Optimization of preparation conditions for Salsola laricifolia protoplasts using response surface methodology and artificial neural network modeling.

BACKGROUND: Salsola laricifolia is a typical C3-C4 typical desert plant, belonging to the family Amaranthaceae. An efficient single-cell system is crucial to study the gene function of this plant. In this study, we optimized the experimental conditions by using Box-Behnken experimental design and Response Surface Methodology (RSM)-Artificial Neural Network (ANN) model based on the previous studies. RESULTS: Among the 17 experiment groups designed by Box-Behnken experimental design, the maximum yield (1.566 × 106/100 mg) and the maximum number of viable cells (1.367 × 106/100 mg) were obtained in group 12, and the maximum viability (90.81%) was obtained in group 5. Based on these results, both the RSM and ANN models were employed for evaluating the impact of experimental factors. By RSM model, cellulase R-10 content was the most influential factor on protoplast yield, followed by macerozyme R-10 content and mannitol concentration. For protoplast viability, the macerozyme R-10 content had the highest influence, followed by cellulase R-10 content and mannitol concentration. The RSM model performed better than the ANN model in predicting yield and viability. However, the ANN model showed significant improvement in predicting the number of viable cells. After comprehensive evaluation of the protoplast yield, the viability and number of viable cells, the optimal results was predicted by ANN yield model and tested. The amount of protoplast yield was 1.550 × 106/100 mg, with viability of 90.65% and the number of viable cells of 1.405 × 106/100 mg. The corresponding conditions were 1.98% cellulase R-10, 1.00% macerozyme R-10, and 0.50 mol L-1 mannitol. Using the obtained protoplasts, the reference genes (18SrRNA, ß-actin and EF1-α) were screened for expression, and transformed with PEG-mediated pBI121-SaNADP-ME2-GFP plasmid vector. There was no significant difference in the expression of ß-actin and EF1-α before and after treatment, suggesting that they can be used as internal reference genes in protoplast experiments. And SaNADP-ME2 localized in chloroplasts. CONCLUSION: The current study validated and evaluated the effectiveness and results of RSM and ANN in optimizing the conditions for protoplast preparation using S. laricifolia as materials. These two methods can be used independently of experimental materials, making them suitable for isolating protoplasts from other plant materials. The selection of the number of viable cells as an evaluation index for protoplast experiments is based on its ability to consider both protoplast yield and viability. The findings of this study provide an efficient single-cell system for future genetic experiments in S. laricifolia and can serve as a reference method for preparing protoplasts from other materials.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray.

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.

DR1-CSE/H2S pathway upregulates autophagy and inhibits H9C2 cells damage induced by high glucose.

In the cardiovascular system, long-term high glucose (HG) can lead to cardiomyocyte damage. Hydrogen sulfide (H2S) reduces cell autophagy in cardiomyocytes. Dopamine 1 receptors (DR1), a specific binding receptor for dopamine, which has a significant regulatory effect on cardiomyocytes. However, it is unclear whether DR1 inhibits HG-induced cardiomyocyte damage by regulating endogenous H2S production and the level of cell autophagy. The present data indicated that the expression of DR1 and cystathionine-γ-lyase (CSE, a key enzyme for endogenous H2S production) and H2S content were significantly reduced in HG-induced cardiomyocytes, which was reversed by SKF38393 (an agonist of DR1). NaHS (an exogenous H2S donor) only increased H2S content and the expression of CSE with no effect on DR1 expression. HG reduced cell viability, the expression of Bcl-2 and Beclin1, the production of autophagosomes and LC3 II/I ratio and increased the cell apoptotic ratio, the expression of cleaved caspase-3, cleaved caspase-9, cytochrome c, P62, and p-mTOR/t-mTOR ratio. SKF38393 and NaHS reversed the effects of HG. PPG (an inhibitor of CSE) and 3MA (an inhibitor of autophagy) abolished the beneficial effect of SKF38393. In addition, AICAR (an agonist of AMPK) and Rapamycin (an inhibitor of mTOR) increased the production of autophagosomes but decreased the p-mTOR/t-mTOR ratio, which was similar to the effects of SKF38393 and 3MA. Our findings suggest that DR1 reduces the HG-induced cardiomyocyte damage via up-regulating the CSE/H2S pathway, which increases cell autophagy by inhibiting the activation of mTOR.

