Hydrogen Sulfide Ameliorates Lipopolysaccharide-Induced Memory Impairment in Mice by Reducing Apoptosis, Oxidative, and Inflammatory Effects.

Hydrogen sulfide (H2S) is reported to have a neuroprotective activity; however, the role of H2S in neuroinflammation-induced neuronal damage is ambiguous. Here, we aimed to evaluate the underlying mechanisms for the neuroprotective effect of NaHS, a known H2S donor, against lipopolysaccharide (LPS)-induced memory impairment (MI). All the treatments were administered for 28 days, and LPS (0.25 mg/kg i.p.) was co-administered intermittently for 7 days from days 15 to 21. Morris water maze (MWM) and Y-maze tests were performed to evaluate MI. Neurodegeneration was histopathologically examined, and the brain homogenates were characterized for reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor (TNF)-α, interleukin (IL)-6, caspase-3, c-Jun, and acetylcholinesterase (AChE) by biochemical analysis. H2S administration significantly improved spatial and working memory in MWM and Y-maze tasks, respectively. Exogenous H2S significantly reversed LPS-induced oxidative stress as evidenced by improved GSH, MDA, and SOD levels. H2S pretreatment significantly attenuated LPS-induced apoptosis and inflammation by decreasing c-Jun and caspase-3 levels and inhibiting TNF-α and IL-6, respectively. The decrease in these markers was supported by H&E and Nissl staining, which confirmed the anti-necrotic activity of H2S. However, there was no significant improvement in LPS-induced increase in AChE activity. These results indicate that chronic systemic inflammation leads to neurodegeneration and MI and H2S exerts its neuroprotective effect due to its anti-oxidative, anti-inflammatory, and anti-apoptotic potential via modulation of JNK and extrinsic apoptosis pathways.

Metal-organic frameworks for therapeutic gas delivery.

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are gaseous signaling molecules (gasotransmitters) that regulate both physiological and pathological processes and offer therapeutic potential for the treatment of many diseases, such as cancer, cardiovascular disease, renal disease, bacterial and viral infections. However, the inherent labile nature of therapeutic gases results in difficulties in direct gases administration and their controlled delivery at clinically relevant ranges. Metal-organic frameworks (MOFs) with highly porous, stable, and easy-to-tailor properties have shown promising therapeutic gas delivery potential. Herein, we highlight the recent advances of MOF-based platforms for therapeutic gas delivery, either by endogenous (i.e., direct transfer of gases to targets) or exogenous (i.e., stimulating triggered release of gases) means. Reports that involve in vitro and/or in vivo studies are highlighted due to their high potential for clinical translation. Current challenges for clinical requirements and possible future innovative designs to meet variable healthcare needs are discussed.

Activation of Endogenous H2S Biosynthesis or Supplementation with Exogenous H2S Enhances Adipose Tissue Adipogenesis and Preserves Adipocyte Physiology in Humans.

Aims: To investigate the impact of exogenous hydrogen sulfide (H2S) and its endogenous biosynthesis on human adipocytes and adipose tissue in the context of obesity and insulin resistance. Results: Experiments in human adipose tissue explants and in isolated preadipocytes demonstrated that exogenous H2S or the activation of endogenous H2S biosynthesis resulted in increased adipogenesis, insulin action, sirtuin deacetylase, and PPARγ transcriptional activity, whereas chemical inhibition and gene knockdown of each enzyme generating H2S (CTH, CBS, MPST) led to altered adipocyte differentiation, cellular senescence, and increased inflammation. In agreement with these experimental data, visceral and subcutaneous adipose tissue expression of H2S-synthesising enzymes was significantly reduced in morbidly obese subjects in association with attenuated adipogenesis and increased markers of adipose tissue inflammation and senescence. Interestingly, weight-loss interventions (including bariatric surgery or diet/exercise) improved the expression of H2S biosynthesis-related genes. In human preadipocytes, the expression of CTH, CBS, and MPST genes and H2S production were dramatically increased during adipocyte differentiation. More importantly, the adipocyte proteome exhibiting persulfidation was characterized, disclosing that different proteins involved in fatty acid and lipid metabolism, the citrate cycle, insulin signaling, several adipokines, and PPAR, experienced the most dramatic persulfidation (85-98%). Innovation: No previous studies investigated the impact of H2S on human adipose tissue. This study suggests that the potentiation of adipose tissue H2S biosynthesis is a possible therapeutic approach to improve adipose tissue dysfunction in patients with obesity and insulin resistance. Conclusion: Altogether, these data supported the relevance of H2S biosynthesis in the modulation of human adipocyte physiology. Antioxid. Redox Signal. 35, 319-340.

