Role Of Prohibitin In Endothelial Cell Apoptosis Caused By Glycated Low-density Lipoproteins

Part OneRole of prohibitin in endothelial cell apoptosis caused by glycated low-density lipoproteinsBackgroundWith the improvement of living standards and changes in diet, there are more patients of diabetes mellitus. Long-term hyperglycemia and metabolic disorders caused by diabetes lead to serious complications, among which vascular complications are the major cause of mortality and morbidity in patients with diabetes. Vascular complications of diabetes can be divided into two categories:macro-vascular complications and micro-vascular complications. Diabetes is a major risk factor for myocardial infarction, stroke and chronic renal disease, not only affecting patients’ quality of life, but also bringing huge economic burden to society. Therefore, to explore the pathogenesis of diabetic vascular complications and to find effective treatments for diabetes are of great importance.Diabetes is characterized by hyperglycemia and dyslipidemia. Hyperglycemia causes non-enzymatic glycosylation of proteins from plasma and tissue, leading to production of glycation end-products (AGEs). Low-density lipoproteins (LDL) are carriers of cholesterol in the blood. Elevated level of LDL is a known risk factor for coronary heart disease. In diabetes, LDL are irreversibly glycated by chronic hyperglycemia. High levels of glycated LDL (glyLDL) are detected in diabetic patients and contribute to accelerated atherosclerosis. Endothelium is a mono-cellular barrier located between blood flow and other tissue. Endothelial dysfunction is a critical step in the pathophysiology of atherosclerosis. Under pathological conditions of atherosclerosis, endothelial cells are activated, inducing local vascular inflammation and leading to plaque formation. Large attention was attached to glyLDL-caused thrombosis, lipid loading and pro-inflammation. However, the pro-apoptotic potential and apoptotic mechanism of glyLDL on endothelial cells have hardly been documented.Prohibitins (PHBs) are a family of evolutionarily conserved proteins, including prohibitin (PHB or PHB1) and prohibitin 2 (PHB2), both of which comprise a large protein complex at the inner membrane of mitochondria and exert multiple biological functions related to mitochondria. PHB is reported to be involved in various cellular processes including cell cycle control, differentiation, apoptosis and senescence. Our previous study showed that PHB was implicated in endothelial dysfunction caused by AGEs. Here in this study, we aimed to study the interactions among PHB, glyLDL and endothelial apoptosis. Meanwhile, we used grape seed procyanidin B2 (GSPB2) as treatment, to investigate its effects on endothelial cells. This study is of great importance to the prevention and treatment of diabetic vascular complications.Objectives1. To explore effects of glyLDL on endothelial viability and apoptosis, and the protective effects of GSPB2.2. To further study role of PHB in endothelial cell apoptosis and the potential mechanism of GSPB2.Methods1. Human umbilical vein endothelial cells (HUVEC) were cultured in RPMI 1640 medium containing 10% FBS. Cell treatment and groups:untreated HUVEC used as normal control (CC), HUVEC treated with 50μg/ml glyLDL for 48 h (glyLDL), HUVEC pre-incubated with 10μmol/l GSPB2 followed by treatment with 50μg/ml glyLDL for 48 h (glyLDL+GSPB2). CCK-8 and TUNEL assay were used to detect the viability and apoptosis of HUVEC. Electron microscope was used to examine the ultrastructural morphology of HUVEC. Western Blot was used to determine the protein expression of PHB.2. PHB overexpression plasmids and knock-down siRNAs were constructed. Empty vector pEGFP-C1 and negative siRNA were used as negative control for each. Cell treatment and groups:untreated HUVEC used as normal control (CC), HUVEC treated with 50μg/ml glyLDL for 48 h (glyLDL), HUVEC pre-incubated with 1 Oμmol/1 GSPB2 or transfected with PHB overexpression plasmids or empty vector followed by treatment with 50μg/ml glyLDL for 48 h (glyLDL+GSPB2, glyLDL+VP, glyLDL+V), HUVEC transfected with negative control siRNA (NC), HUVEC transfected with PHB siRNA followed by incubation with or without GSPB2 for 48 h (siPHB, siPHB+GSPB2). CCK-8 and TUNEL assay were used to detect the viability and apoptosis of HUVEC. Electron microscope was used to examine the ultrastructural morphology of mitochondria. Western Blot was used to determine the efficiency of transfection, protein expression of Bax/Bcl-2 and p-Akt.Results1. Effects of glyLDL and GSPB2 on cell viabilityAfter exposure to glyLDL (50μg/ml) for 48 h, cell viability decreased nearly 50% compared to CC (P< 0.05). Pre-incubation with GSPB2 (10μmol/L) remarkably