic transplantation could alleviate brain edema and inflammation and AQP4 protein expression contributed to the alleviation of brain edema. In addition, mitogen-activated protein kinase (MAPK) pathways, including p38/MAPK pathway and c-Jun N-terminal kinase pathway, were involved in AQP4 modulation by ADSCs transplantation in ICH. In conclusion, ADSCs transplantation could alleviate the nervous tissue injury, reduce cell apoptosis, and relieve brain edema in ICH. And the edema regulation effect of ADSCs transplantation is associated with inhibition of inflammation and AQP4 protein expression.

Effects of Hemin on cognitive function and the expression of neuroglobin in rats with vascular dementia

Objective To observe the influence of Hemin on both the expression of neuroglobin (Ngb) and Tau protein phosphorylation in rats with vascular dementia (VD), and explore the effect and mechanism of Ngb in VD. Methods VD model was established in rats with a permanent bilateral common carotid arteries occlusion(2-VO). Fifty four healthy male SD rats were randomly divided into 3 groups: sham operation group, VD group and Hemin group. According to ischemic time, the animals in each group were randomly allocated into 1 week subgroup, 4 weeks subgroup and 8 weeks subgroup(n=6 in each subgroup). The learning and memory ability of rats were evaluated by Morris water maze test.The brain histopathological changes were observed by HE staining and the expression of Ngb and phosphorylated Tau (p-Tau) was determined by Western blot. Results Compared with the sham operation group, after 2-VO the escape latency extended (all P<0.05) and cross-platform performance reduced in rats in both VD group and Hemin group(all P<0.05). The learning and memory ability of rats in Hemin group was better than that in VD group at the corresponding time point: the increase in the number of crossing platforms(1 week: Hemin group (7.00±0.89) times vs VD group (5.50±1.22) times, t=2.42, P<0.05; 4 weeks: Hemin group (5.33±0.52) times vs VD group (3.50±1.04) times, t=3.84, P<0.01; 8 weeks: Hemin group (6.50±0.55) times vs VD group (4.50±1.05) times, t=4.14, P<0.01). The expression of Ngb and phosphorylated Tau (p-Tau) increased in VD group and Hemin group (P<0.01). Compared with VD group, the expression of Ngb in Hemin group was increased(1 week: Hemin group (3.45±0.23) vs VD group (2.56±0.08), t=5.67, P<0.01; 4 weeks: Hemin group (3.08±0.13) vs VD group (2.28±0.08), t=7.96, P<0.01; 8 weeks: Hemin group (2.82±0.12) vs VD group (2.05±0.10), t=7.28, P<0.01), while p-Tau was decreased (1 week: Hemin group (1.57±0.12) vs VD group (2.40±0.15), t=7.62, P<0.01; 4 weeks: Hemin group (2.14±0.21) vs VD group (3.18±0.14), t=8.23, P<0.01; 8 weeks: Hemin group (1.83±0.13) vs VD group (2.79±0.19), t=4.91, P<0.01). In VD group and Hemin group, the learning and memory ability exhibited a negative correlation with Ngb (r=-0.892, P<0.01), whereas a positive correlation with p-Tau (r=0.932, P<0.01). Conclusion Hemin induces high expression of Ngb and inhibits Tau phosphorylation in VD rats. Ngb may play a neuroprotective role in the development of VD by regulating Tau phosphorylation. Key words: Hemin; Neuroglobin; Tau; Vascular dementia; Cognitive function; Rat

Pathophysiological changes in experimental polycystic ovary syndrome in female albino rats: Using either hemin or L-arginine.

Polycystic ovary syndrome (PCOS), one of the important endocrine disorders affecting females in the reproductive age, is caused mainly by an abnormal oxidation status that subsequently causes inflammatory conditions. Thus, this study aims to examine the possible individual prophylactic effects of gasotransmitters, hemin, or L-arginine in letrozole-induced PCOS. Fifty adult female albino rats were used and separated into a control group, which received the vehicle; a letrozole-induced PCOS group (L), which received letrozole orally at a dose level of 1 mg/kg for 21 days; a letrozole+hemin (L+H) group, which received letrozole plus hemin at a dose level of 25 mg/kg injected IP twice per week for 21 days; and a letrozole+L-arginine (L+A) group, which received letrozole plus L-arginine at a dose level of 200 mg/kg orally for 21 days. During PCO induction, the body weight and Lee index were measured. Serum glucose, insulin, lipid profile, gonadotrophic hormones, testosterone, estrogen, and tumor necrosis factor alpha were assayed, while ovarian tissues were analyzed to measure the oxidative state and histopathological changes. Our results proved that either hemin or L-arginine administration could improve the oxidative state, the inflammatory reaction, the hormonal imbalance, and the metabolic disturbances in PCO rats, which was confirmed by a histopathological examination of the rats' ovaries. In conclusion, either hemin or L-arginine had protective effects against PCOS with better pathophysiological changes with hemin.