In vitro anticancer activity of hydrogen sulfide and nitric oxide alongside nickel nanoparticle and novel mutations in their genes in CRC patients.

This study was carried out to assess the impact of nickel nanoparticles (NiNPs) as well as scorpion venom on colorectal cancer (CRC) cells in the presence and/or absence of 5-fluorouracil (5-FU), hydrogen sulfide (H2S), and nitric oxide (NO) donors and to determine alterations in endothelial NO synthase (eNOS) and cystathionine γ-lyase (CSE) enzyme-producing genes in CRC patients. The IC50 of both H2S and NO donors, along with NiNPs, were determined. The CRC cells were treated for 24hrs, and the cytotoxic activities were assessed using the MTT test. Moreover, the apoptosis was determined after 24hrs and 48hrs using TUNEL assay. Furthermore, the mutations in the eNOS gene (intron 4, -786T>C and 894 G>T) and CSE gene (1364GT) were determined using direct sequencing. The IC50 values for sodium disulfide (Na2S) and sodium nitroprusside (SNP) at 24hrs treatment were found to be 5 mM and 10-6 M, respectively, while the IC50 value for 5-FU was reached after 5-days of treatment in CRC cell line. Both black and yellow scorpion venoms showed no inhibition of cell proliferation after 24hrs treatment. Furthermore, Na2S showed a significant decrease in cell proliferation and an increase in apoptosis. Moreover, a co-treatment of SNP and 5-FU resulted in inhibition of the cytotoxic effect of 5-FU, while a combination treatment of NiNPs with Na2S, SNP, and 5-FU caused highly significant cytotoxicity. Direct sequencing reveals new mutations, mainly intronic variation in eNOS gene that has not previously been described in the database. These findings indicate that H2S promotes the anticancer efficiency of 5-FU in the presence of NiNPs while NO has antiapoptotic activity in CRC cell lines.

Hydrogen sulfide is a regulator of mammary gland development in prepubescent female mice.

The present study aimed to investigate the effects of exogenous H2S on mammary gland development in pubescent mice and to explore the underlying mechanism. The mouse mammary epithelial cell line HC11, along with C57BL/6J mice, were treated with different concentrations of sodium hydrosulfide (NaHS), which is a donor of H2S. The HC11 cell viability, pubescent mammary gland development, and the involvement of proliferative proteins and pathways were assessed by CCK­8 assay, EdU assay, whole mount staining, H&E staining, western blotting and reverse transcription­quantitative PCR. Both in vitro and in vivo, a low concentration of NaHS (100 µM in vitro; 9 mg/kg in vivo) significantly promoted the viability of HC11 cells and the development of mammary glands by increasing the expression of the proliferative markers cyclin D1/3 and proliferating cell nuclear antigen. However, a high concentration of NaHS (1,000 µM in vitro; 18 mg/kg in vivo) inhibited HC11 cell viability, mammary gland development and the expression levels of proteins involved in proliferation. Subsequent experiments revealed that NaHS regulated the phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt)­mammalian target of rapamycin (mTOR) signaling pathway during this process. In vivo, intraperitoneal injection of low concentration NaHS (9 mg/kg) activated the PI3K/Akt­mTOR pathway in mammary glands of pubescent mice, increased the secretion of insulin­like growth factor 1 (IGF­1) and estradiol (E2), and then stimulated mammary gland ductal development. Whereas a high concentration of NaHS (18 mg/kg) elicited the opposite effects to those of low­dose NaHS. In conclusion, the present study demonstrated that exogenous H2S supplied by NaHS may exert bidirectional effects on mammary gland ductal development; promoting ductal development at a low concentration and inhibiting it at a high concentration. The effects of H2S may occur via the intracellular PI3K/Akt­mTOR signaling pathway, or by regulation of the secretion of IGF­1 and E2.

