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Diabetic nephropathy is a serious chronic complication affecting at least 25% of diabetic patients. Hyperglycemia associated advanced glycation end-products (AGEs) increase tubular epithelial-myofibroblast transdifferentiation (TEMT) and extracellular matrix synthesis and thereby causes renal fibrosis. The chalcone isoliquiritigenin, found in many herbs of Glycyrrhiza family, is known for potential health-promoting effects. However, their effects on AGE-associated renal proximal tubular fibrosis are not known yet. In this study, the effect of isoliquiritigenin on AGE-induced renal proximal tubular fibrosis was determined in cultured HK-2 cell line. The results show that 200 μg/mL of AGE-induced TEMT and the formed myofibroblasts synthesized collagen to increase extracellular matrix formation thereby lead to renal tubular fibrosis. However, treatment with 200 nM of isoliquiritigenin considerably inhibited the TEMT and suppressed the TGFβ/STAT3 mechanism to inhibit collagen secretion. Therefore, isoliquiritigenin effectively suppressed AGE-induced renal tubular fibrosis.

Different stress condition stimulates the expression level of insulin-like growth factor receptor II (IGF-IIR) in cardiomyoblasts that lead to apoptosis. Tanshinone IIA (TSN), a pharmacologically active component from Danshen, has been shown cardioprotective effects against cardiac apoptosis induced by several stress conditions. Therefore, this study was conducted to assess the cardioprotective effects of TSN IIA mediated through the estrogen receptor (ER) in order to inhibit the Leu27IGF-II-enhanced IGF-IIR-mediated cardiac apoptosis. The estrogenic activity of TSN IIA was examined after myocardial cells were pretreated with the ER antagonist, and inhibited the phospho-inositide-3 kinase (PI3K). Here, we found that TSN IIA significantly induced ER that phosphorylated Akt. Further, Akt activation considerably suppressed the Leu27IGF-II induced IGF-IIR expression level and the downstream effectors, including Gαq and calcineurin as well as mitochondrial dependent apoptosis proteins including Bad, cytochrome c, and active caspase-3 that result in cardiac apoptosis resistance. However, the western blot analysis, JC-1 staining, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay revealed that TSN IIA attenuated Leu27IGF-II-induced IGF-IIR mediated cardiac apoptosis was reversed by an ER antagonist such as ICI 182780, and PI3K inhibition. All these findings demonstrate that TSN IIA exerts estrogenic activity, which can activate PI3K-Akt pathway, and thereby inhibits Leu27IGFII induced IGF-IIR mediated cardiac apoptosis. Thus, TSN IIA can be considered as an effective therapeutic strategy against IGF-IIR signaling cascade to suppress cardiac apoptosis.

Diabetes-induced cardiovascular complications are mainly associated with high morbidity and mortality in patients with diabetes. Insulin-like growth factor II receptor α (IGF-IIRα) is a cardiac risk factor. In this study, we hypothesized IGF-IIRα could also deteriorate diabetic heart injury. The results presented that both in vivo transgenic Sprague-Dawley rat model with specific IGF-IIRα overexpression in the heart and in vitro myocardium H9c2 cells were used to investigate the negative function of IGF-IIRα in diabetic hearts. The results showed that IGF-IIRα overexpression aided hyperglycemia in creating more myocardial injury. Pro-inflammatory factors, such as Tumor necrosis factor-alpha, Interleukin-6, Cyclooxygenase-2, Inducible nitric oxide synthase, and Nuclear factor-kappaB inflammatory cascade, are enhanced in the diabetic myocardium with cardiac-specific IGF-IIRα overexpression. Correspondingly, IGF-IIRα overexpression in the diabetic myocardium also reduced the PI3K-AKT survival axis and activated mitochondrial-dependent apoptosis. Finally, both ejection fraction and fractional shortening were be significantly decrease in diabetic rats with cardiac-specific IGF-IIRα overexpression. Overall, all results provid clear evidence that IGF-IIRα can enhance cardiac damage and is a harmful factor to the heart under high-blood glucose conditions. However, the pathophysiology of IGF-IIRα under different stresses and its downstream regulation in the heart still require further research.

This study reports the effect of cardiac-specific insulin-like growth factor-II receptor α (IGF-IIRα) overexpression on the development of liver dysfunction in transgenic rats via STZ-induced diabetic hepatocyte damage. The cardio-hepatic syndrome comprises a number of heart and liver illnesses in which an acute or chronic disease in one organ can lead to acute or chronic disease in the other. However, the molecular mechanism involved in such a set of conditions is unclear. In this study, we developed a transgenic rat model with cardiac-specific overexpression of IGF-IIRα, which is a supplementary splicing variant of insulin-like growth factor-II receptor (IGF-IIR), expressed in pathological hearts, to investigate the relationship between late fetal gene expression in diabetic hearts and their influence on diabetic hepatopathy. STZ (55 mg/kg) was intraperitoneally delivered into IGF-IIR overexpressed transgenic (TG) and non-transgenic (NTG) animal models developed in Sprague-Dawley (SD) rats after an overnight fast. The relationship among IGF-IIRα overexpression and hepatocyte damages have been determined based on the complexity of damage in the liver. Our findings revealed that overexpression of the cardiac-specific IGF-IIRα enhances diabetes-induced morphological alterations and hepatic inflammation in the livers. The diabetic transgenic rats demonstrated the development of pathological conditions such as thick collagen fiber deposition, bridging fibrosis, and elevation of α-SMA and MMP1 related liver fibrosis mechanisms. Our data suggest that IGF-IIRα overexpression in the heart during a pathological state may worsen diabetic hepatopathy in rats.