Exogenous hydrogen sulfide inhibits the senescence of cardiomyocytes through modulating mitophagy in rats.

Hydrogen sulfide (H2S), a gaseous molecule, has been shown to be involved in the regulation of body pathophysiological processes. Aging is related to structural and functional alterations within the heart. There is evidence that diminished mitophagy accelerates the aging process. Studies in recent years have revealed that plasma levels of H2S in humans and old rats decrease with age, and H2S acts as a cytoprotective mediator in the aging process. However, it is unclear whether H2S can delay the senescence of cardiomyocytes by regulating mitophagy. Our present results showed that exogenous H2S inhibited mitochondrial damage, oxidative stress and cell apoptosis, and enhanced mitophagy through upregulating the SIRT1-PINK1-parkin pathway in myocardial tissues of aged rats and cultured aged cardiomyocytes. Furthermore, the effect of exogenous H2S on the above indicators was the same as that of SRT1720 (a SIRT1 agonist) and kinetin (a PINK1 activator). Our findings suggest that exogenous H2S inhibits the senescence of cardiomyocytes by increasing mitophagy via upregulation of the SIRT1-PINK1-parkin pathway in rats.

DR1 Activation Inhibits the Proliferation of Vascular Smooth Muscle Cells through Increasing Endogenous H2S in Diabetes.

Tissue ischemia and hypoxia caused by the abnormal proliferation of smooth muscle cells (SMCs) in the diabetic state is an important pathological basis for diabetic microangiopathy. Studies in recent years have shown that the chronic complications of diabetes are related to the decrease of endogenous hydrogen sulfide (H2S) in diabetic patients, and it has been proven that H2S can inhibit the proliferation of vascular SMCs (VSMCs). Our study showed that the endogenous H2S content and the expression of cystathionine gamma-lyase (CSE), which is the key enzyme of H2S production, were decreased in arterial SMCs of diabetic mice. The expression of PCNA and Cyclin D1 was increased, and the expression of p21 was decreased in the diabetic state. After administration of dopamine 1-like receptors (DR1) agonist SKF38393 and exogenous H2S donor NaHS, the expression of CSE was increased and the change in proliferation-related proteins caused by diabetes was reversed. It was further verified by cell experiments that SKF38393 activated calmodulin (CaM) by increasing the intracellular calcium ([Ca2+]i) concentration, which activated the CSE/H2S pathway, enhancing the H2S content in vivo. We also found that SKF38393 and NaHS inhibited insulin-like growth factor-1 (IGF-1)/IGF-1R and heparin-binding EGF-like growth factor (HB-EGF)/EGFR, as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways. Taken together, our results suggest that DR1 activation up-regulates the CSE/H2S system by increasing Ca2+-CaM binding, which inhibits the IGF-1/IGF-1R and HB-EGF/EGFR pathways, thereby decreasing their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways to achieve the effect of inhibiting HG-induced VSMCs proliferation.

Dopamine 1 receptors inhibit apoptosis via activating CSE/H2 S pathway in high glucose-induced vascular endothelial cells.