Exogenous hydrogen sulfide reduces atrial remodeling and atrial fibrillation induced by diabetes mellitus via activation of the PI3K/Akt/eNOS pathway.

Diabetes mellitus (DM) facilitates atrial fibrosis and increases the risk of atrial fibrillation (AF). The underlying mechanism of DM in causing AF remains mostly unknown and potential therapeutic targets for DM­induced AF are rarely reported. Hydrogen sulfide (H2S) has drawn considerable attention in recent years for its potential as a cardiovascular protector. Thus, the aim of the present study was to investigate the effect of H2S on DM­induced AF and the mechanism of action. Sprague­Dawley rats were divided into four groups, including the control group, the DM group, the H2S group and the DM+H2S group. The DM group and the DM+H2S group were administered streptozotocin to induce DM, whereas the other two groups were given citrate buffer as a control. The H2S group and the DM+H2S group were administered with an intraperitoneal injection of sodium hydrosulfide (precursor of H2S). AF inducibility, AF duration, atrial fibrosis and vital protein expression of oxidative stress were compared among the four groups. The DM group showed significantly higher AF incidence rates and duration (P<0.05). Histology results demonstrated severe atrial fibrosis in the DM group, and the PI3K/Akt/endothelial nitric oxide synthase (eNOS) pathway was significantly downregulated (P<0.05). However, when H2S was administered, the rats showed lower AF incidence and duration compared with the DM group. Additionally, H2S was able to mitigate the atrial fibrosis induced by DM, as well as the proliferation and migration of cardiac fibroblasts, as demonstrated by an MTT assay and real­time cell analyzer migration experiment. Western blotting showed that the expression levels of the PI3K/Akt/eNOS pathway in the DM+H2S group were significantly upregulated compared with those of the DM group (P<0.05). In summary, DM status can lead to the structural remodeling of atrial fibrosis, facilitating AF incidence and persistence. Administration of H2S does not affect the glucose level, but can significantly mitigate atrial fibrosis and reduce the incidence of AF induced by DM, probably via activation of the PI3K/Akt/eNOS pathway.

Hydrogen Sulfide Alleviates Anxiety, Motor, and Cognitive Dysfunctions in Rats with Maternal Hyperhomocysteinemia via Mitigation of Oxidative Stress.

Hydrogen sulfide (H2S) is endogenously produced from sulfur containing amino acids, including homocysteine and exerts neuroprotective effects. An increase of homocysteine during pregnancy impairs fetal growth and development of the offspring due to severe oxidative stress. We analyzed the effects of the H2S donor-sodium hydrosulfide (NaHS) administered to female rats with hyperhomocysteinemia (hHcy) on behavioral impairments and levels of oxidative stress of their offspring. Rats born from females fed with control or high methionine diet, with or without H2S donor injections were investigated. Rats with maternal hHcy exhibit increased levels of total locomotor activity and anxiety, decreased muscle endurance and motor coordination, abnormalities of fine motor control, as well as reduced spatial memory and learning. Oxidative stress in brain tissues measured by activity of glutathione peroxidases and the level of malondialdehyde was higher in rats with maternal hHcy. Concentrations of H2S and the activity and expression of the H2S generating enzyme-cystathionine-beta synthase-were lower compared to the control group. Administration of the H2S donor to females with hHcy during pregnancy prevented behavioral alterations and oxidative stress of their offspring. The acquisition of behavioral together with biochemical studies will add to our knowledge about homocysteine neurotoxicity and proposes H2S as a potential agent for therapy of hHcy associated disorders.