In aging hypertensive conditions, deterioration of insulin-like growth factor 1 receptor (IGF1R) cause a pathological impact on hypertensive hearts with an increased Ang II level. Recovering these adverse conditions through transplanted adipose-derived stem cells is a challenging approach. Moreover, Danggui, a Traditional Chinese medicine (TCM), is used for the treatment of cardioprotective effects. In this study, to evaluate whether the combined effect of MSCs and TCM can recover the cardiac function in late-stage hypertension rats. We observed that lower dose of Danggui crude extract treatment showed an increased level of cell viability with maintained stemness properties and growth rate in rat adipose-derived stem cells (rADSCs). Further, we cocultured the H9c2 cells with rADSCs and the results revealed that Danggui-treated MSCs enhanced the IGF1R expression and attenuated the hypertrophy in H9c2 cells against Ang II challenge by immunoblot and rhodamine-phalloidin staining. In addition, Danggui crude extract was also quantified and characterized by HPLC and LC-MS analysis. Furthermore, the in vivo study was performed by considering 11 months old rats (n = 7). Importantly, the oral administration of Danggui crude extract with stem cells intravenous injection in SHR-D-ADSCs group showed a combination effect to augment the cardiac function through enhancement of ejection fraction, fractional shortening, contractility function in the late-stage hypertension conditions. We have also observed a decreased apoptosis rate in the heart tissue of SHR-D-ADSCs group. Taken together, these results indicate that the combinatorial effects of Danggui crude extract and stem cell therapy enhanced cardiac function in late-stage SHR rats.

Neurodegenerative diseases have become increasingly prevalent in the aged population. Rheumatoid arthritis (RA) is an autoimmune disease that causes systemic inflammation, damaging the neurons. However, only a few treatment options can reduce RA-induced neurodegeneration. This study aimed to evaluate whether adipose-derived stem cells (ADSCs) pretreated with curcumin could ameliorate RA-induced neurodegenerative illness in an RA rat model. Wistar rats were randomly classified into the following four groups: control, RA, RA + ADSC (1 × 106 cells per rat), and RA + curcumin-pretreated ADSC (1 × 106 cells per rat). After treatment for two months, the effects were specifically evaluated in the brains collected from the rats. Our results demonstrated that the transplantation of curcumin-pretreated ADSCs substantially reduced inflammation and apoptosis in the cortices of RA rats compared to those of other groups. Thus, the combination of ADSCs and curcumin exerts a synergistic effect in enhancing neuronal protection in RA rats. In the future, this combination therapeutic strategy can potentially be used as a novel treatment method to reduce RA-induced neurodegenerative disorders.

Oxidative stress is a major cause of aging related skin injuries. Hydrogen peroxide related ROS accumulation triggers increase in matrix metalloproteinases and elevated collagen degradation, which is a characteristic of skin aging. In this study, we investigated the protective effect of Poria cocos, used widely in the treatment of inflammatory diseases, against H2 O2 induced oxidative stress. The aqueous extract of dried P. cocos was obtained by heating 10 g in 500 ml of distilled water. The mixture was evaporated up to 400 ml and the remaining 100 ml was filtered through muslin cloth repeatedly to obtain a clear aqueous extract of the P. cocos. Hs68 human dermal fibroblast cells were challenged with 100 μM of H2 O2 for 24 h. Following H2 O2 challenge, the cells were treated with increasing concentration of P. cocos extract (100-400 μg/ml) for 24 h. P. cocos extract hindered the H2 O2 induced cell death significantly that was correlated with reduction in ROS accumulation. Western blot analysis show that P. cocos extract suppressed the expression of metallomatrix proteinases, inflammatory markers and skin aging markers, but increased TGF-β1 levels and antioxidant related proteins. These data suggest that P. cocos is effective in attenuating oxidative stress associated skin aging effects and may be a potential agent in cosmetics products.

Alcalase potato protein hydrolysate (APPH) might have a very important role in therapeutic effects. This study aims to examine the beneficial effects of bioactive peptides (DIKTNKPVIF [DI] and IF) from APPH supplement in the regulation of cardiac apoptosis, autophagy, and mitochondrial biogenesis pathway in spontaneously hypertensive rats (SHR). We have investigated ejection fraction, fractional shortening, Tunel assay, apoptosis, autophagy, and mitochondrial biogenesis pathway marker expression to show the efficacy of bioactive peptides in an SHR model. Bioactive peptides significantly upregulate ejection fraction and fractional shortening in SHR rats. SHR rats exhibited higher protein expression of apoptotic markers such as BAD, cytochrome c, and caspase 3. Finally, the bioactive peptides upregulate survival proteins (p-AKT/p-PI3K), autophagy (Beclin1/LC3B), and mitochondrial biogenesis (p-AMPKα/SIRT1/PGC1α/p-Foxo3a/Nrf2/CREB) marker expressions compared with the SHR groups. In summary, the bioactive peptides protect the heart tissues through the activation of autophagy and mitochondrial biogenesis pathway and thereby attenuate cardiac apoptosis in a spontaneously hypertensive rat model.

Parkinson's disease (PD) is a neurodegenerative disorder involving the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Cellular clearance mechanisms, including the autophagy-lysosome pathway, are commonly affected in the pathogenesis of PD. The lysosomal CaThis study aims is to investigate the neuroprotective effects of A. argyi in promoting the TRPML1-mediated autophagy/mitophagy-enhancing effect METHODS: In this study, we used 1-methyl-4-phenyl-pyridinium (MPP+)-induced PD model established in an SH-SY5Y human neuroblastoma cell line as well as in a 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-induced PD model in C57BL/6 J mice. MTT assay was conducted to measure the cell viability and further MitoSoX and DCFDA assay were used to measure the ROS. Western blot analysis was used to access levels of TRPML1, p-DRP1 (ser616), p-AKT, PI3K, and β-catenin, Additionally, IF and IHC analysis to investigate the expression of TRPML1, LC3B, β-catenin, TH+, α-synuclein. Mitotracker stain was used to check mitophagy levels and a lysosomal intracellular activity kit was used to measure the lysosomal dysfunction. Behavioral studies were conducted by rotarod and grip strength experiments to check motor functions.In our in vitro study, A. argyi rescued the MPP+-induced loss of cell viability and reduced the accumulation of mitochondrial and total reactive oxygen species (ROS). Subsequently, it increased the expression of TRPML1 protein, thereby inducing autophagy, which facilitated the clearance of toxic accumulation of α-synuclein. Furthermore, A. argyi played a neuroprotective role by activating the PI3K/AKT/β-catenin cell survival pathway. MPP+-mediated mitochondrial damage was overcome by upregulation of mitophagy and downregulation of the mitochondrial fission regulator p-DRP1 (ser616) in SH-SY5Y cells. In the in vivo study, A. argyi ameliorated impaired motor function and rescued TH+ neurons in the SNpc region. Similar to the results of the in vitro study, TRPML1, LC3B, and β-catenin expression was enhanced in the SNpc region in the A. argyi-treated mice brain.Thus, our results first demonstrate that A. argyi can exert neuroprotective effects by stimulating TRPML1 and rescuing neuronal cells by boosting autophagy/mitophagy and upregulating a survival pathway, suggesting that A. argyi can further be exploited to slow the progression of PD.

