Epigallocatechin-3-gallate alleviates ethanol-induced endothelia cells injury partly through alteration of NF-κB translocation and activation of the Nrf2 signaling pathway.

Ethanol (alcohol) is a risk factor that contributes to non-communicable diseases. Chronic abuse of ethanol is toxic to both the heart and overall health, and even results in death. Ethanol and its byproduct acetaldehyde can harm the cardiovascular system by impairing mitochondrial function, causing oxidative damage, and reducing contractile proteins. Endothelial cells are essential components of the cardiovascular system, are highly susceptible to ethanol, either through direct or indirect exposure. Thus, protection against endothelial injury is of great importance for persons who chronic abuse of ethanol. In this study, an in vitro model of endothelial injury was created using ethanol. The findings revealed that a concentration of 20.0 mM of ethanol reduced cell viability and Bcl-2 expression, while increasing cell apoptosis, intracellular ROS levels, mitochondrial depolarization, and the expression of Bax and cleaved-caspase-3 in endothelial cells. Further study showed that ethanol promoted nuclear translocation of NF-κB, increased the secretion of TNF-α，IL-1ß, IL-6 in the culture medium, and inhibited Nrf2 signaling pathway. The aforementioned findings suggest that ethanol has a harmful impact on endothelial cells. Nevertheless, the application of epigallocatechin-3-gallate (EGCG) to the cells can effectively mitigate the detrimental effects of ethanol on endothelial cells. In conclusion, EGCG alleviates ethanol-induced endothelial injury partly through alteration of NF-κB translocation and activation of the Nrf2 signaling pathway. Therefore, EGCG holds great potential in safeguarding individuals who chronically abuse ethanol from endothelial dysfunction.

Transcutaneous Auricular Vagus Nerve Stimulation Ameliorates Preeclampsia-Induced Apoptosis of Placental Trophoblastic Cells Via Inhibiting the Mitochondrial Unfolded Protein Response.

Preeclampsia is a serious obstetric complication. Currently, there is a lack of effective preventive approaches for this disease. Recent studies have identified transcutaneous auricular vagus nerve stimulation (taVNS) as a potential novel non-pharmaceutical therapeutic modality for preeclampsia. In this study, we investigated whether taVNS inhibits apoptosis of placental trophoblastic cells through ROS-induced UPRmt. Our results showed that taVNS promoted the release of acetylcholine (ACh). ACh decreased the expression of UPRmt by inhibiting the formation of mitochondrial ROS (mtROS), presumably through M3AChR. This reduced the release of pro-apoptotic proteins (cleaved caspase-3, NF-κB-p65, and cytochrome C) and helped preserve the morphological and functional integrity of mitochondria, thus reducing the apoptosis of placental trophoblasts, improving placental function, and relieving preeclampsia. Our study unravels the potential pathophysiological mechanism of preeclampsia. In-depth characterization of the UPRmt is essential for developing more effective therapeutic strategies for preeclampsia targeting mitochondrial function.

Icariin ameliorates osteoporosis by activating autophagy in ovariectomized rats.