Tumor pH-responsive metastable-phase manganese sulfide nanotheranostics for traceable hydrogen sulfide gas therapy primed chemodynamic therapy.

Manganese-based nanomaterials have piqued great interest in cancer nanotheranostics, owing to their excellent physicochemical properties. Here we report a facile wet-chemical synthesis of size-controllable, biodegradable, and metastable γ-phase manganese sulfide nanotheranostics, which is employed for tumor pH-responsive traceable gas therapy primed chemodynamic therapy (CDT), using bovine serum albumin (BSA) as a biological template (The final product was denoted as MnS@BSA). The as-prepared MnS@BSA can be degraded in response to the mildly acidic tumor microenvironment, releasing hydrogen sulfide (H2S) for gas therapy and manganese ions for magnetic resonance imaging (MRI) and CDT. In vitro experiments validated the pH-responsiveness of MnS@BSA at pH 6.8 and both H2S gas and •OH radicals were detected during its degradation. In vivo experiments showed efficiently tumor turn-on T1-weighted MRI, significantly suppressed tumor growth and greatly prolonged survival of tumor-bearing mice following intravenous administration of MnS@BSA. Our findings indicated that MnS@BSA nanotheranostics hold great potential for traceable H2S gas therapy primed CDT of cancer.

Dose-Dependent Effects of Long-Term Administration of Hydrogen Sulfide on Myocardial Ischemia-Reperfusion Injury in Male Wistar Rats: Modulation of RKIP, NF-κB, and Oxidative Stress.

Decreased circulating levels of hydrogen sulfide (H2S) are associated with higher mortality following myocardial ischemia. This study aimed at determining the long-term dose-dependent effects of sodium hydrosulfide (NaSH) administration on myocardial ischemia-reperfusion (IR) injury. Male rats were divided into control and NaSH groups that were treated for 9 weeks with daily intraperitoneal injections of normal saline or NaSH (0.28, 0.56, 1.6, 2.8, and 5.6 mg/kg), respectively. At the end of the study, hearts from all rats were isolated and hemodynamic parameters were recorded during baseline and following IR. In isolated hearts, infarct size, oxidative stress indices as well as mRNA expression of H2S-, nitric oxide (NO)-producing enzymes, and inflammatory markers were measured. In heart tissue following IR, low doses of NaSH (0.28 and 0.56 mg/kg) had no effect, whereas an intermediate dose (1.6 mg/kg), improved recovery of hemodynamic parameters, decreased infarct size, and decreased oxidative stress. It also increased expression of cystathionine γ-lyase (CSE), Raf kinase inhibitor protein (RKIP), endothelial NO synthase (eNOS), and neuronal NOS (nNOS), as well as decreased expression of inducible NOS (iNOS) and nuclear factor kappa-B (NF-κB). At the high dose of 5.6 mg/kg, NaSH administration was associated with worse recovery of hemodynamic parameters and increased infarct size as well as increased oxidative stress. This dose also decreased expression of CSE, RKIP, and eNOS and increased expression of iNOS and NF-κB. In conclusion, chronic treatment with NaSH has a U-shaped concentration effect on IR injury in heart tissue. An intermediate dose was associated with higher CSE-derived H2S, lower iNOS-derived NO, lower oxidative stress, and inflammation in heart tissue following IR.

ZYZ-803, a novel hydrogen sulfide-nitric oxide conjugated donor, promotes angiogenesis via cross-talk between STAT3 and CaMKII.