Habitual chewing of the areca nut increases the risk of mortality owing to cardiovascular disease, but few reports have revealed the cardiotoxicity mechanism of the areca nut. Arecoline has been reported to be the primary toxic constituent in the areca nut. In order to study the acute cardiotoxicity of the areca nut in the development of pathologic heart hypertrophy, we induced heart injury in rats using arecoline. Arecoline at a low dosage (5 mg/kg/day) or a high dosage (50 mg/kg/day) was intraperitoneally injected to Sprague-Dawley rats for 21 days. The change of heart function and biochemical pathways were investigated with echocardiography and Western blot. The results were presented that heart functions were weakened by arecoline stimulation, and western blotting analysis revealed an elevation in BNP levels in the heart after arecoline exposure. Arecoline induced IL-6-mediated activation of the MEK5/ERK5 and JAK2/STAT3 pathways, as well as mitogen-activated protein kinase signaling cascades. Further, arecoline increased the calcineurin and NFATc3 levels in the heart. In summary, our results suggest that arecoline causes significantly cardiotoxicity and heart damage by inducing several hypertrophy-related signaling pathways, including IL-6-induced MEK5/ERK5, JAK2/STAT3, mitogen-activated protein kinases, and calcineurin signaling pathways. The study elucidated, for the first time, the possible cardiac hypertrophy mechanisms underlying the cardiotoxicity of the areca nut.

It has been well-documented that the consumption of deep sea water (DSW) has beneficial effects on myocardial hypertrophy and cardiac apoptosis induced by hypercholesterolemia. However, the molecular mechanisms for the anti-inflammatory effects of DSW on diabetic cardiomyopathy are still largely unclear. The main purpose of this present study was to test the hypothesis that DSW exerts anti-inflammatory effects through the suppression of the TNF-α-mediated signaling pathways. IP injection of streptozotocin (STZ) at the dose of 65 mg/kg was used to establish a diabetes rat model. DSW mineral extracts that diluted in desalinated water were prepared in three different dosages and administered to the rats through gavages for 4 weeks. These dosages are DSW-1X (equivalent to 37 mg Mg2+ /kg/day), 2X (equivalent to 74 mg Mg2+ /kg/day) and 3X (equivalent to 111 mg Mg2+ mg/kg/day). Immunofluorescence staining and Western blot showed that the protein expression level of TNF-α was markedly higher in the STZ-induced diabetic rat hearts than in the control group. Consequently, the phosphorylation levels of the TNF-α-modulated downstream signaling molecules and P38 mitogen-activated protein kinases (MAPKs) were notably elevated in heart tissues of STZ-induced diabetes. These higher phosphorylation levels subsequently upregulated NF-κB-modulated inflammatory mediators, such as cyclooxygenase (COX)-II and inducible nitric oxide synthase (iNOS). However, treatment with DSW as well as MgSO4 , the main mineral in DSW, significantly reversed all the alterations. These findings suggest that DSW has potential as a therapeutic agent for preventing diabetes-related cardiovascular diseases.

We previously reported that deep sea water (DSW) prolongs the life span of streptozotocin (STZ)-induced diabetic rats by the compensatory augmentation of the insulin like growth factor (IGF)-I survival signaling and inhibition of apoptosis. Here, we investigated the effects of DSW on cardiac hypertrophy in diabetic rats. Cardiac hypertrophy was induced in rats by using STZ (65 mg/kg) administered via IP injection. DSW was prepared by mixing DSW mineral extracts and desalinated water. Different dosages of DSW-1X (equivalent to 37 mg Mg2+·kg−1·day−1), 2X (equivalent to 74 mg Mg2+·kg−1·day−1) and 3X (equivalent to 111 mg Mg2+·kg−1·day−1) were administered to the rats through gavage for 4 wk. Cardiac hypertrophy was evaluated by the heart weight-to-body weight ratio and the cardiac tissue cross-sectional area after hematoxylin and eosin staining. The protein levels of the cardiac hypertrophy signaling molecules were determined by Western blot. Our results showed that the suppressive effects of the DSW treatment on STZ-induced cardiac hypertrophy were comparable to those of MgSO4administration and that the hypertrophic marker brain natriuretic peptide (BNP) was decreased by DSW. In addition, DSW attenuated both the eccentric hypertrophy signaling pathway, IL-6-MEK-STAT3, and the concentric signaling pathway, IGF-II-PKCα-CaMKII, in DM rat hearts. The cardiac hypertrophy-associated activation of extracellular signal-regulated kinase (ERK) and the upregulation of the transcription factor GATA binding protein 4 (GATA4) were also negated by treatment with DSW. The results from this study suggest that DSW could be a potential therapeutic agent for the prevention and treatment of diabetic cardiac hypertrophy.NEW & NOTEWORTHY Deep sea water, containing high levels of minerals, improve cardiac hypertrophy in diabetic rats through attenuating the eccentric signaling pathway, IL-6-MEK5-STAT3, and concentric signaling pathway, IGF2-PKCα-CaMKII. The results from this study suggest that deep sea water could be a potential therapeutic agent for the prevention and treatment of diabetic cardiac hypertrophy.