Effect of caveolin-1 scaffolding domain peptides on heme oxygenase-1 activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages induced by lipopolysaccharide

To investigate the effect of caveolin-1 scaffolding domain (CSD) peptides on heme oxygenase-1 (HO-1) activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages (AMs) induced by lipopolysaccharide (LPS). Bioinformatics was used to analyze the binding of full-length wild-type CSD polypeptide and 101 amino acid deleted truncated mutant CSD polypeptide (Δ101CSD) to HO-1. Primary AMs were isolated from rats, when cell fusion reached 80%, they were synchronized with serum-free medium and divided into five groups: no treatment was given to the blank control group; LPS group was treated with 100 μg/L LPS for 16 hours; LPS + hemin group was treated with 100 μg/L LPS and 20 μmol/L hemin for 16 hours; wild-type CSD polypeptide + LPS + hemin group was pretreated with 10 μmol/L wild-type CSD polypeptide 6 hours before LPS treatment; Δ101CSD + LPS + hemin group was pretreated with 10 μmol/L Δ101CSD polypeptide 6 hours before LPS treatment. After treatment for 16 hours, the co-localization between caveolin-1 (Cav-1) and HO-1 was displayed by confocal microscope; the mRNA expressions of inflammatory cytokines interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) and M1/M2 polarization cytokines tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), leukocyte differentiation antigen 206 (CD206) and IL-10 were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR); the HO-1 activity and nitric oxide (NO) production were determined by spectrophotometry. Bioinformatics analysis showed: both wild-type CSD and Δ101CSD peptides could bind to HO-1, and there was no significant difference in the binding ability between the two peptides, but the deletion of 101 Arg resulted in the disappearance of part of the binding region between Δ101CSD and HO-1. The results of laser confocal microscopy showed: the expressions of Cav-1 and HO-1 were lowed in the blank control group, and Cav-1 was bound to HO-1 in LPS group and LPS + hemin group. Both wild-type CSD and Δ101CSD peptides pretreatment could significantly reduce the binding of HO-1 to Cav-1 induced by LPS. HO-1 activity analysis showed: after LPS stimulation, the activity of HO-1 was significantly higher than that of the blank control group; the activity of HO-1 induced by LPS was increased by hemin; after pretreatment with two kinds of CSD peptides, the activity of HO-1 was further increased, and the effect of wild-type CSD peptide was more significant, which showed a statistically significant difference as compared with that of LPS + hemin group (pmol×mg-1×h-1: 3 683±266 vs. 2 408±132, P < 0.05). RT-qPCR results showed: LPS could induce elevation of cytokines and M1 markers and decrease of M2 markers, while hemin could inhibit LPS-induced inflammatory response and M1/M2 phenotypic polarization. Compared with LPS + hemin group, after pretreatment with wild-type CSD peptide, the levels of inflammatory factors in AMs were decreased, and the mRNA expression levels of TNF-α and iNOS, M1 markers, were decreased [TNF-α mRNA (2-ΔΔCt): 6.82±0.05 vs. 8.70±0.24, iNOS mRNA (2-ΔΔCt): 331.50±32.05 vs. 506.70±0.10, both P < 0.05], and IL-10 mRNA expression level was increased (2-ΔΔCt: 269.09±6.54 vs. 119.05±3.30, P < 0.05). The deletion of 101 site partially weakened the inhibitory effect of CSD peptides on inflammatory factors and only reduced the expression of iNOS mRNA (2-ΔΔCt: 429.11±8.92 vs. 506.70±0.10, P < 0.05), indicating that its ability to transform AMs from M1 phenotype to M2 phenotype was poor. The two peptides had no effect on the expression of CD206. Wild-type CSD had beneficial effects of anti-inflammation by reducing Cav-1 binding to HO-1 induced by LPS, restoring the HO-1 activity and driving M2 phenotype in alveolar macrophages.