BACKGROUND: Osteoporosis (OP) is a major problem that increases the mortality and disability rate worldwide. With an increase in the aging population, OP has become a major public threat to human health. Searching for effective and suitable targets for drug treatment in OP has become an urgent need. OBJECTIVES: Osteoporosis is a metabolic bone disease characterized by reduced bone mass and density as well as micro-architectural deterioration. Icariin is a flavonoid extracted from plants of the genus Epimedium and has been shown to exert potential anti-OP activity. The present study was designed to observe the effect of icariin on OP and to clarify the underlying mechanisms in ovariectomized (OVX) rats. MATERIAL AND METHODS: Hematoxylin and eosin (H&E) staining, von Kossa staining and micro-computed tomography (micro-CT) confirmed significant bone loss in the OVX group. Protein expression level was detected with western blot analysis. RESULTS: Icariin reversed a trend of increased bone turnover by reducing serum alkaline phosphatase (ALP), procollagen type I N-terminal propeptide (PINP), tartrate-resistant acid phosphatase isoform 5b (TRACP-5b), and C-telopeptide of type I collagen (CTX-I). Furthermore, icariin decreased sequestosome 1 (p62) and increased microtubule-associated protein 1 light chain 3II/microtubule-associated protein 1 light chain 3I (LC3II/LC3I), autophagy-related protein 7 (Atg7), and Beclin 1 in the femur of OVX rats, improving the indicators of impaired autophagy in OP. CONCLUSIONS: Icariin reversed the significant upregulation of the serine/threonine protein kinase (Akt), mammalian target of rapamycin (mTOR) and unc-51-like autophagy activating kinase 1 (ULK1) at Ser757, and the downregulation of p-AMP-activated protein kinase (p-AMPK) and ULK1 phosphorylated at Ser555 in the OVX rats, suggesting that the mechanism of icariin action in OP treatment involves the activation and suppression of the AMPK/ULK1 and AKT/mTOR/ULK1 autophagy pathways, respectively.

Paeonol alleviates lipopolysaccharide­induced hepatocytes injury through alteration of mitochondrial function and NF­κB translocation.

Sepsis is a severe disease, with high mortality. Permanent organ damage caused by sepsis reduces the quality of life of surviving patients. The liver is an easily damaged organ in sepsis and sepsis­associated liver injury foretells a poor prognosis. Unfortunately, there are no effective treatments or drugs to solve this problem. Therefore, strategies or novel drugs are urgently required to protect against liver dysfunction in sepsis. In the present study, lipopolysaccharide (LPS) was used to establish a model of liver injury in vitro. The data demonstrated that pretreatment of L02 human normal hepatocytes with paeonol (PAE) alleviated LPS­induced cell injury and decreased the levels of alanine aminotransferase and aspartate transaminase, indicating a protective effect of PAE. Further experiments demonstrated that PAE increased LPS­decreased L02 cell viability, the levels of superoxide dismutase and Bcl­2 expression. PAE decreased LPS­increased cell apoptosis, intracellular reactive oxygen species and the expression levels of Bax and cleaved­caspase­3. PAE decreased LPS­promoted mitochondrial depolarization and nuclear translocation of NF­κB. In conclusion, PAE alleviated LPS­induced liver injury via alteration of mitochondrial function and NF­κB translocation. Therefore, PAE has potential for the treatment of sepsis.

Septic serum mediates inflammatory injury in human umbilical vein endothelial cells via reactive oxygen species, mitogen activated protein kinases and nuclear factor­κB.

Sepsis­induced blood vessel dysfunction is mainly caused by microvascular endothelial cell injury. However, the mechanism underlying sepsis­induced endothelial cell injury remains unclear. The present study hypothesized that sepsis­induced inflammatory injury of endothelial cells may be the first step of endothelial barrier dysfunction. Therefore, the present study aimed to uncover the mechanism underlying the inflammatory effects of sepsis. A rat model of cecal ligation and puncture­induced sepsis was established, and septic serum was collected. Subsequently, human umbilical vein endothelial cells (HUVECs) were treated with the isolated septic or normal serum. HUVEC viability was assessed using a Cell Count Kit­8 assay. Furthermore, transmission electron microscopy and reverse transcription­quantitative PCR (RT­qPCR) analysis were carried out to observe the cell morphology and determine the mRNA expression levels in septic serum­induced HUVECs. The protein expression levels were evaluated by western blot analysis, and the secretion of the inflammatory factors interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α was determined by ELISA. Additionally, reactive oxygen species (ROS) generation and nuclear factor (NF)­κB nuclear translocation were observed under a fluorescence microscope. The results of the present study demonstrated that HUVEC viability was significantly decreased following 12­ or 24­h treatment with septic serum. In addition, chromatin condensation, mitochondrial vacuolization and endoplasmic reticulum degranulation were observed following treatment with septic serum. Furthermore, the secretion levels of IL­1ß, IL­6 and TNF­α were increased in septic serum­stimulated HUVECs. Septic serum treatment also enhanced superoxide anion generation, promoted extracellular signal regulated kinase 1/2 (ERK1/2), N­terminal kinase (JNK) and p38 mitogen­activated protein kinase (p38) phosphorylation, and increased NF­κB levels in the nuclei of HUVECs. Finally, pre­treatment of HUVECs with the antioxidant N­acetylcysteine, the ERK1/2 inhibitor PD98059, the p38 inhibitor SB203580, the JNK inhibitor SP610025 or the NF­κB inhibitor pyrrolidine dithiocarbamate restored the septic serum­induced IL­1ß, IL­6 and TNF­α expression. In conclusion, the results of the current study suggested that the septic serum­induced endothelial cell injury may be mediated by increasing ROS generation, activation of mitogen­activated protein kinases and NF­κB translocation.