In aging hypertensive conditions, deterioration of insulin-like growth factor 1 receptor (IGF1R) cause a pathological impact on hypertensive hearts with an increased Ang II level. Recovering these adverse conditions through transplanted adipose-derived stem cells is a challenging approach. Moreover, Danggui, a Traditional Chinese medicine (TCM), is used for the treatment of cardioprotective effects. In this study, to evaluate whether the combined effect of MSCs and TCM can recover the cardiac function in late-stage hypertension rats. We observed that lower dose of Danggui crude extract treatment showed an increased level of cell viability with maintained stemness properties and growth rate in rat adipose-derived stem cells (rADSCs). Further, we cocultured the H9c2 cells with rADSCs and the results revealed that Danggui-treated MSCs enhanced the IGF1R expression and attenuated the hypertrophy in H9c2 cells against Ang II challenge by immunoblot and rhodamine-phalloidin staining. In addition, Danggui crude extract was also quantified and characterized by HPLC and LC-MS analysis. Furthermore, the in vivo study was performed by considering 11 months old rats (n = 7). Importantly, the oral administration of Danggui crude extract with stem cells intravenous injection in SHR-D-ADSCs group showed a combination effect to augment the cardiac function through enhancement of ejection fraction, fractional shortening, contractility function in the late-stage hypertension conditions. We have also observed a decreased apoptosis rate in the heart tissue of SHR-D-ADSCs group. Taken together, these results indicate that the combinatorial effects of Danggui crude extract and stem cell therapy enhanced cardiac function in late-stage SHR rats.

Hypoxic preconditioning is a well-known strategy to improve the survival and therapeutic potential of stem cells against various challenges including hemodynamic and neurohormonal modulations. However, the mechanism involved in hypoxia-induced benefits on stem cells is still ambiguous. In pathological hypertension, the elevation of the neurohormonal mediator Angiotensin II (Ang II) causes the adverse effects to stem cells. In this study, we investigate the effect and mechanism of action of short term hypoxia-inducible miRNA in suppressing the effects of AngII on stem cells. According to the results obtained, Ang II affects the normal cell cycle and triggers apoptosis in rADSCs with a corresponding increase in the expression of cell death-inducing p53 target 1 (CDIP1) protein. However, the short term hypoxia-inducible miRNA-miR-210-3p was found to target CDIP1 and reduce their levels upon the Ang II challenge. CDIP1 induces stress-mediated apoptosis involving the extrinsic apoptosis pathway via Bid/Bax/cleaved caspase3 activation. Administration of mimic miR-210-3p targets CDIP1 mRNA by binding to the 3' UTR region as confirmed by dual luciferase assay and also reduced Ang II-induced mitochondrial ROS accumulation as analyzed by MitoSOX staining. Moreover, the present study demonstrates the mechanism of miR-210-3p in the regulation of Ang II-induced CDIP1-associated apoptotic pathway in rADSCs.

Aging is the most important current issue and is usually accompanied by complications, such as cardiovascular disorders and neurodegenerative diseases, which are the leading causes of death worldwide and the second major cause of death in Taiwan. In this study, we have investigated the protective effect of adipose-derived mesenchymal stem cells (ADSCs) and the role of epigallocatechin gallate (EGCG) in enhancing this effect in aging cerebral cortex of rats. Further, we attempted to elucidate the molecular mechanism through which EGCG influences the protective effects of ADSC. ADSCs, co-cultured with EGCG, were injected into 20-month-old Wistar rats. Hematoxylin and eosin staining of the cerebral cortex revealed noticeable neurogenic activity and visible improvements in the integrity of the pre-frontal cortex tissue, compared to that in rats treated with ADSCs alone. Western blot analysis confirmed that ADSC, co-cultured with EGCG, enhanced cell survival via the p-Akt pathway and improved mitochondrial biogenesis via the SIRT-1 pathway. Moreover, it increased the available brain-derived neurotrophic factor to a higher degree than that in the ADSC group. Furthermore, western blotting showed that EGCG improved the antioxidant activity of the ADSCs in the cortex tissues via the Nrf-2 and HO-1 pathway. Based on these findings, we propose that this variation in stem cell treatment may facilitate functional recovery and enhanced neuroprotection in aged brains.

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKC&delta, activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKC&delta, activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKC&delta, mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKC&delta, wild type (WT). Inhibition of PKC&delta, by PKC&delta, kinase-deficient (KD) or rottlerin not only inhibited cardiac PKC&delta, activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKC&delta, WT plasmids reversed the inhibitory effects of DATS on PKC&delta, activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKC&delta, activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKC&delta, signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

Carthamus tinctorius L. (Compositae) is used in Chinese medicine to treat heart disease and inflammation. In our previous study, we found that C. tinctorius L. inhibited lipopolysaccharides (LPS)-induced tumor necrosis factor-alpha (TNF-α) activation, JNK expression, and apoptosis in H9c2 cardiomyoblast cells. The present study was performed to investigate the protective effect of C. tinctorius extract (CTF) on LPS-challenged H9c2 myocardioblast cell and to explore the possible underlying mechanism. Cell viability assay showed that LPS treatment decreased the cell viability of H9c2 cell, whereas CTF treatment reversed LPS cytotoxicity in a dose-dependent manner, especially in the LPS + CTF 25 (μg/mL) group. LPS treatment-induced apoptosis was determined by transferase-mediated dUTP nick end labeling assay, and by Western blot. LPS-induced apoptotic bodies were decreased following CTF treatment. Expression of TNF-α, FAS-L, FAS, FADD, caspase-8, BID, and t-BID was significantly increased in LPS-treated H9c2 cells. In contrast, it was significantly suppressed by the administration of CTF extract. In addition, CTF treatment activates antiapoptotic proteins, Bcl-2 and p-Bad, and downregulates Bax, cytochrome-c, caspase-9, caspase-3, and apoptosis-inducing factor expression. Furthermore, CTF exerted cytoprotective effects by activating insulin-like growth factor-I (IGF-I) signaling pathway leading to downregulation of the apoptotic proteins involved in FAS death receptor pathway. In addition, AG1024 and IGF-I receptor (IGF-IR) inhibitor and siRNA silencing reverses the effect of CTF implying that CTF functions through the IGF-IR pathway to inhibit LPS-induced H9c2 apoptosis. These results suggest that treatment with CTF extract prevented the LPS-induced apoptotic response through IGF-I pathway.

