Bioinformatic Identification of the Pyroptosis-Related Transcription Factor-MicroRNA-Target Gene Regulatory Network in Angiotensin II-Induced Cardiac Remodeling and Validation of Key Components.

BACKGROUND: Accumulative evidence suggests that pyroptosis plays a key role in mediating angiotensin II (Ang II)-induced cardiac remodeling However, the potential role of pyroptosis-related transcription factor (TF)-microRNA (miRNA)-gene regulatory networks in mediating Ang II-associated cardiac remodeling remains largely unknown. Therefore, we identified the pyroptosis-related hub genes and constructed a transcription factor (TF)-miRNA-target gene regulatory network using bioinformatic tools to elucidate the pathogenesis of Ang II-induced cardiac remodeling. METHODS: The pyroptosis-related differentially expressed genes (DEGs) were identified from the cardiac remodeling-related dataset GSE47420. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and protein-protein interaction (PPI) analysis were performed to identify the pyroptosis-related hub DEGs. A TF-miRNA-target gene network was constructed and further validated by quantitative real-time polymerase chain reaction (qRT-PCR) in animal experiments. The correlation between the pyroptosis-related hub DEGs and cardiac remodeling was evaluated using comparative toxicogenomics database. The drug-gene interaction analysis was performed to identify potential drugs that target the pyroptosis-related hub DEGs. RESULTS: A total of 32 pyroptosis-related DEGs were identified and enriched in the inflammation-related pathways by KEGG analysis. 13 of the 32 pyroptosis-related DEGs were identified as hub DEGs. Furthermore, a TF-miRNA-target gene regulatory network containing 16 TFs, 6 miRNAs, and 5 hub target genes was constructed. The five pyroptosis-related hub target genes (DDX3X, ELAVL1, YWHAZ, STAT3, and EED) were identified as crucial cardiac remodeling-related genes using the comparative toxicogenomics database (CTD) database. Five drugs including celecoxib were identified as potential drugs for the treatment of cardiac remodeling. Finally, the expression levels of two top-ranked TF-miRNA-target genes axis were verified by qRT-PCR in mice with Ang II-induced cardiac remodeling and found to be generally consistent with the microarray results. CONCLUSIONS: This study constructed a pyroptosis-related TF-miRNA-target gene regulatory network for Ang II-induced cardiac remodeling. Five pyroptosis-related genes (DDX3X, ELAVL1, YWHAZ, STAT3, and EED) can be considered the core genes associated with pyrotposis-related cardiac remodeling. The findings of this study provide new insights into the molecular mechanisms of Ang II-induced cardiac remodeling and may serve as potential biomarkers or therapeutic targets for Ang II-induced cardiac remodeling.

Xanthohumol relieves arthritis pain in mice by suppressing mitochondrial-mediated inflammation.

Chronic pain is the most common symptom for people who suffer from rheumatoid arthritis and it affects approximately 1% of the global population. Neuroinflammation in the spinal cord induces chronic arthritis pain. In this study, a collagen-induced arthritis (CIA) mice model was established through intradermally injection of type II collagen in complete Freund's adjuvant solution. Following CIA inducement, the paws and ankles of mice were found to swell, mechanical pain and spontaneous pain were induced, and their motor coordination was impaired. The spinal inflammatory reaction was triggered, which presented as severe infiltration of inflammatory cells, and the expression levels of GFAP, IL-1ß, NLRP3, and cleaved caspase-1 increased. Oxidative stress in the spinal cord of CIA mice was manifested as reduced Nrf2 and NDUFB11 expression and SOD activity, and increased levels of DHODH and Cyto-C. At the same time, spinal AMPK activity was decreased. In order to explore the potential therapeutic options for arthritic pain, Xanthohumol (Xn) was intraperitoneally injected into mice for three consecutive days. Xn treatment was found to reduce the number of spontaneous flinches, in addition to elevating mechanical pain thresholds and increasing latency time. At the same time, Xn treatment in the spinal cord reduced NLRP3 inflammasome-mediated inflammation, increased the Nrf2-mediated antioxidant response, and decreased mitochondrial ROS level. In addition, Xn was found to bind with AMPK via two electrovalent bonds and increased AMPK phosphorylation at Thr174. In summary, the findings indicate that Xn treatment activates AMPK, increases Nrf2-mediated antioxidant response, reduces Drp1-mediated mitochondrial dysfunction, suppresses neuroinflammation, and can serve to relieve arthritis pain.

