[Effects of high glucose induced primary cardiomyocytes injury on necroptosis and the related mechanism].

OBJECTIVE: To observe whether necroptosis was happened in high glucose (HG) - induced primary cardiomyocytes injury and to investigate the likely mechanism. METHODS: The primary cultured cardiomyocytes were divided into 4 groups (n=9): control group (the cardiomyocytes were incubated with 5.5 mmol/L glucose for 48 h), HG group (the cardiomyocytes were incubated with 30 mmol/L glucose for 48 h), HG + necrostatin-1 (Nec-1) group (the cardiomyocytes was co-incubated with necroptosis inhibitor Nec-1 at 100 µmol/L and HG for 48 h) and hypertonic pressure group (HPG, the cardiomyocytes was co-incubated with 5.5 mmol/L glucose and 24.5 mmol/L mannitol for 48 h). Cell viability was measured by MTT method, reactive oxygen species (ROS) generation was measured by DHE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) were tested by ELISA method. The mRNA and protein expressions of necroptosis related genes receptor interacting serine/threonine protein kinase 1 (RIP1), RIP3, mixed lineage kinase domain-like protein (MLKL) were tested by quantitative real-time PCR and Western blot. RESULTS: The results showed HG intervention decreased cardiomyocytes viability, increased ROS generation, up-regulated the levels of TNF-α, IL-6 and IL-1ß, increased RIP1, RIP3, MLKL expressions at mRNA and protein levels. Nec-1 treatment attenuated HG-induced increased cardiomyocytes viability, reduced ROS generation, down-regulated the levels of TNF-α, IL-6 and IL-1ß, decreased RIP1, RIP3, MLKL expressions at mRNA and protein levels. CONCLUSION: Necroptosis was happened in high glucose-induced primary cardiomyocytes injury. Inhibition of necroptosis can reduce high glucose-induced cardiomyocytes damage, may be related to inhibition of oxidative stress and depression of inflammative factors releasing.

Exogenous H2S mitigates myocardial fibrosis in diabetic rats through suppression of the canonical Wnt pathway.

Hydrogen sulfide (H2S) has antifibrotic activity in the kidneys, heart, lungs, and other organs. The present study investigated the protective activity of exogenous H2S against myocardial fibrosis in a rat model of diabetes. Animals were assigned to normal control, diabetes mellitus (DM), DM + sodium hydrosulfide (NaHS; DM + NaHS) and NaHS groups. Fasting blood glucose (FBG), cardiac function and hydroxyproline were monitored. Heart histomorphology and ultrastructure were additionally evaluated. Wnt1­inducible signaling pathway protein (WISP)­1 protein expression in the myocardium was determined by immunohistochemical staining. Matrix metalloprotease (MMP)­2, tissue inhibitor of metalloproteinase (TIMP)­2, collagens, and canonical Wnt and transforming growth factor (TGF)­ß1/SMAD family member 3 (Smad3) pathway­related proteins were assessed by western blotting. Cardiac function was decreased, and myocardial injury, hypertrophy and fibrosis were increased in the diabetes model rats. MMP­2 expression was decreased, and the expressions of WISP­1, TIMP­2, collagens, and canonical Wnt and TGF­ß1/Smad3 pathway­related proteins were increased in the myocardia of the diabetes model rats. The present results indicated that the canonical Wnt pathway promoted diabetic myocardial fibrosis by upregulating the TGF­ß1/Smad3 pathway. Except for FBG, exogenous H2S ameliorated the changes in diabetes­associated indices in rats in the DM + NaHS group. The results are consistent with H2S protection of streptozotocin­induced myocardial fibrosis in the diabetes model rats by downregulation of the canonical Wnt and TGF­ß1/Smad3 pathway and decreased myocardial collagen deposition.

Expression and localization of absent in melanoma 2 in the injured spinal cord.

In traumatic brain injury, absent in melanoma 2 (AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day (post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+ (neurons), GFAP+ (astrocytes), CNPase+ (oligodendrocytes) and CD11b+ (microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+ (leukocytes) and CD68+ (activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.

Genistein attenuates renal fibrosis in streptozotocin­induced diabetic rats.