Hypertension, which is known as a silent killer, is the second leading cause of kidney failure worldwide. Elevated blood pressure causes approximately 7.6 million deaths, which account for ~13.5% of the total deaths and will continue to rise. High blood pressure is the prime risk factor associated with complications in major organs, including the heart, brain and kidney. High blood pressure accelerates oxidative stress and thereby causes organ dysfunction through the production of reactive oxygen species. In this study, we investigated the renal-protective effects of the bioactive peptide IF from alcalase potato protein hydrolysate in spontaneously hypertensive rat kidney. Sixteen-week-old spontaneously hypertensive rats were divided into three groups (n = 6), and Sixteen-week-old Wistar Kyoto rats (n = 6) served as the control group. The rats were administered IF and captopril via oral gavage for 8 weeks and then sacrificed, and their kidneys were harvested. The kidney sections from the rats treated with IF showed restoration of the structure of the glomerulus and Bowman’s capsule. The expression levels of Nrf2-mediated antioxidants were also increased, as confirmed by 4-hydroxynonenal immunohistochemical staining. The TUNEL assay revealed a significant reduction in the number of apoptotic cells in the IF-treated groups, which was consistent with the western blot results. Thus, the bioactive peptide IF exerts potential protective effects against hypertension-associated ROS-mediated renal damage via the Nrf2-dependent antioxidant pathway along the DJ-1 and AKT axes. Hence, we speculate that IF might have promising therapeutic effects on renal damage associated with hypertension.

Severe and potentially fatal hypotension and cardiac contractile dysfunction are common symptoms in patients with sepsis. LPS was previously found to dramatically upregulate expression of fibrosis-related factors FGF-2, uPA, MMP-2, and MMP-9 in primary cardiac fibroblasts. MMPs are capable of denaturing and degrading fibrillar collagens and other components of the extracellular matrix (ECM). Studies have shown that dysregulation of expression of MMPs is associated with development of myocardial extracellular matrix remodeling and cardiac fibrosis, which contribute to progression of heart failure. In this study, H9c2 cells and cardiac fibroblasts were divided into five treatment groups: control, LPS (1 μg/mL) and three concentrations of FCEtOH (Carthami Flos ethanolic extract) (31.25, 62.5, and 125 μg/mL). Phosphorylation of ERK-1/2 was observed to be rapidly induced upon treatment with LPS. In contrast, it was significantly suppressed by the administration of FCEtOH (125 μg/mL). Effects of FCEtOH on LPS-induced MMP-2 and MMP-9 expression in H9c2 cells occurred directly through ERK1/2 were determined. H9c2 cells were therefore pretreated with EGF-R to activate ERK pathway. Both protein levels of MMP-2 and MMP-9 and immunefluorescent signals of MMP-9 were significantly enhanced by EGFR. In contrast, MMP-2 and MMP-9 were significantly reduced after FCEtOH administration. Based on these findings, the authors concluded that FCEtOH elicits a protective effect against LPS-induced cardio-fibrosis through the ERK1/2 pathway. Carthamus tinctorius L may potentially serve as a cardio-protective agent against LPS- induced cardiac fibrosis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 754-763, 2017.

Extreme hypoxia often leads to myocardial apoptosis and causes heart failure. Angiotensin-(1-7)Ang-(1-7) is well known for its cardio-protective effects. However, the effects of Ang-(1-7) on long-term hypoxia (LTH)-induced apoptosis remain unknown. In this study, we found that Ang-(1-7) reduced myocardial apoptosis caused by hypoxia through the Mas receptor. Activation of the Ang-(1-7)/Mas axis down-regulated the hypoxia pro-apoptotic signaling cascade by decreasing the protein levels of hypoxia-inducible factor 1α (HIF-1α) and insulin-like growth factor binding protein-3 (IGFBP3). Moreover, the Ang-(1-7)/Mas axis further inhibited HIF-1α nuclear translocation. On the other hand, Ang-(1-7) activated the IGF1R/PI3K/Akt signaling pathways, which mediate cell survival. However, the above effects were abolished by A779 treatment or silencing of Mas expression. Taken together, our findings indicate that the Ang-(1-7)/Mas axis protects cardiomyocytes from LTH-stimulated apoptosis. The protective effect of Ang-(1-7) is associated with the inhibition of HIF-1α nuclear translocation and the induction of IGF1R and Akt phosphorylation.

Hypertension triggers oxidative stress, causing dysfunction in organs, including the brain, heart, and kidney. In this study, we examined the potential renal benefit of the soybean bioactive peptide VHVV by in silico analysis and in spontaneously hypertensive rats. Sixteen-week-old spontaneously hypertensive rats were divided into 3 groups and treated with or without VHVV or captopril. Age-matched Wistar-Kyoto rats were used as normotensive control group. The rats were sacrificed after 8 weeks of treatment, and kidneys were analyzed to demonstrate the efficacy of VHVV. In silico analysis confirmed the antihypertensive activity of VHVV. In animal study, VHVV induced antioxidant defense and modulated mitochondrial homeostasis in the hypertensive kidney via SIRT1-PGC1α/Nrf2 pathway. Hence, improvements in renal cells were attributed to the suppression of inflammation and apoptosis, and the architecture of the hypertensive kidney was restored after peptide treatment. Overall, VHVV exhibited therapeutic effects by decreasing renal damage caused by hypertension-induced free radicals.

Fatty acids and glucose are the main energy sources for the heart. Energy metabolic imbalance can cause many heart dysfunctions. Cardiomyocytes can switch to using fatty acids or glucose as an energy source. Palmitic acid (PA) treatment induces lipoptoxity and loss of mitochondrial function. We utilised PA treatment to simulate hyperlipidaemia and subsequently investigated the protective effects of tetramethylpyrazine (TMP) on the PAinduced cluster of differentiation 36 (CD36) and glucose transporter type 4 (GLUT4) signalling switch. The results showed that TMP can protect H9c2 cells and neonatal rat cardiomyocytes from PA-induced lipotoxicity similar to resveratrol significantly activated sirtuin1 to increase CD36 and decrease GLUT4 pathway proteins following treatment with PA, and switch the energy signalling GLUT4 pathway back to the CD36 pathway to ameliorate cardiac myocyte lipotoxicity and rescue the impaired energy metabolism. Therefore, we recommend TMP as a more efficient herbal component for maintaining the energy metabolism of the heart.