Identification of key genes and pathways in atherosclerosis using integrated bioinformatics analysis.

BACKGROUND: Atherosclerosis (AS) is a chronic inflammatory disease that might induce severe cardiovascular events, such as myocardial infarction and cerebral infarction. These risk factors in the pathogenesis of AS remain uncertain and further research is needed. This study aims to explore the potential molecular mechanisms of AS by bioinformatics analyses. METHODS: GSE100927 gene expression profiles, including 69 AS samples and 35 healthy controls, were downloaded from Gene Expression Omnibus database and indenfied for key genes and pathways in AS. RESULTS: A total of 443 differentially expressed genes (DEGs) between control and AS were identified, including 323 down-regulated genes and 120 up-regulated genes. The Gene ontology terms enriched by the up-regulated DEGs were associated with the regulation of leukocyte activation, endocytic vesicle, and cytokine binding, while the down-regulated DEGs were associated with negative regulation of cell growth, extracellular matrix, and G protein-coupled receptor binding. KEGG pathway analysis showed that the up-regulated DEGs were enriched in Osteoclast differentiation and Phagosome, while the down-regulated DEGs were enriched in vascular smooth muscle contraction and cGMP-PKG signaling pathway. Using the modular analysis of Cytoscape, we identified 3 modules mainly involved in Leishmaniasis and Osteoclast differentiation. The GSEA analysis showed the up-regulated gene sets were enriched in the ribosome, ascorbated metabolism, and propanoate metabolism. The LASSO Cox regression analysis showed the top 3 genes were TNF, CX3CR1, and COL1R1. Finally, we found these immune cells were conferred significantly higher infiltrating density in the AS group. CONCLUSIONS: Our data showed the pathway of Osteoclast differentiation and Leishmaniasis was involved in the AS process and we developed a three-gene model base on the prognosis of AS. These findings clarified the gene regulatory network of AS and may provide a novel target for AS therapy.

Increased Neuromedin B is Associated with a Favorable Prognosis in Glioblastoma.

BACKGROUND: Neuromedin B (NMB) is a neuropeptide that plays a key role in many physiological processes and is involved in the pathology of various diseases. Increased levels of NMB have been reported in solid tumors. Therefore, we investigated the prognostic value of NMB in glioblastoma (GBM). METHODS: Expression profiles of NMB mRNA were investigated in GBM and normal tissues using data from the cancer genome atlas (TCGA). NMB protein expression was obtained using data from the Human Protein Atlas. Receiver operating characteristic (ROC) curves were evaluated in GBM and normal tissues. The survival effect of NMB in GBM patients was evaluated using the Kaplan-Meier method. Protein-protein interaction networks were constructed using STRING, and the functional enrichment analyses were performed. The relationship between NMB expression and tumor-infiltrating lymphocytes was analyzed using the Tumor Immune Estimation Resource (TIMER) and the Tumor-Immune System Interaction database (TISIDB). RESULTS: NMB was overexpressed in GBM relative to normal biopsy specimens. The ROC analysis showed that the sensitivity and specificity of NMB in GBM were 96.4% and 96.2%, respectively. Kaplan-Meier survival analysis showed that GBM patients with high NMB expression had a better prognosis than those with low NMB expression (16.3 vs. 12.7 months, p = 0.002). Correlation analysis showed that NMB expression was associated with tumor-infiltrating lymphocytes and tumor purity. CONCLUSIONS: High expression of NMB was associated with increased GBM patient survival. Our study indicated that the NMB expression may be a biomarker for prognosis and that NMB may be an immunotherapy target in GBM.

LY294002 alleviates bone cancer pain by reducing mitochondrial dysfunction and the inflammatory response.