The present study aimed to investigate the antifibrogenic effects of genistein (GEN) on the kidney in streptozotocin (STZ)­induced diabetic rats and to determine the associated mechanisms. Rats were randomized into four groups: Normal control (N), STZ (S), L (STZ + low­dose GEN) and H (STZ + high­dose GEN). After 8 weeks, the fasting blood glucose (FBG) level, the ratio of kidney weight to body weight (renal index), 24­h urine protein, blood urea nitrogen (BUN), serum creatinine (SCr), renal total antioxidant capacity (T­AOC), superoxide dismutase (SOD), lipid peroxidation (LPO), malondialdehyde (MDA) and hydroxyproline (Hyp) contents were measured. The histomorphology and ultrastructure of the kidney were also assessed. In addition, mRNA expression levels of transforming growth factor­ß1 (TGF­ß1) and protein expression levels of nuclear factor erythroid 2­related factor 2 (Nrf2), heme oxygenase­1 (HO­1), NAD(P)H:quinone oxidoreductase 1 (NQO1), TGF­ß1, mothers against decapentaplegic homolog 3 (Smad3), phosphorylated (p)­Smad3 and collagen IV were estimated. Compared with group N, the levels of FBG, renal index, 24­h urine protein, BUN, SCr, LPO, MDA and Hyp were increased, whereas the levels of T­AOC and SOD were decreased in group S. The structure of renal tissue was damaged, and the expression of Nrf2, HO­1 and NQO1 were reduced, whereas the expression of TGF­ß1, Smad3, p­Smad3 and collagen IV were increased in group S. Compared with group S, the aforementioned indices were improved in groups L and H. In conclusion, GEN exhibited reno­protective effects in diabetic rats and its mechanisms may be associated with the inhibition of oxidative stress by activating the Nrf2­HO­1/NQO1 pathway, and the alleviation of renal fibrosis by suppressing the TGF­ß1/Smad3 pathway.

[Protective Effects of Genistein on Myocardial Injury in Diabetic Rats].

OBJECTIVE: To investigate the effects of genistein (Gen) on myocardial injury in diabetic rats and explore its mechanisms. METHODS: Male SD rats were randomly divided into normal (N) group, diabetic (D) group, Gen 5 mg/kg treatment (L) group and Gen 25 mg/kg treatment (H) group (n=8 for each group). Intraperitoneal injection of streptozotocin was utilized to establish type 1 diabetic rat model. After successful building models, from the fifth week, the rats in the L and H groups were daily gavaged with 5 mg/kg and 25 mg/kg Gen solution, respectively. After 4 weeks of treatment with Gen, the hemodynamic parameters and fasting blood glucose (FBG) level were measured. The morphological structure and ultrastructure of myocardium were observed using HE staining and transmission electron microscopy, respectively. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), malondialdehyde (MDA), glutathione (GSH) and Caspase-3 in myocardial tissue were measured. The levels of myocardial Bcl-2 and Bax at mRNA expression were detected using RT-PCR. The levels of myocardial thioredoxin (Trx), Trx-interacting protein (TXNIP) and apoptosis signal regulating kinase 1 (ASK1) at protein expression were detected using Western blot. RESULTS: Compared with the N group, the FBG, TNF-α, IL-1ß, IL-6, MDA and Caspase-3 levels were increased (P<0.01), while hemodynamic parameters and GSH content were decreased (P<0.01), the myocardial morphological structure and ultrastructure were damaged in the D group. The levels of Bcl-2 mRNA and Trx protein expression were significantly decreased (P<0.01), while the levels of Bax mRNA, TXNIP and ASK1 protein expression were significantly increased (P<0.01) in the D group. Compared with the D group, in the L and H groups, there was no significant difference in [CM(155mm]FBG, the TNF-α, IL-1ß, IL-6, MDA and Caspase-3 levels were decreased (P<0.05, P<0.01), while the hemodynamic parameters and GSH content were increased (P<0.05, P<0.01); the myocardial morphological structural and ultrastructural damages were alleviated; the levels of Bcl-2 mRNA and Trx protein expression were increased (P<0.05, P<0.01), while the levels of Bax mRNA, TXNIP and ASK1 protein expression were significantly decreased (P<0.05, P<0.01). CONCLUSION: Gen exhibits a protective effect on myocardial injury in diabetic rats, and the mechanisms may be associated with the reduction of inflammatory reaction, the regulation of Trx system expression, and the inhibition of oxidative stress and cell apoptosis.