High glucose (HG)-induced dysfunction of vascular endothelial cells plays a crucial role in the development of diabetic vascular complications. Inhibition of cystathionine γ-synthase/hydrogen sulfide (CSE/H2 S) pathway is one of the causes of vascular endothelial cell injury induced by HG. Dopamine D1 receptors (DR1) are widely expressed and regulate important physiological functions in the vascular system. However, the effect of DR1 inhibition on HG-induced vascular endothelial apoptosis by regulating the CSE/H2 S pathway is unclear. Therefore, we aimed to determine if DR1 can regulate the CSE/H2 S pathway and regulate the effect of DR1 on HG-induced apoptosis in human umbilical vein endothelial cells. In this study, we found that HG treatment significantly decreased the expression of DR1 and CSE and the endogenous content of H2 S; DR1 agonist SKF 38393 reversed these effects, while sodium hydrosulfide (NaHS) only increased CSE expression and the endogenous H2 S production and had no effect on DR1 expression. Meanwhile, HG significantly increased the intracellular calcium concentration ([Ca2+ ]i ), and SKF 38393 further increased HG-induced [Ca2+ ]i . In addition, HG increased the lactate dehydrogenase activity, malondialdehyde and reactive oxygen species contents, apoptotic rate, the expression of cleaved caspase-3, caspase-9, and cytochrome c, and the activity of phosphorylated-inhibitor of nuclear factor-kappaBα (NF-κBα) (p-IκBα) and phosphorylated-NF-κB (p-NF-κB), and reduced cell viability, superoxide dismutase activity, and Bcl-2 expressions. SKF 38393 and NaHS markedly reversed the effect of HG. The effect of SKF 38393 was similar to N-acetyl- l-cysteine (an inhibitor of oxidative stress) or pyrrolidinedithiocarbamate ammonium (an NF-kB inhibitor). Taken together, DR1 upregulates the CSE/H2 S pathway by increasing the [Ca2+ ]i , which inhibits HG-induced apoptosis via downregulating NF-κB/IκBα pathway in vascular endothelial cells.

SKF38393 prevents high glucose (HG)-induced endothelial dysfunction by inhibiting the effects of HG on cystathionine γ-lyase/hydrogen sulfide activity and via a RhoA/ROCK1 pathway.

BACKGROUND: Endothelial dysfunction plays a crucial role in diabetic vascular complications. A decrease in hydrogen sulfide (H2S) levels is increasingly becoming a vital factor contributing to high glucose (HG)-induced endothelial dysfunction. Dopamine D1-like receptors (DR1) activation has important physiological functions in the cardiovascular system. H2S decreases the dysfunction of vascular endothelial cells. However, no studies have reported whether DR1 protects the function of vascular endothelial cells by regulating H2S levels. AIM: The present study aimed to determine whether DR1 regulates the levels of endogenous H2S, which exerts protective effects against HG-induced injury of human umbilical vein endothelial cells (HUVECs) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing kinase 1 (ROCK1) signalling. METHODS: HUVECs were exposed to HG (30 mM) or normal glucose (5.5 mM) after different treatments. Cell viability, proliferation and migration were measured by Cell Counting Kit-8, EdU cell proliferation assay, transwell assay and wound healing assay, respectively. H2S probe (7-Azido-4-Methylcoumarin) was used to detect levels of H2S. The intracellular calcium concentration ([Ca2+]i) were measured using Fluo-4 AM. The protein expressions were quantified by Western blot. RESULTS: We found that HG decreased the expression of DR1 and cystathionine γ-lyase (CSE) and H2S production. The DR1 agonist SKF38393 significantly increased DR1 and CSE expression and H2S production, whereas NaHS (a H2S donor) only increased CSE expression and H2S production but had no effect on DR1 expression. Meanwhile, SKF38393 further increased the [Ca2+]i induced by HG. In addition, HG reduced cell viability and the expression of Cyclin D1 and proliferating cell nuclear antigen and increased the expression of p21C⁢i⁢p/W⁢A⁢F-1, collagen I, collagen III, matrix metalloproteinase 9, osteopontin and α-smooth muscle actin and the activity of phosphorylated RhoA and ROCK1. SKF38393 and NaHS reversed these effects of HG. PPG (a CSE inhibitor) abolished the beneficial effect of SKF38393. These effects of SKF38393 were similar to those of Y-27632 (a ROCK inhibitor). CONCLUSION: Taken together, our results suggest that DR1 activation upregulates the CSE/H2S pathway by increasing the [Ca2+]i, which protects endothelial cells from HG-induced injury by inhibiting the RhoA/ROCK1 pathway.