Endothelial angiogenesis plays a vital role in recovery from chronic ischemic injuries. ZYZ-803 is a hybrid donor of hydrogen sulfide (H2S) and nitric oxide (NO). Previous studies showed that ZYZ-803 stimulated endothelial cell angiogenesis both in vitro and in vivo. In this study, we investigated whether the signal transducer and activator of transcription 3 (STAT3) and Ca2+/CaM-dependent protein kinase II (CaMKII) signaling was involved in ZYZ-803-induced angiogenesis. Treatment with ZYZ-803 (1 µM) significantly increased the phosphorylation of STAT3 (Tyr705) and CaMKII (Thr286) in human umbilical vein endothelial cells (HUVECs), these two effects had a similar time course. Pretreatment with WP1066 (STAT3 inhibitor) or KN93 (CAMKII inhibitor) blocked ZYZ-803-induced STAT3/CAMKII activation and significantly suppressed the proliferation and migration of HUVECs. In addition, pretreatment with the inhibitors significantly decreased ZYZ-803-induced tube formations along with the outgrowths of branch-like microvessels in aortic rings. In the mice with femoral artery ligation, administration of ZYZ-803 significantly increased the blood perfusion and vascular density in the hind limb, whereas co-administration of WP1066 or KN93 abrogated ZYZ-803-induced angiogenesis. By using STAT3 siRNA, we further explored the cross-talk between STAT3 and CaMKII in ZYZ-803-induced angiogenesis. We found that STAT3 knockdown suppressed ZYZ-803-induced HUVEC angiogenesis and affected CaMKII expression. ZYZ-803 treatment markedly enhanced the interaction between CaMKII and STAT3. ZYZ-803 treatment induced the nuclear translocation of STAT3. We demonstrated that both STAT3 and CaMKII functioned as positive regulators in ZYZ-803-induced endothelial angiogenesis and STAT3 was important in ZYZ-803-induced CaMKII activation, which highlights the beneficial role of ZYZ-803 in STAT3/CaMKII-related cardiovascular diseases.

Hydrogen sulfide attenuates cigarette smoke-induced airway remodeling by upregulating SIRT1 signaling pathway.

Airway remodeling is one of the characteristics for chronic obstructive pulmonary disease (COPD). The mechanism underlying airway remodeling is associated with epithelial-mesenchymal transition (EMT) in the small airways of smokers and patients with COPD. Sirtuin 1 (SIRT1) is able to reduce oxidative stress, and to modulate EMT. Here, we investigated the effects and mechanisms of hydrogen sulfide (H2S) on pulmonary EMT in vitro and in vivo. We found that H2S donor NaHS inhibited cigarette smoke (CS)-induced airway remodeling, EMT and collagen deposition in mouse lungs. In human bronchial epithelial 16HBE cells, NaHS treatment also reduced CS extract (CSE)-induced EMT, collagen deposition and oxidative stress. Mechanistically, NaHS upregulated SIRT1 expression, but inhibited activation of TGF-ß1/Smad3 signaling in vivo and in vitro. SIRT1 inhibition by a specific inhibitor EX527 significantly attenuated or abolished the ability of NaHS to reverse the CSE-induced oxidative stress. SIRT1 inhibition also abolished the protection of NaHS against CSE-induced EMT. Moreover, SIRT1 activation attenuated CSE-induced EMT by modifying TGF-ß1-mediated Smad3 transactivation. In conclusion, H2S prevented CS-induced airway remodeling in mice by reversing oxidative stress and EMT, which was partially ameliorated by SIRT1 activation. These findings suggest that H2S may have therapeutic potential for the prevention and treatment of COPD.

Hydrogen Sulfide Attenuates High Glucose-induced Myocardial Injury in Rat Cardiomyocytes by Suppressing Wnt/beta-catenin Pathway.