[Effects of hydrogen sulfide on renal fibrosis in diabetic rats and its mechanism].

OBJECTIVE: To investigate the effects of hydrogen sulfide (H2S) on renal fibrosis in diabetic rats and explore its mechanism. METHODS: Male Sprague-Dawley rats were randomly divided into normal control (NC) group, a diabetic control (DC) group, diabetes mellitus (DM)+sodium hydrosulfide (NaHS) group and DM+DL-propargylglycine (PAG) group, with 8 rats in each group.Type 1 diabetes was induced in the respective groups by a single intraperitoneal (i.p.) injection of streptozotocin.From the fifth week, rats in the DM+NaHS and DM+PAG groups were injected (i.p.) with 56 µmol/kg NaHS and 40 mg/kg PAG once a day, respectively.After treatment for 4 weeks, the levels of fasting blood glucose (FBG), blood urea nitrogen (BUN) and serum creatinine (SCr) were detected.The deposition of renal collagen fibers was observed by Masson staining, and collagen volume fraction (CVF) was calculated.The ultrastructural change of renal tissue was observed by transmission electron microscopy.The levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and hydroxyproline (Hyp) in renal tissues were detected using the kits.The expression levels of TGF-ß1, Smad3, phosphorylated (p)-Smad3 and collagen-IV (col-IV) in renal tissues were detected using Western blot. RESULTS: Compared with the NC group, the levels of FBG, BUN, SCr, CVF, IL-1ß, IL-6, TNF-α and Hyp were increased; the deposition of renal collagen fibers and the ultrastructural damage were aggravated; the levels of TGF-ß1, Smad3, p-Smad3, p-Smad3/Smad3 and col-IV were increased in the DC group.Compared with the DC group, excluding FBG, the aforementioned indices were improved in the DM+NaHS group; the aforementioned indices were further aggravated in the DM+PAG group. CONCLUSIONS: H2S attenuated renal fibrosis in diabetic rats, and the mechanism might be associated with the reduction of the release of proinflammatory cytokines, downregulation of the TGF-ß1/Smad3 pathway, and inhibition of excessive accumulation of col-IV.

Effect of genistein on myocardial fibrosis in diabetic rats and its mechanism.

The aim of the present study was to investigate the effects of genistein (GEN) on myocardial fibrosis in type 1 diabetic rats and explore the underlying mechanisms. Rats were divided into 4 groups: Normal control (N), diabetic control (D), low­dose GEN treatment (L) and high­dose GEN treatment (H) groups. Following 8 weeks, the ventricular hemodynamic parameters, fasting blood glucose (FBG), heart­weight to body­weight ratio (HW/BW), myocardial hydroxyproline (Hyp) content, serum creatine kinase MB isozyme (CK­MB), lactate dehydrogenase (LDH), tumor necrosis factor­α (TNF­α), interleukin­1ß (IL­1ß) and interleukin­6 (IL­6) levels were measured. The histomorphology and ultrastructure of the heart were observed. The protein expression of myocardial transforming growth factor­ß1 (TGF­ß1), mothers against decapentaplegic homolog (Smad)­3, phosphorylated (p)­Smad3, Smad4, collagen­I and collagen­III were estimated. Compared with the N group, while the cardiac function was decreased, the levels of FBG, HW/BW, Hyp content, CK­MB, LDH, TNF­α, IL­1ß and IL­6 were increased in the D group. The myocardial histomorphological alterations and ultrastructure were damaged, and the protein expression of myocardial TGF­ß1, Smad3, p­Smad3, Smad4, collagen­I and collagen­III were increased in the D group. Compared with the D group, there were no differences in the ventricular hemodynamic parameters, FBG and p­Smad3 expression in the L group, while HW/BW, Hyp content, CK­MB, LDH, TNF­α, IL­1ß and IL­6 levels were decreased. The myocardial histomorphological damage was alleviated and the protein expression of TGF­ß1, Smad3, Smad4, collagen­I and collagen­III was decreased in the L group. Compared with L group, excluding FBG, the aforementioned indices were improved in the H group. In conclusion, GEN can attenuate myocardial fibrosis in type 1 diabetic rats, and the underlying mechanisms may be associated with the reduction of CK­MB and LDH leakage, inhibition of the inflammatory reaction, and suppression of the TGF­ß1/Smad3 signaling pathway to regulate collagen expression.