Aging is the most important risk factor in cardiovascular disease (CVD), which is the leading causes of death worldwide and the second major cause of death in Taiwan. The major factor in heart failure during aging is heart remodeling, including long-term stress-induced cardiac hypertrophy and fibrosis. Exercise is good for aging heart health, but the impact of exercise training on aging is not defined. This study used 3-, 12- and 18-month-old rats and randomly divided each age group into no exercise training control groups (C3, A12 and A18) and moderate gentle swimming exercise training groups (E3, AE12 and AE18). The protocol of exercise training was swimming five times weekly with gradual increases from the first week from 20 to 60 min for 12 weeks. Analyses of protein from rat heart tissues and sections revealed cardiac inflammation, hypertrophy and fibrosis pathway increases in aged rat groups (A12 and A18), which were improved in exercise training groups (AE12 and AE18). There were no heart injuries in young rat hearts in exercise group E3. These data suggest that moderate swimming exercise training attenuated aging-induced cardiac inflammation, hypertrophy and fibrosis injuries of rat hearts.

Obesity is one of the major risk factors for nonalcoholic fatty liver disease (NAFLD), and NAFLD is highly associated with an increased risk of cardiovascular disease (CVD). Scholars have suggested that certain probiotics may significantly impact cardiovascular health, particularly certain Lactobacillus species, such as Lactobacillus reuteri GMNL-263 (Lr263) probiotics, which have been shown to reduce obesity and arteriosclerosis in vivo. In the present study, we examined the potential of heat-killed bacteria to attenuate high fat diet (HFD)-induced hepatic and cardiac damages and the possible underlying mechanism of the positive effects of heat-killed Lr263 oral supplements. Heat-killed Lr263 treatments (625 and 3125 mg/kg-hamster/day) were provided as a daily supplement by oral gavage to HFD-fed hamsters for eight weeks. The results show that heat-killed Lr263 treatments reduce fatty liver syndrome. Moreover, heat-killed Lactobacillus reuteri GMNL-263 supplementation in HFD hamsters also reduced fibrosis in the liver and heart by reducing transforming growth factor β (TGF-β) expression levels. In conclusion, heat-killed Lr263 can reduce lipid metabolic stress in HFD hamsters and decrease the risk of fatty liver and cardiovascular disease.

In this study, the antitumor activity of KHC-4 was analyzed using human prostate cancer (CaP) cells and the underlining anticancer mechanisms of KHC-4 were identified. KHC-4 inhibited cell proliferation and induced cytotoxicity in the castration-resistant CaP DU145 cell line. The most effective concentration of KHC-4 was 0.1 μM. Cell cycle analysis demonstrated that KHC-4 treatment caused G2/M arrest and a subsequent increase in the sub-G1 population. Furthermore, KHC-4 is up-regulated p21, p27, and p53 in a time- and concentration-dependent manner. The exposure of cells to KHC-4 induced Cdk1/cyclin B1 complex activity, which led to cell cycle arrest. Moreover, KHC-4 inhibited the activities of MMP-2 and MMP-9 to inhibit tumor cell metastasis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1879-1887, 2016.

Myocardial infarction (MI) usually results in myocardial ischemia, remodeling and hypoxia that lead to cell death. To date, the insulin-like growth factor binding protein-3 (IGFBP3) is known to play an important role in insulin growth factor (IGF) bioavailability. Previous studies have found that hypoxia results in cell apoptosis. However, the detailed mechanism and roles of IGFBP3 in long-term hypoxia (LTH) regulated heart cell apoptosis remains unknown. In this study H9c2 cardiomyoblast cells were treated with investigated long-term hypoxic exposure with the possible mechanisms involved. The results showed that LTH enhanced IGFBP3 protein synthesis and induced its secretion. The accumulated IGFBP3 sequestered Insulin growth factor 1 (IGF-1) away from the type I IGF receptor (IGF-1 R), which blocked the IGF1R/PI3K/Akt survival signaling pathway, resulting in cell apoptosis. According to our findings, IGFBP3 could be a valuable target for developing treatments for cardiac diseases in long-term hypoxia exposure patients.

Objectives Prolactin (PRL) is a multifunctional hormone that influences multiple physiological processes. It has been shown to have a protective effect on the cardiovascular system; however, the mechanisms of this effect are poorly understood. The purpose of the study was to elucidate the role of PRL in intermittent hypoxia (IH)-induced apoptosis in the cardiovascular system. Method and results We established a hyperprolactinemic rat model by implanting two anterior pituitary (AP) glands into the renal capsule of male Sprague–Dawley rats. The rats were kept under normoxic conditions for 4weeks after implantation in order to reach the expression plateau of PRL in the plasma, and then treated with IH for 7 or 14days. Their hearts were then removed for histological and protein expression analyses. Cerebral cortex (CX)-grafted control rats challenged with IH displayed unique phenotypes such as a thicker heart wall, an abnormal myocardial architecture and an increased interstitial space of the left ventricle. They exhibited reduced expressions of p -JAK2, p -STAT5, cell cycle-dependent proteins (cyclin D1, cyclin E and cyclin A), IGF-IRα, PI3Kα, p -AKT and p -ERK1/2 in cardiomyocytes at 7days. Conclusions Our comprehensive analysis suggested that high plasma PRL can protect rat cardiomyocytes against IH through (1) the p -JAK2 and p -STAT5 pathways for transient cell proliferation, (2) the PI3Kα/AKT and MAPK survival pathways through IGF-I, and (3) the downregulation of IGF-II and ERK5, which inhibit cell hypertrophy.