Diabetic cardiomyopathy (DCM) is one of the major heart complications of diabetic patients. Hydrogen sulfide (H2S) is now recognized as an important signaling molecule and has been shown to attenuate the development of diabetic cardiomyopathy. However, the underlying mechanisms linking H2S and the development of DCM have not been fully elucidated. In the present study, we therefore sought to explore the role and mechanism of H2S in the pathogenesis of DCM by establishing high glucose-induced injury model in neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Using cystathionine gamma-lyase (CSE) overexpression and CSE interference vectors transfection, the cell viability, cell apoptosis. and oxidative stress were determined and compared between the treatment of high glucose induction and exgenous NaHS administration. Meanwhile, the relationship between the CSE/H2S system and Wnt/beta-catenin pathway was analyzed and discussed in the high glucose-induced cardiomyocytes. Our results indicated that H2S played an important protective role in high glucose-induced apoptosis and oxidative stress in cardiomyocytes, as shown by the decreased reactive oxygen species and malondialdehyde levels, and the increased activities of superoxide dismutase, catalase and glutathione peroxidase. Moreover, H2S could attenuate the Wnt/ß-catenin signalling pathway and up-regulate the expression of haem oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in the diabetic myocardium cells. Together, these results demonstrated that H2S could attenuate high glucose-induced myocardial injury in rat cardiomyocytes by suppressing Wnt/ß-catenin pathway.

Hydrogen sulfide stimulates activation of hepatic stellate cells through increased cellular bio-energetics.

Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extracellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.

Application of signaling molecules in reducing metal accumulation in alfalfa and alleviating metal-induced phytotoxicity in Pb/Cd-contaminated soil.

The utilization of forages grown on metal-contaminated soil can increase the risk of heavy metals entering the food chain and affecting human health because of elevated toxic metal concentrations. Meanwhile, hydrogen sulfide (H2S) and nitric oxide (NO) as signaling molecules are known to promote plant growth in metal-contaminated soils. However, the regulatory mechanisms of such molecules in plant physiology and soil biochemistry have not been well-documented. Hence, we investigate the role of the exogenous application of H2S and NO on alfalfa growth in lead/cadmium (Pb/Cd)-contaminated soil. Our results indicate that the signaling molecules increase the alfalfa chlorophyll and biomass content and improve alfalfa growth. Further, H2S and NO reduce the translocation and bioconcentration factors of Pb and Cd, potentially reducing the risk of heavy metals entering the food chain. These signaling molecules reduce metal-induced oxidative damage to alfalfa by mitigating reactive oxygen species accumulation and increasing antioxidant enzyme activities. Their exogenous application increases soil enzymatic activities, particularly of catalase and polyphenol oxidase, without significantly changing the composition and structure of rhizosphere bacterial communities. Interestingly, H2S addition enriches the abundance of plant-growth-promoting rhizobacteria in soil, including Nocardioides, Rhizobium, and Glycomyces. H2S is more effective than NO in improving alfalfa growth and reducing heavy-metal contamination of the food chain. These results provide new insights into the exogenous application of signaling molecules in alleviating metal-induced phytotoxicity, including an efficient strategy for the safe use of forages.

Hyaluronated liposomes containing H2S-releasing doxorubicin are effective against P-glycoprotein-positive/doxorubicin-resistant osteosarcoma cells and xenografts.

Doxorubicin (dox) is one of the first-line drug in osteosarcoma treatment but its effectiveness is limited by the efflux pump P-glycoprotein (Pgp) and by the onset of cardiotoxicity. We previously demonstrated that synthetic doxs conjugated with a H2S-releasing moiety (Sdox) were less cardiotoxic and more effective than dox against Pgp-overexpressing osteosarcoma cells. In order to increase the active delivery to tumor cells, we produced hyaluronic acid (HA)-conjugated liposomes containing Sdox (HA-Lsdox), exploiting the abundance of the HA receptor CD44 in osteosarcoma. HA-Lsdox showed favorable drug-release profile and higher toxicity in vitro and in vivo than dox or the FDA-approved liposomal dox Caelyx® against Pgp-overexpressing osteosarcoma, displaying the same cardiotoxicity profile of Caelyx®. Differently from dox, HA-Lsdox delivered the drug within the endoplasmic reticulum (ER), inducing protein sulfhydration and ubiquitination, and activating a ER stress pro-apoptotic response mediated by CHOP. HA-Lsdox also sulfhydrated the nascent Pgp in the ER, reducing its activity. We propose HA-Lsdox as an innovative tool noteworthy to be tested in Pgp-overexpressing patients, who are frequently less responsive to standard treatments in which dox is one of the most important drugs.