Effects of hydrogen sulfide on inducible nitric oxide synthase activity and expression of cardiomyocytes in diabetic rats.

The aim of the present study was to investigate the effects of hydrogen sulfide (H2S) on the activity and expression of inducible nitric oxide synthase (iNOS) in the myocardial tissue of type 1 diabetic rats. Rats were divided randomly into four groups: Normal control (NC), diabetes mellitus (DM), DM+DL­Proparglygylcine (DM+PAG) and DM+sodium hydrosulfide (DM+NaHS) groups. Type 1 diabetes was induced in the respective groups by a single intraperitoneal (i.p.) injection of streptozotocin. Rats in the DM+PAG and DM+NaHS groups were injected with PAG and NaHS (i.p.) once a day, respectively. The level of fasting blood glucose (FBG), the heart­weight to body­weight (HW/BW) ratio and the ventricular hemodynamic parameters were measured. The activities of serum total NOS (tNOS), iNOS, lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase MB isozyme (CK­MB), and the content of nitric oxide (NO) were detected. The contents of myocardial malondialdehyde (MDA) and NO, and the activities of superoxide dismutase (SOD), tNOS and iNOS were determined. The myocardial tissue was examined for histological and ultrastructural alterations. The expression level of iNOS at the transcriptional and translational levels in the myocardial tissue was estimated. The level of FBG was increased in the DM group compared with the NC group, verifying the diabetic condition of the rats. The function of the left ventricle, the myocardial histological alterations and ultrastructures were damaged in the DM group. The DM group additionally demonstrated an increase in the serum NO content and tNOS, iNOS, LDH, CK and CK­MB activities. The myocardial MDA, NO content and tNOS levels were additionally increased in this group. The iNOS activity was increased significantly whereas the myocardial SOD activity was decreased. The increase in the iNOS activity was supported by an enhanced expression level of myocardial iNOS mRNA and protein in the DM group. In the DM+PAG group, in the absence of H2S, the dysfunction of the left ventricle and the oxidative stress injury were increased compared with the DM group. The activity and the expression of tNOS and iNOS were increased significantly. However, the rats in the DM+PAG group demonstrated the opposite effects. In conclusion, H2S exhibits a protective effect on the myocardium in type 1 diabetic rats, which may be associated with the suppression of iNOS activity and expression, a decrease in the NO content and the inhibition of oxidative stress injury.

[Effects of hydrogen sulfide on NO content and iNOS activity in diaphragm from type 1 diabetic rats].

OBJECTIVE: To investigate the effects of hydrogen sulfide (H2S) on nitric oxide (NO) content and inducible nitric oxide syn-thase (iNOS) activity in diaphragm from type 1 diabetic rats. METHODS: Thirty-two male SD rats were randomly divided into four groups:nor-mal control group (NC), diabetes mellitus group (DM), DM treatment group (DM + NaHS) and NaHS control group (NaHS) (n=8). Rats were treated with streptozotocin 55 mg/kg by intraperitoneal injection to establish type 1 diabetic rat model. The fourth week after the model-ing, the rats in the DM + NaHS and NaHS groups were treated with 14µmol/kg NaHS solution by intraperitoneally injected. After treatment for 5 weeks, fasting blood glucose (FBG) and diaphragm weight (DW)/body weight (BW) were measured. The pathological changes of draphrag-matic tissues were observed by HE staining. The activity of iNOS was analyzed by spectrophotometric method, while the content of NO was measured by nitric acid reductase method. The iNOS expressions at mRNA and protein levels in diaphragmatic tissues were detected by RT-PCR and Western blot respectively. RESULTS: Compared with the NC group, there was no significant difference in the various indexes in the NaHS group. While FBG was increased significantly, DW/BW was decreased obviously in the DM group. HE staining revealed obvious changes in diaphragmatic tissues. The activity of iNOS and the content of NO were increased. The levels of iNOS mRNA and protein were in-creased significantly. Compared with the DM group, DW/BW and pathological damages were improved in the DM + NaHS group. The activity of iNOS and NO content were decreased significantly. The levels of iNOS mRNA and protein were decreased obviously. CONCLUSIONS: Exoge-nous H2S can suppress iNOS activity and expression to decrease the content of NO, which improving the capacity of diaphragm in type 1 diabet-ic rats.