The DR1­CSE/H2S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine­Î³­lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)­induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α­smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1­CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

DR1 activation promotes vascular smooth muscle cell apoptosis via up-regulation of CSE/H2 S pathway in diabetic mice.

The important role of hydrogen sulfide (H2 S) as a novel gasotransmitter in inhibiting proliferation and promoting apoptosis of vascular smooth muscle cells (VSMCs) has been widely recognized. The dopamine D1 receptor (DR1), a G protein coupled receptor, inhibits atherosclerosis by suppressing VSMC proliferation. However, whether DR1 contributes to VSMC apoptosis via the induction of endogenous H2 S in diabetic mice is unclear. Here, we found that hyperglycemia decreased the expressions of DR1 and cystathionine-γ-lyase (CSE, a key enzyme for endogenous H2 S production) and reduced endogenous H2 S generation in mouse arteries and cultured VSMCs. DR1 agonist SKF38393 increased DR1 and CSE expressions and stimulated endogenous H2 S generation. Sodium hydrosulfide (NaHS, a H2 S donor) increased CSE expressions and H2 S generation but had no effect on DR1 expression. In addition, high glucose (HG) increased VSMC apoptosis, up-regulated IGF-1-IGF-1R and HB-EGF-EGFR, and stimulated ERK1/2 and PI3K-Akt pathways. Overexpression of DR1, the addition of SKF38393 or supply of NaHS further promoted VSMC apoptosis and down-regulated the above pathways. Knock out of CSE or the addition of the CSE inhibitor poly propylene glycol diminished the effect of SKF38393. Moreover, calmodulin (CaM) interacted with CSE in VSMCs; HG increased intracellular Ca2+ concentration and induced CaM expression, further strengthened the interaction of CaM with CSE in VSMCs, which were further enhanced by SKF38393. CaM inhibitor W-7, inositol 1,4,5-trisphosphate (IP3 ) inhibitor 2-APB, or ryanodine receptor inhibitor tetracaine abolished the stimulatory effect of SKF38393 on CaM expression and intracellular Ca2+ concentration. Taken together, these results suggest that DR1 up-regulates CSE/H2 S signaling by inducing the Ca2+ -CaM pathway followed by down-regulations of IGF-1-IGF-1R and HB-EGF-EGFR and their downstream ERK1/2 and PI3K-Akt, finally promoting the apoptosis of VSMCs in diabetic mice.

[Effects of exogenous H2S on hepatic fibrosis in diabetic mice and its mechanism].

OBJECTIVE: To investigate the effects of exogenous hydrogen sulfide (H2S) on the hepatic fibrosis in diabetic mice and its mechanism. METHODS: Twenty-four C57 male mice (weight 22±2 g) were randomly divided into three groups (n=8): ① Normal control group (Control): Mice were intraperitoneally injected equal amount of normal saline, the injection time was the same as that of the experimental groups; ② Diabetes model groups (HG): Streptozotocin (STZ) was injected intraperitoneally once according to body weight (150 mg/kg) to establish diabetes model; ③ NaHS treatment groups (HG + NaHS): Mice were intraperitoneally injected with NaHS (100 µmol/L·kg·d) once a day for 12 consecutive weeks. The hepatocyte injury was detected by HE staining; the hepatic fibrosis was observed through Masson staining; the protein expressions of cystathionine - ß - synthetase (CBS), collagen-I (CoL-I), collagen-III (CoL-III) and matrix metalloproteinase-9 (MMP-9) were detected by Western blot. RESULTS: Compared with the control group, the damage and fibrosis of hepatocyte were significantly aggravated, the expression of CBS proteins was decreased (P＜0.01), and the expression levels of CoL-I, CoL-III and MMP-9 proteins were increased (P＜0.01) in the diabetic model group. Compared with the diabetic model group, the damage and fibrosis of hepatocyte were significantly lightened, the expression of CBS proteins was obviously increased (P＜0.01), and the expression levels of CoL-I, CoL-III and MMP-9 proteins were markedly decreased (P＜ 0.01). CONCLUSION: H2S inhibits the hepatic fibrosis in diabetic mice, and its mechanism is related to the decrease of collagen and matrix metalloproteinase-9.