Bone cancer pain (BCP) is mainly caused by bone metastasis and markedly impairs the functional capacity and daily functions of patients. Neuroinflammation plays a pivotal role in the pathogenesis and maintenance of chronic pain. Oxidative stress in the mitochondria is a key contributor to neuroinflammation and neuropathic pain. Herein, a rat model of BCP was established which was characterized by bone destruction, pain hypersensitivity and motor disability. In the spinal cord, phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt) signaling was activated, and the inflammatory response and mitochondrial dysfunction were also observed. The intrathecal injection of LY294002, a selective inhibitor of PI3K/Akt signaling, decreased mechanical pain sensitivity, suppressed spontaneous pain and recovered the motor coordination of rats with BCP. Second, LY294002 treatment blocked spinal inflammation by reducing astrocytic activation and downregulating the expression levels of inflammatory factors, such as NF­κB, IL­1ß and TNF­α. Moreover, LY294002 treatment recovered mitochondrial function by activating the manganese superoxide dismutase enzyme, increasing NADH:ubiquinone oxidoreductase subunit B11 expression, and decreasing BAX and dihydroorotate dehydrogenase expression. LY294002 treatment also increased the mitochondrial membrane potential and decreased the mitochondrial reactive oxygen species levels in C6 cells. On the whole, the results of the present study suggest that the inhibition of PI3K/Akt signaling by LY294002 restores mitochondrial function, suppresses spinal inflammation and alleviates BCP.

GSK-3ß inhibition alleviates arthritis pain via reducing spinal mitochondrial reactive oxygen species level and inflammation.

Pain is the main symptom of osteoarthritis, which severely reduces the patients' quality of life. Stimulated neuroinflammation and elevated mitochondrial oxidative stress are associated arthritis pain. In the present study, arthritis model was established by intra-articular injection of complete Freund's adjuvant (CFA) on mice. Knee swelling, pain hypersensitivity and motor disability were observed in CFA-induced mice. In spinal cord, neuroinflammation was triggered and presented as severe infiltration of inflammatory cells and up-regulated expressions of glial fibrillary acidic protein (GFAP), nuclear factor-kappaB (NF-κB), PYD domains-containing protein 3 (NLRP3), cysteinyl aspartate specific proteinase (caspase-1) and interleukin-1 beta (IL-1ß). Mitochondrial function was disrupted and characterized as elevated expressions of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), dihydroorotate dehydrogenase (DHODH) and cytochrome C (Cyto C), and reduced expressions of Bcl-2 and Mn-superoxide dismutase (Mn-SOD) activity. Meanwhile, as a potential target for pain management, glycogen synthase kinase-3 beta (GSK-3ß) activity was up-regulated in CFA induced mice. To explore potential therapeutic options for arthritis pain, GSK-3ß inhibitor TDZD-8 was intraperitoneally injected for three days on CFA mice. Animal behavioral tests found that TDZD-8 treatment elevated mechanical pain sensitivity, suppressed spontaneous pain and recovered motor coordination. Morphological and protein expression analysis indicated that TDZD-8 treatment decreased spinal inflammation score and inflammatory related protein levels, recovered mitochondrial related protein levels, and increased Mn-SOD activity. In summary, TDZD-8 treatment inhibits GSK-3ß activity, reduces mitochondrial mediated oxidative stress, suppresses spinal inflammasome response, and alleviates arthritis pain.

Integrin subunit ß-like 1 mediates angiotensin II-induced myocardial fibrosis by regulating the forkhead box Q1/Snail axis.