The Protective Effect of Low-Dose Ethanol on Myocardial Fibrosis through Downregulating the JNK Signaling Pathway in Diabetic Rats.

Objective. To investigate the effects of low dose ethanol feeding in diabetic rats and analyze its underlying mechanisms. Methods. Male Sprague-Dawley rats were divided into 4 groups: control (Con), diabetes at 4 weeks (DM4W), diabetes at 8 weeks (DM8W), and EtOH + DM8W. After 8 weeks, hemodynamic parameters were recorded and heart weight/body weight (H/B) and hydroxyproline (Hp) content in myocardium were measured. Morphology of collagen in myocardial tissue was observed with Masson's trichrome staining method and collagen volume fraction (CVF) was analysed. The mRNA expression of ALDH2 was assessed with Real-Time PCR. The protein expressions of p-JNK and JNK were evaluated using western blot. Results. In contrast to Con group, there was no difference in hemodynamic parameters in DM4W group, but mean arterial pressure and heart rate were decreased in DM8W group, and the ratios of H/B, Hp, and CVF were markedly increased. ALDH2 mRNA expression was decreased, while the ratio of p-JNK/JNK were increased. Compared with DM8W group, the above indexes were improved in EtOH + DM8W group. Conclusion. With low dose ethanol intervention, enhanced ALDH2 expression can antagonize the happening of myocardial fibrosis in diabetic rats, which may be relevant with downregulating the JNK pathway.

[Effect of hydrogen sulfide on oxidative stress and endoplasmic reticulum stress in diabetic cardiomyopathy].

OBJECTIVE: To investigate the effects of hydrogen sulfide (H2S) on oxidative stress and endoplasmic reticulum stress (ERS) in a rat model of diabetic cardiomyopathy (DCM). METHODS: Thirty male SD rats were randomly divided into control group, diabetes group and treatment group( n = 10). Intraperitoneal injection of streptozotocin was utilized to establish a rat model of DCM. The rats with DCM in treatment group were intraperitoneally injected with NaHS solution. After treated for 12 weeks, the hearts isolated from rats were perfused on a langendorff apparatus. The ventricular hemodynamic parameters were measured. The ultrastructures of myocardium were observed using electron microscopy. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in myocardial tissue were determined by spectrophotometry. The expressions of C/EBP homologous protein( CHOP), glucose-regulated protein 78 (GRP78) and Caspase 12 at mRNA level in myocardium were detected using RT-PCR. RESULTS: Compared with control group, the cardiac function and myocardial ultrastructure were damaged obviously in diabetic rats. In myocardial tissue, the content of MDA was increased, while the activities of SOD and GSH-Px were decreased. CHOP, GRP78 and Caspase 12 mRNA expressions were increased significantly. Compared with diabetes group, cardiac function and myocardial ultrastructure damage were improved in treatment group. The content of MDA was decreased, while the activities of SOD and GSH-Px were increased significantly. The mRNA levels of CHOP, GRP78 and Caspase 12 were increased. CONCLUSION: H2S can protect myocardium in diabetic rats, maybe it is related to reduce oxidative stress damage and inhibition of the ERS-induced apoptosis pathway.

[Protective effect of hydrogen sulfide on kidneys of type 1 diabetic rats].