Background/Aims: Numerous epidemiological studies have associated elevated serum phosphorus levels with cardiovascular disease and the risk of death in the general population as well as in chronic kidney disease (CKD) and dialysis patients. In this study, we explored whether elevated phosphate conditions induce cardiac hypertrophy and attempted to identify the molecular and cellular mechanisms in the hypertrophic response. Methods: H9c2 myocardial cells were incubated in high-phosphate conditions to induce hypertrophy. Pathological hypertrophic responses were measured in terms of cell size, arrangement of actin filaments, and hypertrophy markers such as atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in myocardial cells. Several transcriptional factors involved in cardiac hypertrophy development were measured to investigate the molecular pathways involved in elevated phosphate-induced cardiac hypertrophy. Results: High-phosphate conditions induced cellular hypertrophy, marked by increased cell size, reorganization of actin filaments, and upregulation of both ANP and BNP in H9c2 cells. Both upstream calcineurin and downstream transcription factors, including GATA-4 and NFAT-3, were significantly increased under hyperphosphate conditions. Moreover, both MEK1/2 and ERK1/2 expression increased significantly, and cellular hypertrophy was markedly attenuated by U0126, an ERK1/2 inhibitor. Conclusions: These results suggest that hyperphosphate conditions induce myocardial hypertrophy through the ERK signaling pathway in H9c2 cells. Our findings provide a link between the hyperphosphate-induced response and the ERK/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy. In view of the potent and selective activity of the ERK inhibitor U0126, this agent warrants further investigation as a candidate for preventing hyperphosphate-induced cardiac hypertrophy in CKD and dialysis patients.

Background/Aims: The risk of heart disease is higher in males than in females. However, this advantage of females declines with increasing age, presumably a consequence of decreased estrogen secretion and malfunctioning of the estrogen receptor. We previously demonstrated that 17β-estradiol (E2) prevents cardiomyocyte hypertrophy, autophagy and apoptosis via estrogen receptor α (ERα), but the effects of ERβ on myocardial injury remained elusive. The present paper thus, investigated the cardioprotective effects of estrogen (E2) and ERβ against hypoxia-induced cell death. Methods: Transient transfection of Tet-On ERβ gene construct was used to overexpress ERβ in hypoxia-treated H9c2 cardiomyoblast cells. Results: Our data revealed that IGF1R, Akt phosphorylation and Bcl-2 expression are enhanced by ERβ in H9c2 cells. Moreover, ERβ overexpression reduced accumulation of hypoxia-related proteins, autophagy-related proteins and mitochondria-apoptotic proteins and enhanced the protein levels of Bcl-2, pAkt and Bad under hypoxic condition. In neonatal rat ventricular myocytes (NRVMs), we observed that hypoxia induced cell apoptosis as measured by TUNEL staining, and E2 and/or ERβ could totally abolish hypoxia-induced apoptosis. The suppressive effects of E2 and/or ERβ in hypoxia-treated NRVMs were totally reversed by ER antagonist, ICI. Taken together, E2 and/or ERβ exert the protective effect through repressed hypoxia-inducible HIF-1α, BNIP3 and IGFBP-3 levels to restrain the hypoxia-induced autophagy and apoptosis effects in H9c2 cardiomyoblast cells. Conclusion: The results suggest that females probably could tolerate better prolonged hypoxia condition than males, and E2/ERβ treatment could be a potential therapy to prevent hypoxia-induced heart damage.”

IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy.

Post-menopausal women show dramatically increased cardiovascular disease morbidity (CVD). Danshen is used widely in China for the treatment of cardiovascular disorders, including coronary heart disease. Danshen possesses lipid-soluble biologically active components with a structure similar to 17β-estrodiol (E2). This study assesses whether the cardio-protection exerted by Danshen is mediated through the ERs within H9c2 cardiomyoblast cells. Cardiomyoblast cells pretreated with Fulvestrant (ICI 182,780), an estrogen receptor antagonist was applied to investigate the estrogenic activity of Danshen. The Danshen extract preventive effects on Leu27IGF-II-induced IGF-IIR signaling activator and H9c2 cell apoptosis were identified using TUNEL assay, JC-1 staining and Western blot assay. We found that Danshen extract treatments significantly enhanced phosphorylated Akt through estrogen receptor activation to inhibit Leu27IGF-II-induced calcineurin activation and block H9c2 cell apoptosis. Danshen extracts suppressed the IGF-IIR signaling proteins, pro-apoptotic proteins and reversed the mitochondrial membrane instability induced by Leu27IGF-II. However, the cardioprotective properties of Danshen to inhibit Leu27IGF-II-induced cell apoptosis and promote cell survival were attenuated by applying ICI, which suggests that the Danshen cardioprotective effect is mediated through estrogen receptors. All our data indicated that Danshen exerts strong estrogenic activity which can be considered a novel selective estrogen receptor modulator (SERM) against IGF2R signaling that blocks cardiac apoptosis.

High-calorie diet-induced obesity leads to cardiomyocyte dysfunction and apoptosis. Impaired regulation of epididymal fat content in obese patients has been known to increase the risk of cardiac injury. In our study, a lactic acid bacteria, Lactobacillus reuteri GMNL-263, was evaluated for its potential to reduce body weight and body fat ratio and to prevent heart injury in rats with high-fat diet-induced obesity. Lactic acid bacteria supplementation restored the cardiac function and decreased the physiological changes in the heart of the obese rats. In addition, the Fas/Fas-associated protein pathway-induced caspase 3/e Poly polymerase mediated apoptosis in the cardiomyocytes of the obese rats was reversed in the Lr263-treated rats. These results reveal that fed with Lr-263 reduces body fat ratio, inhibits caspase 3-mediated apoptosis and restores cardiac function in obese rats through recovery of ejection fraction and fractional shortening. Our results indicated that the administration of Lr263 lactic acid bacteria can significantly down-regulate body fat and prevent cardiomyocyte injury in obese rats.