Effect of hemin and ascorbic acid on colonic pre-neoplastic lesions induced by azoxymethane in mice

Colorectal cancer (CRC) is the third most prevalent cancer in the world, and heme iron is known to promote the CRC in an animal model. This study was conducted to investigate the effects of ascorbic acid in the presence of hemin on the formation of pre-neoplastic lesions induced by azoxymethane (AOM)/disodium sulfate (DSS) in mice. After acclimation for 1 week, five-week old mice received three s.c. injections (0-2 weeks of the experiment) of AOM [10 mg/kg body weight (BW)] weekly and were treated with 2% DSS in drinking water for the next week to induce aberrant crypt foci (ACF). All animals were fed the AIN-76A purified rodent diet for experimental period of 6 weeks. Experimental groups were then divided into three groups: carboxymethylcellulose (CMC) alone (control), CMC + Hemin, CMC + Hemin + ascorbic acid (AA). The CMC was used as a solvent for hemin. The daily doses were 534 mg/kg BW hemin and 246 mg/kg BW ascorbic acid administered orally. After the colonic mucosa were stained with methylene blue, aberrant crypt foci (ACF), aberrant crypt (AC) and polyps were counted. Lipid peroxidation in liver was evaluated by the thiobarbituric acid-reactive substances (TBARS) assay. The numbers of ACF, AC and large ACF (≥4 AC/ACF) per colon increased in the hemin group compared to the control group, while they decreased significantly in the hemin + ascorbic acid group compared to the control group or hemin group (p<0.01). The number of polyps/colon in the hemin + AA group was significantly decreased compared to the hemin group (p<0.05). In the liver, the TBARS value of the hemin group was significantly higher than that of the control group (p<0.01). Additionally, the TBARS value of the hemin + AA group decreased slightly compared to that of the hemin group. Taken together, these results suggest that hemin can promote colon carcinogenesis in a mouse model and that ascorbic acid has a protective effect against hemin-promoted colon carcinogenesis.

Relationship between heme oxygenase-1 expression and autophagy during acute lung injury in septic mice

Objective To evaluate the relationship between heme oxygenase-1 (HO-1) expression and autophagy during acute lung injury in septic mice. Methods Forty-eight male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were randomly divided into 4 groups (n=12 each) using a random number table: sham operation group (group SH), sepsis group (group CLP), HO-1 agonist hemin plus sepsis group (group Hemin+ CLP) and HO-1 inhibitor SnPP plus sepsis group (group SnPP+ CLP). Sepsis was induced by cecal ligation and puncture in CLP, Hemin+ CLP and SnPP+ CLP groups.Hemin 50 μmol/kg and SnPP 50 μmol/kg were intraperitoneally injected at 2 h before operation in Hemin+ CLP group and SnPP+ CLP group, respectively.Arterial blood samples were taken at 24 h after operation for blood gas analysis, PaO2 was recorded, and oxygenation index was calculated.Lungs were removed for examination of pathological changes which were scored and for determination of autophagosome count (with an electron microscope), weight/dry weight ratio (W/D ratio) and expression of HO-1, Becling-1 and microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) in lung tissues (by Western blot). Results Compared with group SH, oxygenation index was significantly decreased, and W/D ratio, autophagosome count and pathological scores were increased in CLP, Hemin+ CLP and SnPP+ CLP groups, and the expression of HO-1, Beclin-1 and LC3 Ⅱ was significantly up-regulated in CLP and Hemin+ CLP groups (P<0.05). Compared with group CLP, oxygenation index and autophagosome count were significantly increased, W/D ratio and pathological scores were decreased, and the expression of HO-1, Beclin-1 and LC3 Ⅱ was up-regulated in group Hemin+ CLP, and oxygenation index and autophagosome count were significantly decreased, W/D ratio and pathological scores were increased, and the expression of HO-1, Beclin-1 and LC3 Ⅱ was down-regulated in group SnPP+ CLP (P<0.05). Conclusion Up-regulated expression of HO-1 mediates autophagy, which is involved in the endogenous protective mechanism underlying acute lung injury in septic mice. Key words: Heme oxygenase-l; Autophagy; Sepsis; Respiratory distress syndrome, adult