Curcumin Suppresses Aldosterone-Induced CRP Generation in Rat Vascular Smooth Muscle Cells via Interfering with the ROS-ERK1/2 Signaling Pathway.

Aldosterone regulates the initiation and development of atherosclerosis which is identified as a chronic inflammatory disease by promoting the generation of C-reactive protein in vascular smooth muscle cells. Curcumin is the most active ingredient of turmeric with anti-inflammation and antioxidation effects. Here, the effect of curcumin on aldosterone-induced C-reactive protein generation in vascular smooth muscle and the molecular mechanisms involved were explored. Primary rat vascular smooth muscle cells and hyperaldosteronism model rats were used in this study. The amount of C-reactive protein, reactive oxygen species, and the signaling pathway-related molecules generated were estimated. We found that curcumin inhibited aldosterone-induced C-reactive protein generation in vascular smooth muscle cells by interfering with the reactive oxygen species-ERK1/2 signal pathway. The results provide new evidence for the potential anti-inflammatory and cardiovascular protective effects of curcumin.

Prognostic significance of peritumoural and intratumoural budding in intestinal-type gastric adenocarcinoma.

BACKGROUND AND STUDY AIMS: Tumour budding (TB) at the invasive front, termed peritumoural budding (PTB), is an established prognostic factor for many solid tumours. However, intratumoural budding (ITB) in gastric adenocarcinoma (GAC), which is frequently observed at the tumour centre, particularly in tumour biopsy tissues, remains poorly understood. Hence, we aimed to determine the correlation of ITB with PTB and their connection with clinicopathological characteristics as well as their prognostic value in GAC. PATIENTS AND METHODS: We investigated a total of 153 cases of GAC wherein tissues were primarily resected and their related clinicopathological data. A continuous series of paraffin-embedded tissues was stained by haematoxylin-eosin staining, and budding cells were identified by immunohistochemical staining. PTB and ITB were counted in five fields with the highest density of tumour buds. The selected area was examined under 40 high-power field. Cases were divided into low-grade TB and high-grade TB groups according to the median bud count. RESULTS: Among the 153 patients with GAC, 51 underwent simultaneous observation of ITB and PTB, which were found to have a significant positive correlation. A higher grade of ITB in tumours was associated with positive lymph node metastasis and could predict a worse prognosis. Additionally, patients with simultaneous PTB and ITB had a shorter overall survival than those with PTB alone, suggesting a worse prognosis. CONCLUSION: PTB and ITB were found to be adverse prognostic factors and high-risk indicators of intestinal-type GAC, and ITB plays an important role in evaluating GAC prognosis in gastroscopic biopsy tissues. Additionally, TB might become a useful index for predicting GAC prognosis in the future.

Proapoptotic effects of 2,5­hexanedione on pheochromocytoma cells via oxidative injury.