OBJECTIVE: To explore the protective effects of hydrogen sulfide (H2S) on kidneys of type 1 diabetic rats and its underlying mechanism. METHODS: Thirty-two male SD rats were randomly divided into four groups:normal control (NC) group, diabetes mellitus (DM) group, DM treatment (NaHS+DM) group and NaHS control (NaHS) group. The rats from DM group and NaHS+DM group were injected intraperitoneally with Streptozotocin 55 mg/kg to induce type 1 diabetes mellitus (n=8). After modeling, rats in NaHS+DM group and NaHS group were intraperitoneally injected with NaHS solution at the dosage of 56 µmol/kg. After 8 weeks, urinary protein content was detected in urine samples collected for 24 h. and the ratio of kidney weight/body weight (renal index) was determined in isolated kidneys. Besides, the levels of fasting blood glucose (FBG), blood urea nitrogen (BUN) and serum creatinine (Scr) were measured biochemically. The morphological changes of renal tissue were observed by HE staining. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and Caspase-3 in renal tissue were determined by spectrophotometry. The protein expression levels of Bcl-2 and Bax in renal tissue were detected using Western blot. RESULTS: There was no significant difference in the respective measured indexes in rats between NC group and NaHS group. However, in DM group, the levels of 24 h urinary protein, FBG, BUN, Scr and renal index were increased significantly; HE staining showed that the basement membrane was thickened and the amount of glomerular mesangial matrix was increased; MDA content, Caspase-3 activity and Bax expression levels were increased, while SOD activity and Bcl-2 expression were decreased. Compared with those in DM group, the morphological changes of renal tissue and its function were improved; MDA content, Caspase-3 activity and Bax expression were decreased significantly, while SOD activity and Bcl-2 expression were increased obviously in NaHS+DM group. CONCLUSIONS: H2S can protect the kidneys of type 1 diabetic rats, which is related to suppressing oxidative stress and cell apoptosis.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.

Adoptive transfer of M2 macrophages promotes locomotor recovery in adult rats after spinal cord injury.

Classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) macrophages populate the local microenvironment after spinal cord injury (SCI). The former type is neurotoxic while the latter has positive effects on neuroregeneration and is less toxic. In addition, while the M1 macrophage response is rapidly induced and sustained, M2 induction is transient. A promising strategy for the repair of SCI is to increase the fraction of M2 cells and prolong their residence time. This study investigated the effect of M2 macrophages induced from bone marrow-derived macrophages on the local microenvironment and their possible role in neuroprotection after SCI. M2 macrophages produced anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor ß and infiltrated into the injured spinal cord, stimulated M2 and helper T (Th)2 cells, and produced high levels of IL-10 and -13 at the site of injury. M2 cell transfer decreased spinal cord lesion volume and resulted in increased myelination of axons and preservation of neurons. This was accompanied by significant locomotor improvement as revealed by Basso, Beattie and Bresnahan locomotor rating scale, grid walk and footprint analyses. These results indicate that M2 adoptive transfer has beneficial effects for the injured spinal cord, in which the increased number of M2 macrophages causes a shift in the immunological response from Th1- to Th2-dominated through the production of anti-inflammatory cytokines, which in turn induces the polarization of local microglia and/or macrophages to the M2 subtype, and creates a local microenvironment that is conducive to the rescue of residual myelin and neurons and preservation of neuronal function.

Effects of Olig2-overexpressing neural stem cells and myelin basic protein-activated T cells on recovery from spinal cord injury.

Neural stem cell (NSC) transplantation is a major focus of current research for treatment of spinal cord injury (SCI). However, it is very important to promote the survival and differentiation of NSCs into myelinating oligodendrocytes (OLs). In this study, myelin basic protein-activated T (MBP-T) cells were passively immunized to improve the SCI microenvironment. Olig2-overexpressing NSCs were infected with a lentivirus carrying the enhanced green fluorescent protein (GFP) reporter gene to generate Olig2-GFP-NSCs that were transplanted into the injured site to differentiate into OLs. Transferred MBP-T cells infiltrated the injured spinal cord, produced neurotrophic factors, and induced the differentiation of resident microglia and/or infiltrating blood monocytes into an "alternatively activated" anti-inflammatory macrophage phenotype by producing interleukin-13. As a result, the survival of transplanted NSCs increased fivefold in MBP-T cell-transferred rats compared with that of the vehicle-treated control. In addition, the differentiation of MBP-positive OLs increased 12-fold in Olig2-GFP-NSC-transplanted rats compared with that of GFP-NSC-transplanted controls. In the MBP-T cell and Olig2-GFP-NSC combined group, the number of OL-remyelinated axons significantly increased compared with those of all other groups. However, a significant decrease in spinal cord lesion volume and an increase in spared myelin and behavioral recovery were observed in Olig2-NSC- and NSC-transplanted MBP-T cell groups. Collectively, these results suggest that MBP-T cell adoptive immunotherapy combined with NSC transplantation has a synergistic effect on histological and behavioral improvement after traumatic SCI. Although Olig2 overexpression enhances OL differentiation and myelination, the effect on functional recovery may be surpassed by MBP-T cells.