Cardiac fibrosis is a severe condition with limited therapeutic options and often occurs in chronic cardiovascular diseases such as hypertension and myocardial infarction. There is currently a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of more effective therapeutic strategies targeting cardiac fibrosis. Integrin subunit ß-like 1 (ITGBL1), an extracellular matrix protein, has previously been implicated in various fibrotic diseases. However, the precise role of ITGBL1 in regulating myocardial fibrosis remains unknown. The present study was designed to investigate whether ITGBL1 regulates angiotensin II (Ang II)-induced myocardial fibrosis in vitro and in vivo and the possible mechanism of action. It was found that the protein expressions of ITGBL1, Forkhead box Q1 (FOXQ1), and Snail were all increased significantly in fibrotic heart tissues from Ang II-infused mice and Ang II-stimulated cardiac fibroblasts, all of which were inhibited by the Ang II type I (AT1) receptor antagonist losartan. Silencing the ITGBL1/FOXQ1/Snail axis with specific siRNAs reversed Ang II-induced fibrotic effects and upregulation of FOXQ1 and Snail expressions in cardiac fibroblasts. FOXQ1 siRNA inhibited Snail expression in Ang II-induced cardiac fibroblasts. Furthermore, ITGBL1/FOXQ1 interacted with the TGF-ß1 signaling to form a positive feedback loop. Our findings suggest that the extracellular matrix protein ITGBL1 mediates Ang II-induced cardiac fibrosis via the FOXQ1/Snail axis, which identifies ITGBL1 as a novel mediator of cardiac fibrosis and represents a potential therapeutic target for cardiac fibrosis.

Glycogen synthase kinase-3ß inhibition decreases inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.

Bone is the preferential site of metastasis for breast cancer. Invasion of cancer cells induces the destruction of bone tissue and damnification of peripheral nerves and consequently induced central sensitization which contributes to severe pain. Herein, cancer induced bone pain (CIBP) rats exhibited destruction of tibia, mechanical allodynia and spinal inflammation. Inflammatory response mainly mediated by astrocyte and microglia in central nervous system. Our immunofluorescence analysis revealed activation of spinal astrocytes and microglia in CIBP rats. Transmission electron microscopy (TEM) observations of mitochondrial outer membrane disruption and cristae damage in spinal mitochondria of CIBP rats. Proteomics analysis identified abnormal expression of proteins related to mitochondrial organization and function. Intrathecally, injection of GSK-3ß activity inhibitor TDZD-8 significantly attenuated Drp1-mediated mitochondrial fission and recovered mitochondrial function. Inhibition of GSK-3ß activity also suppressed NLRP3 inflammasome cascade and consequently decreased mechanical pain sensitivity of CIBP rats. For cell research, TDZD-8 treatment significantly reversed TNF-α induced mitochondrial membrane potential (MMP) deficiency and high mitochondrial reactive oxygen species level. Taken together, GSK-3ß inhibition by TDZD-8 decreases spinal inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.

2-Bromopalmitate attenuates inflammatory pain by maintaining mitochondrial fission/fusion balance and function.

Inflammatory pain activates astrocytes and increases inflammatory cytokine release in the spinal cord. Mitochondrial fusion and fission rely on the functions of dynamin-related protein 1 (Drp1) and optic atrophy 1 (OPA1), which are essential for the synaptic transmission and plasticity. In the present study, we aimed to explore the effects of 2-bromopalmitate (2-BP), an inhibitor of protein palmitoylation, on the modulation of pain behavior. Rats were intraplantar injected with complete Freund's adjuvant (CFA) to establish an inflammatory pain model. In the spinal cord of rats with CFA-induced inflammatory pain, the expression of astrocyte-specific glial fibrillary acidic protein (GFAP) and contents of proinflammatory cytokines IL-1ß and TNF-α were increased. Mitochondrial Drp1 was increased, while OPA1 was decreased. Consequently, CFA induced reactive oxygen species (ROS) production and Bcl-2-associated X protein (BAX) expression. The intrathecal administration of 2-BP significantly reversed the pain behaviors of the inflammatory pain in rats. Moreover, 2-BP also reduced the Drp1 expression, elevated the OPA1 expression, and further reduced the GFAP, IL-1ß, and TNF-α expression and ROS production. Furthermore, in vitro study proved a similar effect of 2-BP on the regulation of Drp1 and OPA1 expression. 2-BP also increased the mitochondrial membrane potential and decreased the levels of BAX, ROS, and proinflammatory cytokines. These results indicate that 2-BP may attenuate the inflammatory pain of CFA-treated rats via regulating mitochondrial fission/fusion balance and function.

Roles of klotho and stem cells in mediating vascular calcification (Review).