N­hexanes are prominent environmental pollutants that are able to cause neurotoxicity in vivo and in vitro. Central and peripheral neuropathies induced by n­hexane exposure are a major health concern. 2,5­Hexanedione (2,5­HD) is the most significant neurotoxic metabolite of n­hexane; however, little is known regarding the underlying mechanism of its neurotoxicity. Thus, the aim of the present study was to investigate the damaging effects of 2,5­HD on pheochromocytoma PC12 cells, and to explore the underlying mechanism. Cell viability was tested using a Cell Counting Kit­8 method, and the leakage of lactate dehydrogenase (LDH) from cells was measured using an LDH assay kit. Glutathione peroxidase (GSHPx) and superoxide dismutase (SOD) activities, and the level of malondialdehyde (MDA) were determined using corresponding assay kits. Apoptotic cells were detected using an annexin V­fluorescein isothiocyanate/propidium iodide (PI) apoptosis kit, and were subsequently observed by fluorescence microscopy. The relative expression levels of cleaved­caspase­3, Bcl­associated­X protein (Bax) and Bcl­2 were identified by western blotting. The results revealed that 2,5­HD was able to decrease the viability of PC12 cells and promoted the leakage of LDH in a concentrationdependent manner. Further analysis demonstrated that 2,5­HD decreased the activity of the antioxidative enzymes, SOD and GSHPx, and led to an increase in the levels of MDA in the supernatant of cultured PC12 cells. The annexin V/PI staining results revealed that the numbers of apoptotic cells were increased following treatment with 2,5­HD. In addition, 2,5­HD (5 and 10 mmol/l) led to significant increases in the expression levels of caspase­3 and Bax, with the concomitant downregulation of Bcl­2. The antioxidant N­acetylcysteine was identified to antagonize 2,5­HD­stimulated cleaved­caspase­3 and Bax upregulation, and Bcl­2 downregulation. Collectively, the results of the present study suggested that 2,5­HD exerts proapoptotic effects on PC12 cells via oxidative injury. These findings may be applied in the development of novel therapeutic strategies to treat neurological disorders associated with nhexane exposure.

Fibrinopeptide A induces C-reactive protein expression through the ROS-ERK1/2/p38-NF-κB signal pathway in the human umbilical vascular endothelial cells.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. Inflammation causes endothelial injury and dysfunction, which is an initial step of atherosclerosis. Fibrinopeptide A (FPA) is a biomarker of the activation of the coagulation system, and a high concentration of FPA in the blood occurs in patients with ischemic cardiocerebrovascular diseases. The present research observed that FPA stimulated the generation of C-reactive protein (CRP), IL-1ß, and IL-6 in human umbilical vascular endothelial cells (HUVECs); and anti-IL-1 ß and anti-IL-6 neutralizing antibodies did not alter FPA-induced CRP expression in HUVECs. The subchronic administration of FPA into rats increased the plasma FPA and CRP levels. Further studies showed that FPA stimulated superoxide anion generation, activated ERK1/2 and p38, promoted nuclear factor κB (NF-κB) nuclear translocation, and raised the NF-κB level in the nuclei of HUVECs. Antioxidant N-acetylcysteine (NAC), complex II inhibitor thenoyltrifluoroacetone (TTFA), and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited FPA-stimulated generation of superoxide anion, and NAC reduced FPA-induced expressions of the phosphorylated ERK1/2 and p38. NAC, TTFA, DPI, inhibitors of ERK1/2, p38, and NF-κB all downregulated FPA-induced CRP expression. These results indicate that FPA induces CRP expression in HUVECs via the ROS-ERK1/2/p38-NF-κB signal pathway. Moreover, this is the first report that FPA produces a proinflammatory effect on the vascular endothelial cells.

Rosmarinic acid inhibits nicotine-induced C-reactive protein generation by inhibiting NLRP3 inflammasome activation in smooth muscle cells.