Hydrogen sulphide is an inhibitor of L-type calcium channels and mechanical contraction in rat cardiomyocytes.

AIMS: Hydrogen sulphide (H(2)S) is an endogenously generated gaseous transmitter that has recently been suggested to regulate cardiovascular functions. To date, there is no direct evidence for a potential role of H(2)S in regulating calcium channels in the heart. The present study aims to examine the hypothesis that H(2)S is a novel inhibitor of the L-type calcium channel current (I(Ca,L)). METHODS AND RESULTS: Electrophysiological measurements were performed in cardiomyocytes isolated from Wistar-Kyoto and spontaneously hypertensive rats. Bath application of 100 microM NaHS (a H(2)S donor) significantly reduced the time required for the repolarization of the action potential. Inhibition of the peak I(Ca,L) by NaHS was determined to be concentration-dependent (25, 50, 100, 200, and 400 microM). NaHS inhibited the recovery from depolarization-induced inactivation. Electric field-induced [Ca(2+)]i transients and contraction of single cardiomyocytes and isolated papillary muscles were reduced by NaHS treatment. In contrast, caffeine induced an increase in [Ca(2+)]i that was not altered by NaHS. NaHS had no effect on the K(ATP) current or on the levels of cAMP and cGMP in the current study. CONCLUSION: H(2)S is a novel inhibitor of L-type calcium channels in cardiomyocytes. Moreover, H(2)S-induced inhibition of [Ca(2+)]i appears to be a secondary effect owing to its initial action towards I(Ca,L). The inhibitory effect of H(2)S on I(Ca,L) requires further investigation, particularly in the exploration of new pathways involved in cardiac calcium homeostasis and disease pathology.

[Effect of soybean isoflavones on heart function of rats with adriamycin-induced heart failure].

OBJECTIVE: To observe the protective effect of soybean isoflavones (SI) on the heart function of the rats with adriamycin induced heart failure. METHODS: Thirty adult male SD rats were divided into 5 groups:normal control (NC) group, adriamycin (ADR) group, L-SI group, M-SI group and H-SI group. SI of 30, 60, 120 mg.kg(-1).d(-1) was orally administered through a stomach tube once a day for 6 days in L-SI group, M-SI group and H-SI group, respectively. The other two groups were given the same amount of normal saline the same way. Then ADR of 10 mg/kg was given intraperitoneally once to copy the model of heart-failure. The MedLab-U/4c biological signal collecting system was used to record and analyze the LVSP of the rats. The pathological changes of the cardiomyocytes were observed. RESULTS: As compared with NC group, the LVSP,+/-dp/dt max, Vpm of the ADR group were significantly lower (P<0.05 or P<0.01), but those of the H-SI group were markedly higher than those of the ADR group (P<0.01). Electron microscopic morphometry of the heart samples of the rats in ADR group revealed typical alterations, consisting an increase of collagen content, vacuolation, diminishing of the cardiomyocyte diameter, alteration of myofilaments and Z-lines of myofibers, and myofibrillar degeneration. SI of 120 mg.kg(-1).d(-1) treatment could prevent the loss of myofibrillae and the reduction of myocyte diameter, and the degeneration of myofilaments and Z-lines were reversed by SI. CONCLUSION: SI of 120 mg.kg(-1).d(-1) treatment can relieve the toxic effect of ADR on myocardium, and also obviously improve the cardiac contractility of heart-failure rats.