Overexpression of Saussurea involucrata dehydrin gene SiDHN promotes cold and drought tolerance in transgenic tomato plants.

Dehydrins are late embryogenesis abundant proteins that help regulate abiotic stress responses in plants. Overexpression of the Saussurea involucrata dehydrin gene SiDHN has previously been shown to improve water-use efficiency and enhance cold and drought tolerance of transgenic tobacco. To understand the mechanism by which SiDHN exerts its protective function, we transformed the SiDHN gene into tomato plants (Solanum lycopersicum L.) and assessed their response to abiotic stress. We observed that in response to stresses, the SiDHN transgenic tomato plants had increased contents of chlorophyll a and b, carotenoid and relative water content compared with wild-type plants. They also had higher maximal photochemical efficiency of photosystem II and accumulated more proline and soluble sugar. Compared to those wild-type plants, malondialdehyde content and relative electron leakage in transgenic plants were not significantly increased, and H2O2 and O2- contents in transgenic tomato plants were significantly decreased. We further observed that the production of stress-related antioxidant enzymes, including superoxide dismutase, ascorbate peroxidase, peroxidase, and catalase, as well as pyrroline-5-carboxylate synthetase and lipid transfer protein 1, were up-regulated in the transgenic plants under cold and drought stress. Based on these observations, we conclude that overexpression of SiDHN gene can promote cold and drought tolerance of transgenic tomato plants by inhibiting cell membrane damage, protecting chloroplasts, and enhancing the reactive oxygen species scavenging capacity. The finding can be beneficial for the application of SiDHN gene in improving crop tolerance to abiotic stress and oxidative damage.

H2S restores the cardioprotective effects of ischemic post-conditioning by upregulating HB-EGF/EGFR signaling.

Hydrogen sulfide (H2S) reduces ischemia/reperfusion (I/R) injury and apoptosis and restores the cardioprotective effects of ischemic post-conditioning (PC) in aged cardiomyocytes by inhibiting oxidative stress and endoplasmic reticulum stress and increasing autophagy. However, the mechanism is unclear. In the present study, we observed a loss of PC-mediated cardioprotection of aged cardiomyocytes. NaHS (a H2S donor) exerted significant protective effects against H/R-induced cell damage, apoptosis, production of cleaved caspase-3 and caspase-9, and release of cytochrome c. NaHS also reversed the H/R-induced reduction in cell viability and increased HB-EGF expression, cellular HB-EGF content, and EGFR phosphorylation. Additionally, NaHS increased expression of Bcl-2, c-myc, c-fos and c-jun, and the phosphorylation of ERK1/2, PI3K, Akt and GSK-3ß. PC alone did not provide protection to H/R-treated aged cardiomyocytes, but it was significantly restored by supplementation of NaHS. The beneficial effects of NaHS during PC were inhibited by EGFR knockdown, AG1478 (EGFR inhibitor), PD98059 (ERK1/2 inhibitor) or LY294002 (PI3K inhibitor). These results suggest that exogenous H2S restores PC-mediated cardioprotection by up-regulating HB-EGF/EGFR signaling, which activates the ERK1/2-c-myc (and fos and c-jun) and PI3K-Akt- GSK-3ß pathways in the aged cardiomyocytes.

[Protective effects of exogenous H2S to the recovery of hypoxia post-conditioning on aged H9C2 cells and the underling mechanisms].