Atherosclerosis is widely known to be a chronic inflammatory disease. C-reactive protein (CRP), an important inflammatory factor, plays an essential role in the pathogenesis of atherosclerosis. Nicotine, the main addictive component of cigarette, has been shown to induce the production of CRP. The aim of this study was to investigate the effect of rosmarinic acid (RA), a polyphenol with antiinflammatory activity, on nicotine-induced elevation of CRP in vascular smooth muscle cells (VSMCs). We found that pretreatment of VSMCs with RA attenuated nicotine-induced expression of CRP in a time- and dose-dependant manner. In addition, RA also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and reactive oxygen species (ROS) production resulting from nicotine treatment in VSMCs. To confirm these findings in vivo, we constructed a nicotine-induced atherosclerosis rat model. RA did not significantly reduce the serum nicotine level of the rats, whereas it significantly decreased the levels of serum lipids, including concentrations of cholesterol, triglycerides, and low-density lipoprotein cholesterol, and the serum level of CRP. RA also led to diminished nicotine-induced activation of NLRP3 inflammasome and elevation in the CRP level in the aortic tissue of the model rats. The results of this study suggested a protective role of RA in nicotine-induced atherosclerosis by inhibiting the ROS-NLRP3 inflammasome-CRP axial, and RA therefore represented a potential effective therapeutic approach to atherosclerosis, in particular for those who smoke.

Genistein inhibits Ang II-induced CRP and MMP-9 generations via the ER-p38/ERK1/2-PPARγ-NF-κB signaling pathway in rat vascular smooth muscle cells.

AIMS: C-reactive protein (CRP) and matrix metalloproteinase (MMP)-9 are involved in the inflammation of atherosclerosis lesions. Genistein (Gen) has been demonstrated to exert beneficial effect on the cardiovascular system. However, it remains unclear whether Gen produces anti-inflammatory effect in vascular smooth muscle cells (VSMCs). Therefore, we investigated the effects of Gen on CRP and MMP-9 expressions induced by angiotensin (Ang) II in VSMCs and the related molecular mechanism. MAIN METHODS: Rat VSMCs were cultured, and Ang II was used as a stimulant for CRP and MMP-9 expressions. CRP level was measured by ELISA. The mRNA and protein expressions of related indexes were identified by reverse transcription-polymerase chain reaction and western blot, respectively. KEY FINDINGS: Gen inhibited Ang II-stimulated CRP and MMP-9 mRNA and protein expressions in concentration- and time-dependent manners. Additionally, Gen ameliorated Ang II-induced p-ERK1/2, p-p38 and NF-κB expressions, antagonized Ang II-downregulated peroxisome proliferation-activated receptor (PPAR) γ and estrogen receptor (ER) ß expressions. After treating the VSMCs with GW9662 or ICI182780 in Gen treated groups, inhibitory effect of Gen on CRP and MMP-9 expressions were antagonized in Ang II-stimulated VSMCs. The treatment of VSMCs with ICI182780 abolished downregulations of p-p38/p-ERK1/2, and antagonized upregulation of PPARγ by Gen in Ang II-stimulated VSMCs. Moreover, the inhibitory effect of Gen on Ang II-stimulated NF-κB expression was abolished after preincubation of VSMCs with GW9662 in Gen treated groups. SIGNIFICANCE: Gen exerts anti-inflammatory property via the ER-p38/ERK1/2-PPARγ-NF-κB-CRP/MMP-9 signal pathway in Ang II-stimulated VSMCs.

Resveratrol alleviates ethanol-induced neuroinflammation in vivo and in vitro: Involvement of TLR2-MyD88-NF-κB pathway.

Excessive ethanol (EtOH) intake affects cognitive function and leads to permanent learning and memory deficits. EtOH-induced neuroinflammation plays an important role in EtOH neurotoxicity. Studies have shown that EtOH activates microglia and induces an inflammatory response. Resveratrol (Rsv) is a natural polyphenol found in a wide variety of plants and fruits, and produces the neuroprotective and anti-inflammatory effects in the central nervous system. However, effect of Rsv on EtOH-induced neuroinflammation is still unknown. We investigated the anti-inflammatory effect of Rsv in the context of EtOH-induced neurotoxicity and the molecular mechanisms potentially involved in the effect. The results showed that treatment of rats with Rsv prevented the deficits of spatial reference memory induced by EtOH and mitigated EtOH-induced neuroinflammation by inhibiting microglial activation and decreasing the levels of inflammatory cytokines including interleukin-1ß, interleukin-6 and tumor necrosis factor α. The further studies indicated that Rsv reduced TLR2 expression in vivo and in vitro, and downregulated expression of myeloid differentiation primary response 88 (MyD88) and phosphorylation of nuclear factor κB (NF-κB). These data demonstrate that Rsv alleviates the ethanol-induced neuroinflammation via inhibition of TLR2-MyD88-NF-κB signal pathway.