Vascular calcification, characterized by the active deposition of calcium phosphate in the vascular walls, is commonly observed in aging, diabetes mellitus and chronic kidney disease. This process is mediated by different cell types, including vascular stem/progenitor cells. The anti-aging protein klotho may act as an inhibitor of vascular calcification through direct effects on vascular stem/progenitor cells with osteogenic differentiation potential. A better understanding of the possible effects of klotho on vascular stem/progenitor cells may provide novel insight into the cellular and molecular mechanisms of klotho deficiency-related vascular calcification and disease. The klotho protein may be considered as a promising therapeutic agent for treating vascular calcification and disease and calcification-related vascular diseases.

Curcumin suppresses glioblastoma cell proliferation by p-AKT/mTOR pathway and increases the PTEN expression.

BACKGROUND: Glioblastoma (GB) is the most common neoplasm in the brain. Curcumin, as a known polyphenolic compound extracted from turmeric, is a chemotherapy used in some cancer treatments in China. However, the effect of curcumin on the survivability of GB cells remains to be elucidated. METHODS: We performed a CCK8 assay to detect the viability of GB cells following treatments with curcumin and examined the migration and invasion the ability of these cells using the wound-healing and transwell invasion assays. The cell proliferation and apoptotic proteins were detected by Western blot analyses. We utilized a glioblastoma-xenograft mouse model to assess cell proliferation following curcumin treatment. RESULTS: We found that curcumin inhibited the proliferation, migration, and invasion of U251 and U87 GB cells. We detected that curcumin decreased p-AKT and p-mTOR protein expression, and promoted the apoptosis of U251 and U87 GB cells. Further, we found that curcumin promoted the PTEN and p53 expression, as the tumor suppressor genes. In addition, we administered curcumin to nude mice and found that curcumin decreased the tumor volume, caused necrosis of tumor tissue, and significantly enhanced the PTEN and p53 expression in vivo. CONCLUSIONS: These results indicated that curcumin inhibited proliferation by decreasing the p-AKT/p-mTOR pathway and promoted apoptosis by increasing the PTEN and p53 expression. Our study provided the molecular mechanisms by which curcumin inhibited glioblastoma and its targeted interventions.

Captopril Attenuates the Upregulated Connexin 43 Expression in Artery Calcification.

OBJECTIVE: Vascular calcification is commonly observed in atherosclerosis and diabetes. The renin-angiotensin II system is associated with the regulation of arterial stiffening. The aim of this study was to examine whether the angiotensin-converting enzyme inhibitors captopril attenuates artery calcification. METHODS: The rat model of arterial calcification was established by a combination of warfarin and vitamin K1. Two weeks after the induction of arterial calcification, captopril treatment was initiated. One week after captopril treatment, aortic arteries were examined to determine the calcification morphology and the connexin 43 expression. Matrix Gla protein (MGP), receptor activator of nuclear factor-κB ligand (RANKL) and extracellular regulated protein kinase (ERK) pathways were examined. RESULTS: The morphology of the calcified arteries was significantly attenuated after captopril treatment. Consistently, captopril inhibited the increased connexin 43 expression and enhanced the decreased MGP expression in calcification arteries. Furthermore, captopril enhanced the decreased SM22 expression in calcified arteries by fluorescence assay. Finally, the calcification arteries increased the p38, p-ERK and RANKL expression, which were downregulated by captopril treatment. CONCLUSIONS: We concluded that captopril attenuated the increased connexin 43 expression and enhanced the MGP and SM22 expression levels, which are associated with the inactivation of p-ERK, p38 and RANKL pathways in rat aortic arteries.

Vildagliptin and G-CSF Improved Angiogenesis and Survival after Acute Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) is one of the most important diseases that has stimulated interest in understanding cardiac function recovery. SDF-1 is a chemotactic factor and a pro-angiogenic molecule; SDF-1 degradation is inhibited by dipeptidyl peptidase-4 (DPP4) inhibitors, such as vildagliptin. We investigated whether vildagliptin affects angiogenesis in MI and improves cardiac function recovery. METHODS: We established a therapeutic strategy using vildagliptin and G-CSF treatment to improve cardiac function recovery after MI in mice. RESULTS: Vildagliptin treatment increased the myocardial homing of circulating CXCR4+ stem cells and angiogenesis. The combination of vildagliptin and G-CSF treatment attenuated cardiac remodeling and improved survival and cardiac function after MI. Vildagliptin treatment induced active SDF-1, which preserved the cardiac SDF-1-CXCR4 homing axis for MI injury. CONCLUSION: Vildagliptin and G-CSF induced stem cell mobilization and increased angiogenesis as a therapeutic strategy for improving survival and cardiac function after MI.