OBJECTIVE: To investigate the recovery of protective effects of exogenous hydrogen sulfide (H2S) on hypoxia post-conditioning in aged H9C2 cells and its mechanism. METHODS: H9C2 cells (cardiomyocytes line) were treated with 30 µmol/L hydrogen peroxide (H2O2) for 2 hours, then cultured for 3 days in order to induce cellular aging. Aged H9C2 cells were randomly divided into 5 groups (n=8):Control group (Control), hypoxia/reoxygenation group (H/R), H/R + NaHS group, hypoxia post-conditioning (PC) group, PC+NaHS group. H/R model:the cells were exposed to hypoxic culture medium (serum and sugar free medium, pH=6.8) for 3 hours and then cultured at normal condition for 6 hours. PC model:at the end of hypoxia for 3 hours, the cells were exposed to normoxic culture solution for 5 minutes, then the cells were placed in hypoxic solution for 5 minutes, the cycle above-mentioned was repeated 3 times and followed by reoxygenation for 6 hours. Advanced glycation end products (AGEs) content and caspase-3 activity were detected by ELISA. The cell viability was observed by cell counting kit-8 (CCK-8). The reactive oxygen species (ROS) levels were analyzed using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The apoptotic rate was determined through Hoechst 33342 staining. The mRNA levels of relative gene expression were detected by real-time PCR. RESULTS: Thirty µmol/L H2O2 induced H9C2 cell senescence while did not lead to apoptosis. Compared with control group, cell viability was decreased, the apoptotic rate、levels of ROS and the mRNA of caspase-3, caspase-9 and Bcl-2 were increased in H/R and PC groups (P<0.01). There were no differences in the above indexes between PC group and H/R group. Supplementation of NaHS increased cell viability and decreased apoptotic rate and oxidative stress. The effects of PC + NaHS on the above indexes were better than those of H/R+NaHS group. CONCLUSIONS: Exogenous H2S can restore the protective effect of PC on the aged H9C2 cells, and its mechanism is related to the inhibition of oxidative stress and apoptosis.

Exogenous H2S restores ischemic post-conditioning-induced cardioprotection through inhibiting endoplasmic reticulum stress in the aged cardiomyocytes.

BACKGROUND: A gasotransmitter hydrogen sulfide (H2S) plays an important physiological and pathological role in cardiovascular system. Ischemic post-conditioning (PC) provides cardioprotection in the young hearts but not in the aged hearts. Exogenous H2S restores PC-induced cardioprotection by inhibition of mitochondrial permeability transition pore opening and oxidative stress and increase of autophagy in the aged hearts. However, whether H2S contributes to the recovery of PC-induced cardioprotection via down-regulation of endoplasmic reticulum stress (ERS) in the aged hearts is unclear. METHODS: The aged H9C2 cells (the cardiomyocytes line) were induced using H2O2 and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. Related protein expressions were detected through Western blot. RESULTS: In the present study, we found that 30 µM H2O2 induced H9C2 cells senescence but not apoptosis. Supplementation of NaHS protected against H/R-induced apoptosis, the expression of cleaved caspase-3 and cleaved caspase-9 and the release of cytochrome c. The addition of NaHS also counteracted the reduction of cell viability caused by H/R and decreased the expression of GRP 78, CHOP, cleaved caspase-12, ATF 4, ATF 6 and XBP-1 and the phosphorylation of PERK, eIF 2α and IRE 1α. Additionally, NaHS increased Bcl-2 expression. PC alone did not provide cardioprotection in H/R-treated aged cardiomyocytes, which was significantly restored by the supplementation of NaHS. The beneficial role of NaHS was similar to the supply of 4-PBA (an inhibitor of ERS), GSK2656157 (an inhibitor of PERK), STF083010 (an inhibitor of IRE 1α), respectively, during PC. CONCLUSION: Our results suggest that the recovery of myocardial protection from PC by exogenous H2S is associated with the inhibition of ERS via down-regulating PERK-eIF 2α-ATF 4, IRE 1α-XBP-1 and ATF 6 pathways in the aged cardiomyocytes.