Fibrinopeptide A Induces Expression of C-Reactive Protein via the ROS-ERK1/2/ P38-NF-κB Signal Pathway in Vascular Smooth Muscle Cells.

BACKGROUND/AIMS: Atherosclerosis is a chronic inflammatory disease in the artery walls. Fibrinopeptide A (FPA) is a biomarker of the activation of coagulation system, and a high concentration of FPA in blood occurs in patients with ischemic heart disease etc. However, there exist few studies on the pathological effects of FPA in cardiovascular system. Therefore, the present study examined the effect of FPA on CRP expression in VSMCs and the molecular mechanisms. METHODS: mRNA and protein expression was identified by quantitative real-time PCR and Western blot, respectively. Reactive oxygen species (ROS) and the immunofluorescence staining were observed by a fluorescence microscope. Plasma FPA and CRP level was determined by ELISA. RESULTS: FPA induced the expressions of CRP, IL-1ß and IL-6 in VSMCs, and anti-IL-1ß and anti-IL-6 neutralizing antibodies partially reduced FPA-induced CRP expression in VSMCs. The subchronic administration of FPA to rats increased FPA level in plasma and CRP expression in the aortic artery walls. The further studies showed that FPA promoted superoxide anion generation in VSMCs. Antioxidant NAC antagonized FPA-stimulated superoxide anion generation and inhibited FPA-induced CRP expression in VSMCs. FPA activated ERK1/2 and p38 phosphorylation, and PD98059 and SB203580 reduced FPA-induced CRP expression. Moreover, NAC inhibited the activation of ERK1/2 and p38. In addition, FPA enhanced NF-κB level in the nuclei of VSMCs, and PDTC reduced FPA-induced expression of CRP. CONCLUSIONS: FPA induces CRP expression in VSMCs via ROS-ERK1/2/p38-NF-κB signal pathway. This finding for the first time provides an experimental evidence for pro-inflammatory effect of FPA.

Simvastatin inhibits homocysteine-induced CRP generation via interfering with the ROS-p38/ERK1/2 signal pathway in rat vascular smooth muscle cells.

Inflammation plays a pivotal role throughout the formation and progression of atherosclerosis. As the most representative inflammatory marker, C-reactive protein (CRP) directly participates in the initiation and development of atherosclerosis. The elevated homocysteine (Hcy) level in plasma is an independent risk factor for atherosclerosis. We previously reported that Hcy produces a pro-inflammatory effect by stimulating CRP expression in vascular smooth muscle cells (VSMCs). The present study observed the effect of simvastatin on Hcy-induced CRP expression in VSMCs and the molecular mechanisms. The in vitro experiments revealed that pretreatment of VSMCs with simvastatin decreased Hcy-induced mRNA and protein expression of CRP in a concentration-dependent fashion. The in vivo results showed that simvastatin not only inhibited CRP expression in the vessel walls in mRNA and protein levels, but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further experiments displayed that simvastatin reduced Hcy-induced reactive oxygen species (ROS) generation, ameliorated Hcy-activated phosphorylations of ERK1/2 and p38, and antagonized Hcy-downregulated peroxisome proliferator-activated receptor gamma expression in VSMCs. These data demonstrate that simvastatin is able to inhibit Hcy-induced CRP generation in VSMCs so to relieve the vascular inflammatory response via interfering with the ROS-MAPK signal pathway. The present results provide new evidence for understanding of the potential anti-inflammatory and anti-atherosclerotic effects of simvastatin. As the high level of Hcy in plasma is related to atherosclerosis formation and mediates cardiovascular risk, our findings emphasize the importance and necessity of therapy with statins for hyperhomocysteinemia in atherosclerosis.