Klotho inhibits angiotensin II-induced cardiac hypertrophy, fibrosis, and dysfunction in mice through suppression of transforming growth factor-ß1 signaling pathway.

Recent studies have revealed critical roles of transforming growth factor-ß1 (TGF-ß1) and microRNA-132 (miR-132), a downstream mediator of TGF-ß1, in the pathogenesis of cardiac remodeling. In this study, we tested whether the antiaging protein klotho modifies angiotensin II (Ang II)-induced cardiac remodeling through regulating TGF-ß1-miR-132 axis. We found that both klotho and the TGF-ß1 inhibitor LY364947 significantly inhibited cardiac hypertrophy, fibrosis, and dysfunction in Ang II-infused mice, as evidenced by the ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL), cardiomyocyte cross-sectional area, fibrotic area, and expression of prohypertrophic genes (ANP, ß-MHC) and fibrotic marker genes (α-SMA, collagen I), echocardiographic parameters. Meanwhile, klotho also significantly inhibited Ang II-induced protein expression of TGF-ß1 and phosphorylated Smad2/3 in the heart tissues and cultured cardiomyocytes and cardiac fibroblasts. In vitro experiments demonstrated that Ang II-induced cardiomyocyte hypertrophy and proliferation and activation of cardiac fibroblasts were markedly inhibited by klotho, LY364947 or the miR-132 inhibitor anti-miR-132. Both klotho and the TGF-ß1 inhibitor LY364947 downregulated the miR-132 expression. Additionally, klotho decreased Ang II-induced protein expressions of cardiac fibroblast growth factor (FGF)23 in vivo and in vitro. The decreased protein levels of klotho in serum and renal tissues of Ang II-infused mice were elevated by klotho. Klotho downregulated the protein levels of TGF-ß1 in renal tissues of Ang II-infused mice. In conclusion, our results suggest that klotho prevents Ang II-induced cardiac remodeling and dysfunction through modifying the TGF-ß1-miR-132 axis, providing an experimental basis for clinical treatment on cardiac remodeling.

SIRT1 activation by SRT1720 attenuates bone cancer pain via preventing Drp1-mediated mitochondrial fission.

Bone cancer pain (BCP) is the pain induced by primary bone cancer or tumor metastasis. Increasing evidence and our previous studies have shown that mammalian silent information regulator 2 homolog (SIRT1) is involved in periphery sensitization and central sensitization of BCP, and the underlying mechanism of SIRT1 in bone cancer pain may provide clues for pain treatment. Dynamin-related protein 1 (Drp1) is an essential regulator for mitochondrial fission. In this research, BCP model rats were established by injecting MRMT-1 rat mammary gland carcinoma cells into the left tibia of female Sprague-Dawley rats and validated by tibia radiographs, histological examination and mechanical pain test. As a result BCP rats exhibited bone destruction and sensitivity mechanical pain. BCP increased inflammatory cells infiltration and apoptosis, reduced SIRT1 protein expression and phosphorylation, and elevated Drp1 expression in spinal cord. An agonist of SIRT1 named SRT1720 intrathecal treatment in BCP rats increased SIRT1 phosphorylation, reduced the up-regulated Drp1 expression, and reversed pain behavior. SRT1720 also regulated Bcl-2/BAX and cleaved caspase-3 expressions, and inhibited mitochondrial apoptosis in spinal cord of BCP rats. For in vitro research, SRT1720 treatment decreased Drp1 expression in a dose-dependent manner, blocked CCCP-induced mitochondrial membrane potential change, consequently reduced apoptosis and promoted proliferation. These data suggest that SIRT1 activation by SRT1720 attenuated bone cancer pain via preventing Drp1-mediated mitochondrial fission. Our results provide new targets for therapeutics of bone cancer pain.

Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury.