attenuated the viability decrease caused by glyLDL (P< 0.05).2. Effects of glyLDL and GSPB2 on cell apoptosisTreatment with glyLDL for 48 h resulted in a significant increase in cell apoptosis, with condensate and fragmented nucleus, whereas GSPB2 significantly attenuated glyLDL-induced apoptosis (P< 0.05).3. Effects of glyLDL and GSPB2 on ultrastructural morphology of HUVECIn CC group, most cells showed normal mitochondria with lamellar cristae. GlyLDL group showed aberrant mitochondria, either swollen or fragmented. Regular lamellar cristae were almost completely lost and vesicular or high-density structures were detected within mitochondria. These alterations were significantly ameliorated in glyLDL+GSPB2 group (P< 0.05).4. Effects of glyLDL and GSPB2 on expression of PHBWestern Blot showed that protein expression of PHB was significantly decreased after glyLDL treatment, compared with untreated cells (P< 0.05). While GSPB2 treatment significantly normalized protein level of PHB (P< 0.05).5. Transfection efficiency of PHB overexpression plasmid and siRNATransfection efficiency was evaluated by Western Blot. Protein expression of PHB remarkably elevated at 48 h after transfection with VP (P< 0.05), while transfection with empty vector pEGFP-C1(V) did not show any change in levels of PHB. Conversely, siRNA successfully knocked down PHB by more than 60%(P< 0.05) whereas negative control siRNA (NC) had no significant influence.6. Role of PHB on cell viabilityPHB overexpression notably inhibited the glyLDL-caused viability reduction (P<0.05) while transfection with empty vector had no influence. Moreover, PHB silencing decreased cell viability while GSPB2 alleviated these reductions (P< 0.05).7. Role of PHB on cell apoptosisPHB overexpression significantly reduced glyLDL-induced apoptosis compared with glyLDL+V group (P< 0.05). Moreover, HUVEC with PHB knockdown exhibited a remarkably high percentage of apoptosis while GSPB2 ameliorated these changes, compared with siPHB group (P< 0.05).8. Effects of PHB on mitochondrial morphology of HUVEC treated by glyLDLThe alterations in glyLDL group were significantly ameliorated in glyLDL+VP group (P< 0.05), whereas glyLDL+V group showed no changes versus glyLDL group. By silencing PHB (siPHB group), cells revealed more notably defective mitochondria while GSPB2 significantly restored these abnormalities (P< 0.05).9. Effects of PHB, glyLDL and GSPB2 on Bax/Bcl-2 ratio and p-Akt expression of HUVECStimulation of HUVEC with glyLDL resulted in a significant increase in Bax/Bcl-2 ratio and p-Akt expression (P< 0.05); GSPB2 incubation significantly attenuated these increase, compared with glyLDL group (P< 0.05). PHB overexpression also largely restored the increase of Bax/Bcl-2 ratio and p-Akt expression, compared with glyLDL+V group (P< 0.05). Moreover, PHB knockdown caused a remarkably elevation in Bax/Bcl-2 ratio and p-Akt expression, while GSPB2 significantly inhibited these elevations (P< 0.05).Conclusions1. GlyLDL caused apoptosis of HUVEC, while GSPB2 has anti-apoptotic effects.2. Down-regulation of PHB might involves in endothelial cell apoptosis in response to glyLDL. GSPB2 might exhibit protective effects at least in part through regulating PHB.Part TwoProtective effects of grape seed procyanidin B2 on a diabetic pancreasBackgroundType 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by hyperglycemia and insulin-resistance. Traditional anti-diabetic therapy mainly focuses on glycemic control, including non-medical treatment and medical treatment. Non-medical treatment includes diet-control and exercise, while medical treatment is mainly the treatment of oral anti-diabetic agents and insulin. However, glycemic control of a large population is still not satisfied in diabetic patients, due to some disadvantages of medical treatment. Chronic hyperglycemia gradually causes metabolic disorder, infection or vascular complications, affecting the life quality and life span of diabetic patients. Therefore, to find effective treatments for T2DM is a priority.Insulin-resistance and insulin-deficiency are two important factors in pathogenesis of T2DM, though they have different importances to different patients. Pancreas plays an important role in secreting hormones to maintain the homeostasis of blood glucose. Dysfunction of pancreatic islets, which gradually loses the ability to secrete sufficient insulin against insulin-resistance, is the mainstay in pathogenesis of T2DM, determining the progression and prognosis of diabetic patients. Although molecular mechanisms underlying dysfunction of pancreas and insulin-resistance are not fully understood, accumulated evidence showed that inflammation was implicated. Release of pro-inflammatory cytokines was increased