Wenming Xu

Exogenous Hydrogen Sulfide Protects against Doxorubicin-Induced Inflammation and Cytotoxicity by Inhibiting p38MAPK/NFκB Pathway in H9c2 Cardiac Cells

Background/Aim:We have demonstrated that exogenous hydrogen sulfide (H2S) protects H9c2 cardiac cells against the doxorubicin (DOX)-induced injuries by inhibiting p38 mitogen-activated protein kinase (MAPK) pathway and that the p38 MAPK/nuclear factor-κB (NF-κB) pathway is involved in the DOX-induced inflammatory response and cytotoxicity. The present study attempts to test the hypothesis that exogenous H2S might protect cardiomyocytes against the DOX-induced inflammation and cytotoxicity through inhibiting p38 MAPK/NF-κB pathway. Methods: H9c2 cardiac cells were exposed to 5µM DOX for 24 h to establish a model of DOX cardiotoxicity. The cells were pretreated with NaHS( a donor of H2S) or other drugs before exposure to DOX. Cell viability was analyzed by cell counter kit 8 ( CCK-8), The expression of NF-κB p65 and inducible nitric oxide synthase (iNOS) was detected by Western blot assay. The levels of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-a (TNF-a) were tested by enzyme-linked immunosorbent assay (ELISA). Results: Our findings demonstrated that pretreatment of H9c2 cardiac cells with NaHS for 30 min before exposure to DOX markedly ameliorated the DOX-induced phosphorylation and nuclear translocation of NF-κB p65 subunit. Importantly, the pretreatment with NaHS significantly attenuated the p38 MAPK/NF-κB pathway-mediated inflammatory responses induced by DOX, as evidenced by decreases in the levels of IL-1ß, IL-6 and TNF-a. In addition, application of NaHS or IL-1ß receptor antagonist (IL-1Ra) or PDTC (an inhibitor of NF-κB) attenuated the DOX-induced expression of iNOS and production of nitric oxide (NO), respectively. Furthermore, IL-1Ra also dramatically reduced the DOX-induced cytotoxicity and phosphorylation of NF-κB p65. The pretreatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS) prior to exposure to DOX depressed the phosphorylation of NF-κB p65 induced by DOX. Conclusion: The present study has demonstrated the new mechanistic evidence that exogenous H2S attenuates the DOX-induced inflammation and cytotoxicity by inhibiting p38 MAPK/NF-κB pathway in H9c2 cardiac cells. We also provide novel data that the interaction between NF-κB pathway and IL-1ß is important in the induction of DOX-induced inflammation and cytotoxicity in H9c2 cardiac cells.

Activation of the p38 MAPK/NF-κB pathway contributes to doxorubicin-induced inflammation and cytotoxicity in H9c2 cardiac cells.

A number of studies have demonstrated that inflammation plays a role in doxorubicin (DOX)-induced cardiotoxicity. However, the molecular mechanism by which DOX induces cardiac inflammation has yet to be fully elucidated. The present study aimed to investigate the role of the p38 mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) pathway in DOX-induced inflammation and cytotoxicity. The results of our study demonstrated that the exposure of H9c2 cardiac cells to DOX reduced cell viability and stimulated an inflammatory response, as demonstrated by an increase in the levels of interleukin-1β (IL-1β) and IL-6, as well as tumor necrosis factor-α (TNF-α) production. Notably, DOX exposure induced the overexpression of phosphorylated p38 MAPK and phosphorylation of the NF-κB p65 subunit, which was markedly inhibited by SB203580, a specific inhibitor of p38 MAPK. The inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-κB, significantly ameliorated DOX-induced inflammation, leading to a decrease in the levels of IL-1β and IL-6, as well as TNF-α production in H9c2 cells. The pretreatment of H9c2 cells with either SB203580 or PDTC before exposure to DOX significantly attenuated DOX-induced cytotoxicity. In conclusion, our study provides novel data demonstrating that the p38 MAPK/NF-κB pathway is important in the induction of DOX-induced inflammation and cytotoxicity in H9c2 cardiac myocytes.