OBJECTIVES: Abnormal lung cell death including autophagy and apoptosis is the central feature in acute lung injury (ALI). To identify the cellular mechanisms and the chronology by which different types of lung cell death are activated during lipopolysaccharide (LPS)-induced ALI, we decided to evaluate autophagy (by LC3-II and autophagosome) and apoptosis (by caspase-3) at different time points after LPS treatment in a rat model of LPS-induced ALI. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into two groups: control group and LPS group. ALI was induced by LPS intraperitoneal injection (3 mg/kg). The lung tissues were collected to measure lung injury score by histopathological evaluation, the protein expression of LC3-II and caspase-3 by Western blot, and microstructural changes by electron microscopy analysis. RESULTS: During ALI, lung cell death exhibited modifications in the death process at different stages of ALI. At early stages (1 hr and 2 hr) of ALI, the mode of lung cell death started with autophagy in LPS group and reached a peak at 2 hr. As ALI process progressed, apoptosis was gradually increased in the lung tissues and reached its maximal level at later stages (6 hr), while autophagy was time-dependently decreased. CONCLUSION: These findings suggest that activated autophagy and apoptosis might play distinct roles at different stages of LPS-induced ALI. This information may enhance the understanding of lung pathophysiology at the cellular level during ALI and pulmonary infection, and thus help optimize the timing of innovating therapeutic approaches in future experiments with this model.

Klotho inhibits angiotensin II-induced cardiomyocyte hypertrophy through suppression of the AT1R/beta catenin pathway.

Myocardial hypertrophy is an independent risk factor for cardiac morbidity and mortality. The antiaging protein klotho reportedly possesses a protective role in cardiac diseases. However, the precise mechanisms underlying the cardioprotective effects of klotho remain unknown. This study was aimed to determine the effects of klotho on angiotensin II (Ang II)-induced hypertrophy in neonatal rat cardiomyocytes and the possible mechanism of actions. We found that klotho significantly inhibited Ang II-induced hypertrophic growth of neonatal cardiomyocytes, as evidenced by decreased [(3)H]-Leucine incorporation, cardiomyocyte surface area and ß-myosin heavy chain (ß-MHC) mRNA expression. Meanwhile, klotho inhibited Ang II-stimulated activation of the Wnt/ß-catenin pathway in cardiomyocytes, as evidenced by decreased protein expression of active ß-catenin, downregulated protein and mRNA expression of the ß-catenin target genes c-myc and cyclin D1, and increased ß-catenin phosphorylation. Inhibition of the Wnt/ß-catenin pathway by the specific inhibitor XAV939 markedly attenuated Ang II-induced cardiomyocyte hypertrophy. The further study revealed that klotho treatment significantly downregulated protein expression of Ang II receptor type I (AT1R) but not type II (AT2R). The AT1R antagonist losartan inhibited Ang II-stimulated activation of the Wnt/ß-catenin pathway and cardiomyocyte hypertrophy. Our findings suggest that klotho inhibits Ang II-induced cardiomyocyte hypertrophy through suppression of the AT1R/ß-catenin signaling pathway, which may provide new insights into the mechanism underlying the protective effects of klotho in heart diseases, and raise the possibility that klotho may act as an endogenous antihypertrophic factor by inhibiting the Ang II signaling pathway.

[Effect of tanshinone II(A) on expression of different components in renin-angiotensin system of left ventricles of hypertensive rats].