at both local and systemic levels in the development of β-cells dysfunction and insulin-resistance. Therefore, development of new treatment against inflammation may have great benefits on diabetic therapy.Early intervention to preserve the function of pancreas is crucial for delaying the progression of diabetes. GSPE is extracted from grape seed and possesses various bioactivities such as anti-oxidative stress, anti-inflammation, anti-tumor, and so on. Studies showed that procyanidins treatment in rats modulated the miRNA and proteome profile in pancreatic islets. GSPB2 is a dimeric form of GSPE, one effective components among others. Whether GSPB2 has protective effects on pancreas at the early stage of diabetes remains unknown. In the present study, we aimed to investigate effects of GSPB2 on pancreatic changes in db/db mice, a rodent model for T2DM.Objectives1. To explore effects of GSPB2 on diabetic pancreas in db/db mice.2. To further study the molecular mechanisms underlying the protective effects of GSPB2 on diabetic pancreas in db/db mice.MethodsMale C57BLKS/J db/db mice and db/m mice aged 7-week old were used as subjects. After acclimation for one week, the diabetic db/db mice were randomly divided into two groups:one group was treated with GSPB2 (purity> 95%,30 mg/kg body weight per day, diluted in normal saline) by intragastric administration every morning at 8 a.m. for 10 weeks (DMT, n= 8); the other group was treated with the same amount of normal saline solution (DM, n= 8). The non-diabetic db/m mice were selected as control group (CC, n= 8). Body weight and food intake were monitored every week for all the mice.At the end of the experiment, mice were fasted overnight and anesthetized. Fasting blood was collected for the assay of FBG, AGEs and insulin. Then all mice were perfused with ice-cold normal saline. Pancreases were dissected out for HE staining. Part of the pancreas tissue was used for detecting the protein level of MFG-E8, IL-ip and NLRP3 by Western Blot.Results1. Effects of GSPB2 on body weight and food intakeDuring the experimental period, db/db mice without any treatment showed obvious obesity compared with normal mice. Body weights of DM group were consistently higher than CC group (p< 0.05). After GSPB2 treatment, the increase of body weight in DM group was significantly attenuated from the second week (p< 0.05). Meantime, food intake of DM group was significantly increased, compared with CC group (p< 0.05). GSPB2 treatment effectively inhibited the increasing food intake after the second week (p< 0.05).2. Effects of GSPB2 on FBG and AGEsAt the end of the experiment, serum levels of FBG and AGEs in db/db mice were notably higher than that in db/m mice (p< 0.05). After GSPB2 treatment, levels of AGEs in db/db mice were significantly reduced (p< 0.05). However, levels of FBG were not significantly affected by GSPB2, compared with db/db mice.3. Effects of GSPB2 on morphology of pancreasAt the early stage of diabetes, a compensatory increase of insulin secretion was induced by peripheral insulin-resistance, accompanied by increased β-cell mass. But consistent increase of insulin secretion leads to β-cell exhaust. Therefore, preventing β-cells from over-secretion of insulin might be of great benefits. Our histologic results showed that islet size of db/db mice was strikingly larger than that of db/m mice (p< 0.05). After GSPB2 treatment, the enlargement of islet was significantly ameliorated (p<0.05).4. Effects of GSPB2 on insulin levels and HOMA-IRSerum level of insulin was elevated in db/db mice, whereas GSPB2 treatment decreased these elevations (p< 0.05). HOMA-IR, representing insulin resistance, increased largely in db/db mice, compared with normal mice. However, GSPB2 treatment notably reduced HOMA-IR of db/db mice (p< 0.05).5. Effects of GSPB2 on the protein expression of MFG-E8MFG-E8 was expressed in normal pancreas. In db/db mice, protein level of MFG-E8 was largely increased, compared with CC group (p< 0.05). After treatment with GSPB2, the increased protein level of MFG-E8 was significantly attenuated in DMT group, compared with DM group (p< 0.05).6. Effects of GSPB2 on protein levels of IL-1β and NLRP3Protein level of IL-1β was notably increased in db/db mice, compared with control mice. By GSPB2 treatment, the increased protein level of IL-1β was significantly suppressed, compared with db/db mice (p< 0.05). Meanwhile, NLRP3 was remarkably up-regulated in pancreas of db/db mice, while GSPB2 treatment suppressed the up-regulation of NLRP3 (p< 0.05).Conclusions1. Inflammation is involved in the pancreatic damage in diabetes and GSPB2 exhibits protective effects.2. GSPB2 might exert protective effects through regulating MFG-E8, IL-1β and NLRP3 in diabetic pancreas.