Inhibitory effects of Shenkang injection and its main component emodin on the proliferation of high glucose­induced renal mesangial cells through cell cycle regulation and induction of apoptosis.

Increased mesangial cell proliferation is a major pathological feature of early-stage diabetic nephropathy (DN). The present study investigated the effects of the Traditional Chinese Medicine Shenkang injection (SKI) and its main component emodin (EM) on high glucose­cultured mesangial cells. The proliferation rate, cell cycle distribution, apoptosis and morphology of rat renal mesangial cells (RMCs) cultured in the presence of various concentrations of glucose (5.6 or 25 mM), SKI (25, 50 or 100 mg/l) or EM (10, 20 or 40 µM) were assessed at time­points of 12, 24 or 48 h. High­glucose treatment promoted the proliferation of RMCs, which was significantly inhibited by SKI and EM, while these drugs had no effect on RMCs under normal glucose conditions, as indicated by an MTT assay. Furthermore, flow cytometric analysis revealed that SKI and EM inhibited the cell cycle progression of RMCs and induced apoptosis. Transmission electron microscopy revealed morphological characteristics of apoptosis and western blot analysis demonstrated the upregulation of B­cell lymphoma 2­associated X protein (bax) and activation of caspases in RMCs following treatment with SKI or EM under high­glucose conditions. In conclusion, SKI and its major active component EM were shown to inhibit high­glucose­induced proliferation of RMCs via inducing cell cycle arrest at G1 phase as well as cellular apoptosis via upregulation of pro­apoptotic mediators bax and caspase activation, and may therefore be suitable for the treatment of DN.

The Inhibitory Effect of Rhein on Proliferation of High Glucose-induced Mesangial Cell Through Cell Cycle Regulation and Induction of Cell Apoptosis.

OBJECTIVES: Increased mesangial cell proliferation and accumulation of extracellular matrix (ECM) are the major pathological features of early-stage diabetic nephropathy. This study was sought to investigate the inhibitory effects of rhein (RH) on high glucose (HG)-cultured mesangial cells. Specially, we focus on the analysis of proliferation rate, cell cycle regulation, apoptosis, and the expression of collagen IV and laminin. MATERIALS AND METHODS: The established rat renal mesangial cell (RMC) line was cultured in medium with different concentrations of glucose (5.6 mM or 25 mM) and RH (40 µM, 20 µM, and 10 µM). Pro-treated cells were collected at 12 h, 24 h, and 48 h for cell proliferation analysis and after 24 h for the experiments of flow cytometry, transmission electron microscope, real-time polymerase chain reaction, and Western blotting. RESULTS: Our data shows HG can promote the proliferation of RMCs and RH has an inhibitory effect on HG-induced RMC proliferation and expression of ECM. Based on our data, we hypothesize this inhibitory effect might be a result of cell cycle regulation and the induction of cellular apoptosis. CONCLUSION: RH can inhibit cellular proliferation and downregulate the expression of ECM under the circumstance of HG. The mechanism of growth suppression may be due to cell cycle arrest at G1 phase, induction of cell apoptosis, and upregulation of apoptotic mediators bax and caspase-3. SUMMARY: Rhein (RH) has an inhibitory effect on high glucose.induced rat mesangial cells proliferationRH has an inhibitory effect on the expression of extracellular matrixRH has a growth.suppression effectRH can upregulate the expression of apoptotic mediators bax and caspase-3All above shows RH is one of the main active ingredient in Shenkang injection. Abbreviations used: RH: Rhein, ECM: Extracellular matrix, DN: Diabetic nephropathy, RMC: Renal mesangial cell, SKI: Shenkang injection, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

TSG attenuates LPC-induced endothelial cells inflammatory damage through notch signaling inhibition.