OBJECTIVE: To investigate the effect of tanshinone II(A) on the expression of different components in the renin-angiotensin system of left ventricles of renal hypertensive rats. METHOD: The renal hypertension model was established in rats by the two-kidney-one-clip (2K1C) method. In the experiment, all of the rats were randomly divided into four groups (n = 15 per group) before the operation: the sham-operated (Sham) group, the hypertensive model (Model) group, the low-dose tanshinone II(A) group and the high-dose tanshinone II(A) group. At 5 week after the renal artery narrowing, the third and fourth groups were administered with 35 mg kg(-1) x d(-1) and 70 mg x kg(-1) x d(-1) of tanshinone II(A), respectively. The blood pressure in rats was determined by the standard tail-cuff method in each week after the operation. After the drug treatment for 8 weeks, all the rats were put to death, and their left ventricles were separated to determine the ratio of left ventricle weight to body weight (LVW/BW), the myocardial collagen content, and the expressions of different components in myocardial RAS, including angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE2), angiotensin 1-type receptor (AT1R), Mas receptor mRNA expression and angiotensin II (Ang II) and angiotensin (1-7) [Ang (1-7)] content. RESULT: Compared with the sham group, the hypertensive model group exhibited a markable increase in the content of Ang II and Ang (1-7) and the mRNA expressions of ACE, ACE2, AT1R and Mas (P < 0.01). However, the treatment with tanshinone II(A) showed the does dependence, inhibited left ventricle hypertrophy, decreased myocardial Ang II content and the mRNA expression of ACE and AT, R in renal hypertensive rats (P < 0. 01) , further increased the myocardial Ang (1-7) content and the mRNA expression of ACE2 and Mas (P < 0.01) , but without any change in the blood pressure of hypertensive rats. CONCLUSION: The treatment with tanshinone II(A) could inhibit left ventricle hypertrophy of renal hypertensive rats. Its mechanism may be partially related to the expression of different components in the renin-angiotensin system for regulating myocardial tissues.

Bone mesenchymal stem cells contributed to the neointimal formation after arterial injury.

OBJECTIVES: Recent findings suggest that in response to repair-to-injury bone marrow mesenchymal stem cells (BMSCs) participate in the process of angiogenesis. It is unclear what role BMSCs play in the structure of the vessel wall. In present study, we aimed to determine whether BMSCs had the capacity of endothelial cells (ECs). METHODS: BMSCs were separated and cultured. FACS and RT-PCR analysis confirmed the gene expression phenotype. The capacity of migration and adhesion and the ultrastructure of BMSCs were examined. The effect of BMSCs transplantation on the vascular repair was investigated in a murine carotid artery-injured model. RESULTS: BMSCs could express some markers and form the tube-like structure. The migration and adhesion capacity of BMSCs increased significantly after stimulated. In addition, BMSCs had the intact cell junction. In vivo the local transfer of BMSCs differentiated into neo-endothelial cells in the injury model for carotid artery and contributed to the vascular remodeling. CONCLUSION: These results showed that BMSCs could contribute to neointimal formation for vascular lesion and might be associated with the differentiation into ECs, which indicated the important therapeutic implications for vascular diseases.

Tetramethylpyrazine inhibits angiotensin II-induced cardiomyocyte hypertrophy and tumor necrosis factor-α secretion through an NF-κB-dependent mechanism.

Tetramethylpyrazine (TMP), a bioactive compound isolated from the Chinese herb, Ligusticum wallichii Franchat, has been reported to play a protective role in cardiac diseases. However, the cellular and molecular mechanisms behind the protective effects of TMP on the heart remain to be elucidated. In this study, we aimed to determine the effects of TMP on angiotensin II (Ang II)-induced hypertrophy in neonatal rat cardiomyocytes and its possible mechanisms of action. In addition, we investigated whether TMP regulates tumor necrosis factor-α (TNF-α) secretion and expression. We found that TMP significantly inhibited the Ang II-induced hypertrophic growth of neonatal cardiomyocytes, as evidenced by the decrease in [3H]leucine incorporation and ß-myosin heavy chain (ß-MHC) mRNA expression. TMP inhibited Ang II-stimulated TNF-α protein secretion and mRNA expression in the cardiomyocytes. Further experiments revealed that Ang II increased the level of the phosphorylated form of the transcription factor, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), as well as NF-κB-DNA binding activity in the cardiomyocytes; treatment with TMP significantly inhibited the Ang II-induced activation of NF-κB. Furthermore, the inhibition of NF-κB by the specific inhibitor, pyrrolidine dithiocarbamate (PDTC), markedly attenuated the Ang II-induced increase in [3H]leucine incorporation, ß-MHC mRNA expression and TNF-α protein secretion. Our findings suggest that TMP inhibits Ang II-induced cardiomyocyte hypertrophy and TNF-α production through the suppression of the NF-κB pathway, which may provide new insight into the mechanisms underlying the protective effects of TMP in heart diseases.