Exogenous hydrogen sulfide protects H9c2 cardiac cells against high glucose-induced injury by inhibiting the activities of the p38 MAPK and ERK1/2 pathways

Hyperglycemia is a risk factor for the development of diabetic cardiovascular complications, which are associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrate the inhibitory effects of exogenous hydrogen sulfide (H₂S) on the activation of the MAPK pathway. The aim of the present study was to determine whether exogenous H₂S prevents high glucose (HG)-induced injury by inhibiting the activation of the p38 MAPK and extracellular signal-regulated kinase (ERK)1/2 (members of MAPK) pathways in cardiomyoblasts (H9c2 cells). The findings of the present study demonstrated that the treatment of H9c2 cells with HG (35 mM glucose) for 24 h not only significantly induced injury, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP), but also upregulated the expression levels of phosphorylated (p)-p38 MAPK and p-ERK1/2. The increased expression levels of p-p38 MAPK and p-ERK1/2 were markedly reduced by pre-treatment of the H9c2 cells with 400 µM sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to 35 mM glucose. Importantly, pre-treatment of the cells with 400 µM NaHS or 3 µM SB203580 (a selective inhibitor of p38 MAPK) or 15 µM U0126 (a selective inhibitor of ERK1/2) attenuated the HG-induced cardiomyocyte injury, leading to an increase in cell viability and a decrease in the number of apoptotic cells, preventing ROS generation, as well as the loss of MMP. In addition, pre-treatment of the cells with 1,000 µM N‑acetyl‑L‑cysteine (a ROS scavenger) prior to exposure to HG ameliorated the HG-induced cytotoxicity. Taken together, the data from the present study demonstrate for the first time, to our knowledge, that exogenous H2S exerts a protective effect against HG‑induced injury by inhibiting the activation of the p38 MAPK and ERK1/2 pathways and preventing oxidative stress in H9c2 cells.

Exogenous H2S protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the activation of the NF-κB and IL-1β pathways

Hyperglycemia has been reported to activate the nuclear factor-κB (NF-κB) pathway. We have previously demonstrated that exogenous hydrogen sulfide (H2S) protects cardiomyocytes against high glucose (HG)-induced injury by inhibiting the activity of p38 mitogen-activated protein kinase (MAPK), which can activate the NF-κB pathway and induce interleukin (IL)-1β production. In the present study, we aimed to investigate the hypothesis that exogenous H2S protects cardiomyocytes against HG-induced injury and inflammation through the inhibition of the NF-κB/IL-1β pathway. H9c2 cardiac cells were treated with 35 mM glucose (HG) for 24 h to establish a model of HG-induced damage. Our results demonstrated that treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) or 100 µM pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) for 30 min prior to exposure to HG markedly attenuated the HG-induced increase in the expression levels of the phosphorylated (p)-NF-κB p65 subunit. Notably, pre-treatment of the H9c2 cardiac cells with NaHS or PDTC significantly suppressed the HG-induced injury, including cytotoxicity, apoptosis, oxidative stress and mitochondrial insults, as evidenced by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). In addition, pre-treatment of the cells with NaHS or PDTC ameliorated the HG-induced inflammatory response, leading to a decrease in the levels of IL-1β, IL-6 and tumor necrosis factor-α (TNF-α). Importantly, co-treatment of the H9c2 cells with 20 ng/ml IL-1 receptor antagonist (IL-1Ra) and HG markedly reduced the HG-induced increase in p-NF-κB p65 expression, cytotoxicity, the number of apoptotic cells, as well as the production of TNF-α. In conclusion, the present study presents novel mechanistic evidence that exogenous H2S protects H9c2 cardiac cells against HG-induced inflammation and injury, including cytotoxicity, apoptosis, overproduction of ROS and the dissipation of MMP, by inhibiting the NF-κB/IL-1β pathway. We also provide new data indicating that the positive interaction between the NF-κB pathway and IL-1β is critical in HG-induced injury and inflammation in H9c2 cardiac cells.

Integrative genomic analyses of a novel cytokine, interleukin-34 and its potential role in cancer prediction