The aim of this study was to establish the effective hepatoprotective properties of traditional Chinese medicines (TCMs) in fibrotic rat liver regeneration after partial hepatectomy (PHx). Fibrosis was induced in rats by ethanol (EtOH, 5 ml/kg) administration for 6, 24, 72, and 168 h. The rats were then fed four TCMs (1 g/kg/day, Codonopsis pilosula (CP), Salvia miltorrhiza Bunge (SMB), Bupleurum kasi (BK), and Elephantopus scaber L (ESL)) to Spraque-Dawley rats for 6, 24, 72 and 168 h, respectively. Surgical 70% cirrhotic fibrosis PHx was then conducted at 6, 24, 72, and 168 h. The effects on liver regeneration were examined to estimate and measure hepatocyte growth factor (HGF), focal adhesion kinase (FAK), Cyclin D1, Cyclin E, and retinoblastoma protein (pRb) protein expression using Western blotting analysis. Cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and TIMP-3 mRNA by Reverse transcription polymerase chain reaction (RT-PCR) were analyzed in cirrhotic fibrosis rats. Transforming growth factor-β1 (TGF-β1), Cyclin D1, Cyclin E, pRb and E2F mRNA expression levels were determined in fibrotic rats following PHx using RT-PCR. We found elevated glutamyl oxaloacetic transaminase (GOT), glutamyl pyrubic transaminase (GPT), alkaline phosphatase (ALP), gammaglutamyl transpeptidase (γ-GT), glutathione (GSH), nonprotein sulfhydryl (NPSH) and total bilirubin in serum after 6 h EtOH administration. These levels were progressively decreased over 168 h. Total protein and albumin were reduced in serum after 6 h administration and then progressively increased. In contrast, tissues disorder histology and morphology were determined in liver sections. After rats were fed TCMs we found that SMB extraction not only induced HGF, FAK, Cyclin D1, and pRb protein expression and Cyclin D1 mRNA increases, but also reduced MMP-2 and MMP-9 after 24 and 72 h post injury. In the cell cycle S phase the Cyclin E protein expression was increased by ESL. CP induced TIMP-1, TIMP-2 and TIMP-3 mRNA increases in fibrotic rats. We detected liver regeneration in fibrotic rats. We also found that the liver regeneration index increased from 6 to 168 h post PHx. After 168 h fibrotic liver regeneration rats exhibited reduced TGF-β1 mRNA expression and enhanced Cyclin D1, Cyclin E, pRb and E2F mRNA expression. TCMs play a crucial role in the early mediating process in fibrotic rat liver regeneration after PHx.

The insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3) and human growth hormone (h-GH) have been recognized as therapeutic targets for the heart disease therapy. The bioavailability and actions of insulin-like growth factors-II (IGF-II) and matrix metalloproteinase-9 (MMP9) are important for embryonic development and cardiomyocyte differentiation as well. However, the clinical manifestations following the change in the serum IGF-II and MMP9 in infants with isolated ventricular septal defect (VSD) undergoing surgical repair have not been clearly defined.Serum samples were collected from 72 infants: Twenty normal infants (group I) and 51 consecutive infants with echocardiography established isolated VSD (aged from 3 months to 1 year) were investigated. Among the 51 infants with VSD, 28 with shunt fraction, Qp/Qsor = 1.5 were free of congestive heart failure symptoms (group II); 23 with shunt fraction, Qp/Qsor = 2.0 were in congestive heart failure (group IIIa); and 23 of these 23 infants had undergone VSD repair 6 months before their second study (group IIIb). All insulin-like growth factors-II (IGF-II) and human growth hormone (h-GH), insulin like growth factor binding protein-3 (IGFBP-3) and its specific serum protease-MMP9 concentration were analyzed using ELISA and zymography, respectively.Serum IGF-II and MMP9 exhibited significant decreasing trends among the three groups and significantly lower concentrations of IGF-II, IGF-II/IGFBP-3 ratio and MMP9, were found only in the severe group whereas h-GH/IGF-II ratio became significantly higher in this group. Moreover, there were no significant differences in these parameters between the infants after surgical correction and the normal ones.The improvement in IGF-II and MMP9 serum concentration was identified in infants with VSD after surgical repair. These findings also indicate a significant relationship between IGF-II, MMP9 and VSD which might be used as diagnosis and prognosis indicators for this defect. Slight reductions in IGF-II/IGFBP3 ratio and slight increase in the h-GH/IGF-II ratio indicate mild VSD. The reductions in the MMP9, IGF-II, and IGF-II/IGFBP3 ratio plus high increase in the h-GH/IGF-II ratio indicate severe VSD.

Copper is an essential trace metal for physiological functions, whereas copper overload causes cytotoxicity in living organisms. Genetically determined systems regulate acquisition, distribution and storage for copper maintenance and homeostasis. The Human ATP7A copper transport ATPase modulates intracellular copper distribution, which is critical for copper-dependent enzymes such as superoxide dismutase (SOD1). To investigate the role of zebrafish ATP7A in copper homeostasis, zebrafish atp7a gene expression was reduced for analysis of downstream cellular function. The results demonstrated that zebrafish sod1 has lower expression in atp7a-knockdown fish. Similarly, zebrafish sp1, a transcriptional regulator of sod1, also shows reduced expression in atp7a-knockdown fish. The lower expression of sod1 resulting from atp7a knockdown is independent to p53 gene activation. The knockdown of atp7a and copper chelator NeoC results in hypopigmentation and notochord deformation in zebrafish. Addition of exogenous copper alleviated the impaired development. Interestingly, both sod1 and sp1 transcripts are reduced in the presence of NeoC and increased with exogenous copper, suggesting that the expression of sod1 and sp1 are directly affected by copper status. This is the first report to demonstrate a hierarchic gene expression of copper homeostatic genes between atp7a, sp1 and sod1 in zebrafish.

The surrounding medium of periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of p38 pathway in P. gingivalis conditioned medium-induced H9c2 myocardial cell hypertrophy and apoptosis. DNA fragmentation, cellular morphology, nuclear condensation, p38 protein products, and mitochondrial-dependent apoptotic related proteins in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, immunofluorescence, DAPI, and western blotting following P. gingivalis conditioned medium and/or pre-administration of SB203580 (p38 inhibitor). The p38 protein products and associated activities in H9c2 cells were both upregulated by P. gingivalis conditioned medium. P. gingivalis conditioned medium increased cellular sizes, DNA fragmentation, nuclear condensation, mitochondrial Bcl2-associated death promoter (Bad), cytosolic cytochrome c (cyt c), and the activated form of caspase-9 proteins in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, Bad, cyt c, and caspase-9 activities of H9c2 cells treated with P. gingivalis conditioned medium were all significantly reduced after pre-administration of SB203580. Our findings suggest that the activity of p38 signal pathway may be initiated by P. gingivalis conditioned medium and further activate mitochondrial-dependent apoptotic pathways leading to cell death in cultured H9c2 myocardial cells.