A novel hydrogen sulfide-releasing donor, HA-ADT, suppresses the growth of human breast cancer cells through inhibiting the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling pathways.

Breast cancer is one of the most frequent cancers among women worldwide. Hyaluronic acid (HA) is one of the best biopolymers in terms of safety issues and has been widely used in drug delivery and tissue engineering. 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-thione (ADT-OH) is a commonly used H2S donor. In this study, we designed and synthesized a conjugate, HA-ADT, by connecting HA with ADT-OH through chemical reactions. Our results indicated that HA-ADT could produce more H2S than NaHS and GYY4137. HA-ADT exerted more potent inhibitory effects than NaHS and GYY4137 in the proliferation, viability, migration, and invasion of human breast cancer cells. Similar trends were observed in the apoptosis and the protein levels of phospho (p)-PI3K, p-AKT, p-mTOR, H-RAS, p-RAF, p-MEK, and p-ERK in human breast cancer cells. Furthermore, HA-ADT exhibited more powerful inhibitory effects on the growth of human breast cancer xenograft tumors in nude mice. In conclusion, HA-ADT could suppress the growth of human breast cancer cells through the inhibition of the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK signaling pathways. HA-ADT and its derivatives might be of great potential in the treatment of different types of cancer.

Hydrogen sulfide enhances the effectiveness of mesenchymal stem cell therapy in rats with heart failure: In vitro preconditioning versus in vivo co-delivery.

Stem cell therapy represents a promising therapeutic avenue for cardiac disorders, including heart failure. Although stem cell transplantation showed encouraging preliminary results, the outcomes of clinical studies are still unsatisfactory. This study aimed to compare the outcomes of two therapeutic approaches, in vivo co-delivery of sodium hydrogen sulfide (NaHS) concomitant with bone marrow-derived mesenchymal stem cell (BMSC) transplantation and in vitro preconditioning of BMSCs with NaHS, both of which are intended to promote the success of stem cell therapy in rats with isoprenaline-induced heart failure. Heart failure developed 4 weeks after the subcutaneous injection of isoprenaline (170 mg/kg) for 4 consecutive days. The in vivo approach involved the co-delivery of intraperitoneally administered NaHS concomitant with BMSC transplantation for a period of 14 days. The in vitro approach involved preconditioning BMSCs with NaHS for 30 min before transplantation. Compared to treatment with BMSCs alone, in vitro preconditioning of BMSCs with NaHS improved left ventricular function as measured by echocardiography and electrocardiography and enhanced stem cell homing, proliferation and differentiation as manifested by higher cardiac expression of GATA-4 and myocyte enhancer factor 2. Moreover, the measurement of cardiac transforming growth factor beta 1 levels and histopathological investigation revealed mitigated fibrosis and myocardial injury scores. Compared with BMSC therapy alone, the in vivo approach enhanced stem cell homing and differentiation, alleviated fibrosis and augmented vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression. In conclusion, NaHS can potentiate the efficiency of BMSC therapy for heart failure by in vitro preconditioning or in vivo co-delivery. The in vitro approach is superior with regard to improving cardiac function in addition to enhancing stem cell proliferation, while the in vivo approach is superior with regard to increasing cardiac VEGF and eNOS expression.

Hepatic and Intrahepatic Targeting of Hydrogen Sulfide Prodrug by Bioconjugation.