Interleukin-34 (IL-34) is a novel cytokine, which is composed of 222 amino acids and forms homodimers. It binds to the macrophage colony-stimulating factor (M-CSF) receptor and plays an important role in innate immunity and inflammatory processes. In the present study, we identified the completed IL-34 gene in 25 various mammalian genomes and found that IL-34 existed in all types of vertebrates, including fish, amphibians, birds and mammals. These species have a similar 7 exon/6 intron gene organization. The phylogenetic tree indicated that the IL-34 gene from the primate lineage, rodent lineage and teleost lineage form a species-specific cluster. It was found mammalian that IL-34 was under positive selection pressure with the identified positively selected site, 196Val. Fifty-five functionally relevant single nucleotide polymorphisms (SNPs), including 32 SNPs causing missense mutations, 3 exonic splicing enhancer SNPs and 20 SNPs causing nonsense mutations were identified from 2,141 available SNPs in the human IL-34 gene. IL-34 was expressed in various types of cancer, including blood, brain, breast, colorectal, eye, head and neck, lung, ovarian and skin cancer. A total of 5 out of 40 tests (1 blood cancer, 1 brain cancer, 1 colorectal cancer and 2 lung cancer) revealed an association between IL-34 gene expression and cancer prognosis. It was found that the association between the expression of IL-34 and cancer prognosis varied in different types of cancer, even in the same types of cancer from different databases. This suggests that the function of IL-34 in these tumors may be multidimensional. The upstream transcription factor 1 (USF1), regulatory factor X-1 (RFX1), the Sp1 transcription factor 1, POU class 3 homeobox 2 (POU3F2) and forkhead box L1 (FOXL1) regulatory transcription factor binding sites were identified in the IL-34 gene upstream (promoter) region, which may be involved in the effects of IL-34 in tumors.

Hydrogen sulfide protects PC12 cells against reactive oxygen species and extracellular signal-regulated kinase 1/2-mediated downregulation of glutamate transporter-1 expression induced by chemical hypoxia

Hypoxia and/or ischemia are implicated in neurodegenerative disorders. In these diseases, hypoxia/ischemia may induce oxidative stress, including production of reactive oxygen species (ROS), which result in a decrease in glutamate transporter expression. Hydrogen sulfide (H2S), as the third gasotransmitter, has neuroprotective effects and potent antioxidant properties. In the present study, we investigated the role of glutamate transporter-1 (GLT-1) in the protection of H2S against chemical hypoxia-induced injury in PC12 cells. We found that cobalt chloride (CoCl2), a chemical hypoxia agent, reduced the expression of GLT-1 in a time-dependent manner. Pretreatment with NaHS (a donor of H2S) reversed the CoCl2-induced downregulation of GLT-1 expression. Pretreatment with DHK (a selective inhibitor of GLT-1) for 30 min prior to NaHS preconditioning significantly inhibited the cytoprotection of H2S against CoCl2-induced injuries, leading to an increase in cytotoxicity and apoptosis as well as to a loss of mitochondrial membrane potential (MMP). In addition, we found that similar to the effect of NaHS, pretreatment with NAC (a ROS scavenger) or U0126 (a MEK1/2 inhibitor) blocked the downregulation of GLT-1 expression induced by CoCl2. Collectively, we demonstrated for the first time that ROS and extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated reduction of GLT-1 expression may be involved in chemical hypoxia-induced neural injury and that H2S attenuates this injury partly by upregulating GLT-1 expression in PC12 cells.

Increased expression of heat shock protein 90 under chemical hypoxic conditions protects cardiomyocytes against injury induced by serum and glucose deprivation

Heat shock proteins (HSPs) are critical for adaptation to hypoxia and/or ischemia. Previously, we demonstrated that cobalt chloride (CoCl2), a well-known hypoxia mimetic agent, is an inducer of HSP90. In the present study, we tested the hypothesis that CoCl₂-induced upregulation of HSP90 is able to provide cardioprotection in serum and glucose-deprived H9c2 cardiomyocytes (H9c2 cells). Cell viability was detected using a CCK-8 assay, while HSP90 expression was detected via western blotting. The findings of this study showed that serum and glucose deprivation (SGD) induced significant cytotoxicity, overproduction of reactive oxygen species (ROS) and a loss of mitochondrial membrane potential (MMP) in H9c2 cells. In addition, SGD downregulated the expression of HSP90 in a time-dependent manner. The selective inhibitor of HSP90 17-allylamino-17-demethoxygeldanamycin (17-AAG) aggravated SGD-induced cytotoxicity. CoCl₂ at 100 µM time-dependently enhanced the expression of HSP90. Treatment with CoCl₂ from 50 to 200 µM significantly attenuated cytotoxicity and the downregulation of HSP90 expression induced by SGD for 24 h, respectively. Notably, pretreatment of H9c2 cells with 17-AAG at 2 µM for 60 min before exposure to both CoCl2 (100 µM) and SGD significantly blocked the CoCl2-induced cardioprotective effect, demonstrated by decreased cell viability and MMP loss, as well as increased ROS generation. Taken together, these results suggest that HSP90 may be one of the endogenous defensive mechanisms for resisting ischemia-like injury in H9c2 cells, and that HSP90 plays an important role in chemical hypoxia-induced cardioprotection against SGD-induced injury by its antioxidation and preservation of mitochondrial function.

