[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.

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.

[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.

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 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.

[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.