Lysophosphatidylcholine (LPC) induces inflammation in endothelial cells (ECs) but the mechanism is not fully understood. The Notch signaling pathway is involved in chronic EC inflammation, but its functions in LPC-induced endothelial inflammatory damage and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside's (TSG) protective effect during LPC-induced inflammatory damage in human umbilical vein endothelial cells (HUVECs) is largely unknown. We report that Notch signaling activation contributed to LPC-induced injury in HUVECs, and that TSG protected HUVECs from LPC-induced injury by antagonizing Notch signaling activation by LPC. γ-secretase inhibitor (DAPT), a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to inhibit Notch activity. HUVECs were exposed to LPC in the presence or absence of TSG, DAPT, and Notch1 siRNA. LPC treatment of HUVECs resulted in reduced cell viability, and Notch1 and Hes1 upregulation. Either silencing of Notch1 by siRNA or pharmacological inhibition of Notch signaling by DAPT prevented the loss of cell viability, and induction of apoptosis, and enhanced expression Notch1, Hes1 and MCP-1 by LPC in HUVECs. Similarly, TSG reduced LPC stimulation of Notch1, Hes1, and MCP-1 expression, prevented the release of IL-6 and CRP and rescued HUVECs from LPC-induced cell damage. Our data indicate that the Notch signaling pathway is a crucial mediator of endothelial inflammatory damage and that TSG protects against endothelial inflammatory damage by inhibiting the Notch signaling pathway. Our findings suggest that targeting Notch signaling by natural products such as TSG is a promising strategy for the prevention and treatment of chronic inflammation associated diseases, including atherosclerosis. © 2015 IUBMB Life, 68(1):37-50, 2016.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside protects human umbilical vein endothelial cells against lysophosphatidylcholine-induced apoptosis by upregulating superoxide dismutase and glutathione peroxidase.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside (TSG) has been shown to protect human umbilical vein endothelial cells (HUVECs) from lysophosphatidylcholine (LPC)-induced injury; however, the underlying molecular mechanism remains to be determined. The aim of this study was to investigate the protective mechanism of TSG against LPC-induced injury in HUVECs. We established a stable LPC-induced cell model by treating HUVECs with various concentrations of LPC and found 10.0 µg/mL of LPC to be optimal for inducing HUVECs injury. The effects of TSG on LPC-induced cell injury were assessed by cell counting kit-8, apoptosis assay, transmission electron microscope, and measurement of malondialdehyde (MDA), the antioxidant enzymes superoxide dismutase (SOD), reactive oxygen species (ROS), glutathione peroxidase, and mitochondrial membrane potential. The mRNA and protein levels of caspase-3, Bax, Bcl-2, PARP-1, and cytochrome C were assayed by real-time reverse transcriptase-polymerase chain reaction and immunoblotting, respectively. TSG pretreatment was able to prevent LPC-induced HUVECs injury and restore cell viability in a concentration-dependent manner. LPC treated cells showed typical apoptotic morphological changes including cytoplasmic vacuolation, swollen mitochondria, and characteristic biochemical hallmarks of apoptosis including loss of mitochondrial membrane potential, activation of caspase-3, decrease of Bcl-2, increase of PARP-1, upregulation of Bax, and release of cytochrome C, all of which were apparently inhibited by TSG pretreatment. Treatment of HUVECs with LPC led to decrease of SOD and glutathione peroxidase in addition to rapid increase of MDA and ROS levels. Pretreatment with TSG restored SOD and glutathione peroxidase levels to that of normal levels, and significantly decreased ROS and MDA levels. Our data indicate that TSG inhibits apoptosis of HUVECs mediated by LPC through blocking the mitochondrial apoptotic pathway and suggest that the mechanisms underlying the protective effects of TSG are related to the activation of SOD and glutathione peroxidase, the clearance of intracellular ROS, and reduction of lipid peroxidation.