Integrative genomic analyses of secreted protein acidic and rich in cysteine and its role in cancer prediction

Secreted protein acidic and rich in cysteine (SPARC), also termed osteonectin or basement‑membrane‑40 (BM‑40), is a matrix‑associated protein that elicits changes in cell shape, inhibits cell‑cycle progression and affects the synthesis of extracellular matrix (ECM). The final mature SPARC protein has 286 amino acids with three distinct domains, including an NH2‑terminal acidic domain (NT), follistatin‑like domain (FS) and C terminus domain (EC). The present study identified SPARC genes from 14 vertebrate genomes and revealed that SPARC existed in all types of vertebrates, including fish, amphibians, birds and mammals. In total, 21 single nucleotide polymorphisms (SNPs) causing missense mutations were identified, which may affect the formation of the truncated form of the SPARC protein. The human SPARC gene was found to be expressed in numerous tissues or organs, including in the bone marrow, whole blood, lymph node, thymus, brain, cerebellum, retina, heart, smooth muscle, skeletal muscle, spinal cord, intestine, colon, adipocyte, kidney, liver, pancreas, thyroid, salivary gland, skin, ovary, uterus, placenta, cervix and prostate. When searched in the PrognoScan database, the human SPARC gene was also found to be expressed in bladder, blood, breast, glioma, esophagus, colorectal, head and neck, ovarian, lung and skin cancer tissues. It was revealed that the association between the expression of SPARC and prognosis varied in different types of cancer, and even in the same cancer from different databases. It implied that the function of SPARC in these tumors may be multidimensional, functioning not just as a tumor suppressor or oncogene.

Interleukin-12 and interleukin-2 alone or in combination against the infection in invasive pulmonary aspergillosis mouse model

Aspergillus fumigatus is an intracellular opportunistic fungus causing invasive pulmonary mycosis, characterised by hyphal invasion and destruction of pulmonary tissue. Th1 cytokines could enhance fungicidal activity. The effects from the combination of interleukin‐12 (IL‐12) and IL‐2 are rarely known in invasive pulmonary aspergillosis infection. To assess the cleaning of A. fumigatus infection in the pulmonary tissues by IL‐12 and IL‐2, interferon‐γ (IFN‐γ) was detected in the sera using ELISA, quantification of IFN‐γ mRNA using real‐time RT‐PCR and lung Colony‐forming unit was assayed by cultivation. Morphology was analysed by histopathological examination. Our results showed that IL‐12 and/or IL‐2 could enhance the IFN‐γ expression in the pulmonary tissue, reduce the colony load in the pulmonary tissue and increase the survival rate of mouse. The combination of IL‐12 and IL‐2 could assist in increasing the IFN‐γ expression in the pulmonary tissue, but neither reduce colony load in the pulmonary tissue nor increase the survival rate of mouse significantly. It was demonstrated that IL‐12 and IL‐2 were strong immunomodulatory cytokines as a prerequisite for protecting the host from infectious agents.

The serum glucan level and pathological changes of antifungal treatment for lower respiratory tract infection of Candida albicans

Due to the fact that Candida albicans colonizes in the upper respiratory tracts of healthy people, whether or not its isolation from airway secretions is sufficient to warrant treatment remains controversial. The animal models of immunosuppressive rats with pulmonary candidiasis were established by the intratracheal inoculating suspensions of C. albicans, and the animals were divided into the following three groups: (1) antifungal treatment group, (2) saline control group, and (3) blank control group. We noted the following in our studies: (1) The fungal load of the saline control group gradually increased such that it was higher than those of the antifungal treated group and was significant from the fourth day of treatment (P < 0.01). (2) The serum (1,3)-β-D-glucan (BG) in the saline control group also gradually increased so that it was significantly higher than found with the treated group by the sixth day of treatment (P < 0.05), and in fact, the rank of pulmonary colony count and BG in the two groups at different time points showed an almost perfect linear correlation. (3) The median survival period of the rats in the antifungal treated group and saline control group was 15 and 8 days respectively, no rats died in the blank control group. (4) The lung lesions from the saline control group gradually became more aggravated than those in the antifungal treated group; no significant pathological changes were found in the blank control group. Antifungal treatment (micafungin) is capable of efficaciously decreasing the lung fungal burden, and continuous monitoring of BG is useful for the evaluation of therapeutic effect of antifungals. Infection of C. albicans with associated pathological damage implies the need for antifungal therapy.