Hydrogen sulfide (H2S) is an endogenous gaseous transmitter known to play an important role in biological functions. For the hepatic and intrahepatic targeting of H2S prodrug at the cellular level, we developed two types of sulfo-albumins, in which five sulfide groups (source of H2S) were covalently bound to succinylated (Suc) or galactosylated (Gal) bovine serum albumin (BSA). Sulfo-BSA-Suc and polyethylene glycol (PEG)-Sulfo-BSA-Gal, both released H2S in the 5 mM glutathione solution, but not in the plasma. Sulfo-BSA-Suc and PEG-Sulfo-BSA-Gal were taken up by RAW264.7 cells (mouse macrophage-like cells) and Hep G2 cells (human hepatocellular carcinoma cells), respectively, and H2S was released. These results indicate that Sulfo-BSA-Suc and PEG -Sulfo-BSA-Gal selectively released H2S intracellularly. In a biodistribution study, up to 80% of 111In-labeled Sulfo-BSA-Suc and PEG-Sulfo-BSA-Gal rapidly accumulated in the liver, 30 min after intravenous injection in mice. Furthermore, 111In-labeled Sulfo-BSA-Suc and PEG-Sulfo-BSA-Gal predominantly accumulated in liver nonparenchymal (endothelial cells and Kupffer cells) and parenchymal cells (hepatocytes), respectively. These findings suggest that targeted delivery of H2S prodrug to a specific type of liver cells was successfully achieved by bioconjugation.

First Examples of H2S-Releasing Glycoconjugates: Stereoselective Synthesis and Anticancer Activities.

H2S donors are currently emerging as promising therapeutic agents in a wide variety of pathologies, including tumors. Cancer cells are characterized by an enhanced uptake of sugars, such as glucose. Therefore, novel glycoconjugated H2S donors were synthesized so that high concentrations of H2S can be selectively achieved therein. Dithiolethione portions or isothiocyanate portions were selected for their well-known H2S-releasing properties in the presence of biological substrates. A synthetic procedure employing trichloroacetimidate glycosyl donors was applied to produce, in a stereoselective fashion, C1-glycoconjugates, whereas C6-glycoconjugates were obtained by a Mitsunobu-based transformation. The resulting molecules were then tested for their anticancer effects on human pancreas adenocarcinoma ascites metastasis cell line AsPC-1. The most potent inhibitors of cell viability (6aß and 7b) proved to release H2S inside the AsPC-1 cells and to alter the basal cell cycle.

8e Protects against Acute Cerebral Ischemia by Inhibition of PI3Kγ-Mediated Superoxide Generation in Microglia.

The inflammatory response mediated by microglia plays a critical role in the progression of ischemic stroke. Phosphoinositide 3-kinase gamma (PI3Kγ) has been implicated in multiple inflammatory and autoimmune diseases, making it a promising target for therapeutic intervention. The aim of this study was to evaluate the efficacy of 8e, a hydrogen sulfide (H2S) releasing derivative of 3-n-butylphthalide (NBP), on brain damage and PI3Kγ signaling following cerebral ischemia injury. 8e significantly reduced sensorimotor deficits, focal infarction, brain edema and neural apoptosis at 72 h after transient middle cerebral artery occlusion (tMCAO). The NOX2 isoform of the NADPH oxidase family is considered a major enzymatic source of superoxide. We found that the release of superoxide, together with the expression of NOX2 subunits p47phox, p-p47phox, and the upstream PI3Kγ/AKT signaling were all down-regulated by 8e, both in the penumbral region of the rat brain and in the primary cultured microglia subjected to oxygen-glucose deprivation (OGD). With the use of siRNA and pharmacological inhibitors, we further demonstrated that 8e regulates the formation of superoxide in activated microglia through the PI3Kγ/AKT/NOX2 signaling pathway and subsequently prevents neuronal death in neighboring neurons. Our experimental data indicate that 8e is a potential candidate for the treatment of ischemic stroke and PI3Kγ-mediated neuroinflammation.