Younan Chen

The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo.

Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. The purpose of this study is to confirm the effect of MSCs on tumor cell growth in vitro and in vivo and to elucidate the mechanism. MSCs were isolated from mouse bone marrow and cocultured with murine hepatoma H22, lymphoma (YAC-1 and EL-4) and rat insulinoma INS-1 cell lines. The growth inhibitory effect of MSCs on tumor cells was tested through MTT and 3H-TdR incorporation assay. The apoptosis induction effect of MSCs on tumor cells was assessed with flow cytometry (FCM) and RT-PCR assay. MSCs were inoculated into BALB/c mice alone or coinoculated with ascitogenous hepatoma cells intraperitonealy, respectively. The tumor growth inhibition of MSCs was investigaed through the incidence and volume of ascites formation, and the immunosuppression effect was studied with splenocyte response to ConA stimulation test and T cell subsets analysis (FCM). The results showed that MSCs exhibited a number-dependent growth inhibitory effect on murine tumor cell lines in vitro and inhibited the growth of ascitogenous hepatoma cells in vivo without host immunosuppression. MSCs could upregulate tumor cells mRNA expression of cell cycle negative regulator p21 and apoptosis associated protease caspase 3. The findings of this experimental study demonstrated that MSCs had potential inhibitory effects on tumor cell growth in vitro and in vivo without host immunosuppression, by inducing apoptotic cell death and G0/G1 phase arrest of cancer cells.

The role of Nrf2 in oxidative stress-induced endothelial injuries

Endothelial dysfunction is an important risk factor for cardiovascular disease, and it represents the initial step in the pathogenesis of atherosclerosis. Failure to protect against oxidative stress-induced cellular damage accounts for endothelial dysfunction in the majority of pathophysiological conditions. Numerous antioxidant pathways are involved in cellular redox homeostasis, among which the nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway is perhaps the most prominent. Nrf2, a transcription factor with a high sensitivity to oxidative stress, binds to AREs in the nucleus and promotes the transcription of a wide variety of antioxidant genes. Nrf2 is located in the cytoskeleton, adjacent to Keap1. Keap1 acts as an adapter for cullin 3/ring-box 1-mediated ubiquitination and degradation of Nrf2, which decreases the activity of Nrf2 under physiological conditions. Oxidative stress causes Nrf2 to dissociate from Keap1 and to subsequently translocate into the nucleus, which results in its binding to ARE and the transcription of downstream target genes. Experimental evidence has established that Nrf2-driven free radical detoxification pathways are important endogenous homeostatic mechanisms that are associated with vasoprotection in the setting of aging, atherosclerosis, hypertension, ischemia, and cardiovascular diseases. The aim of the present review is to briefly summarize the mechanisms that regulate the Nrf2/Keap1-ARE signaling pathway and the latest advances in understanding how Nrf2 protects against oxidative stress-induced endothelial injuries. Further studies regarding the precise mechanisms by which Nrf2-regulated endothelial protection occurs are necessary for determining whether Nrf2 can serve as a therapeutic target in the treatment of cardiovascular diseases.

Regulation of SIRT1 in aging: Roles in mitochondrial function and biogenesis

Aging is a degenerative process associated with cumulative damage, which leads to cellular dysfunction, tissue failure, and disorders of body function. Silent information regulator-1, also known as sirtuin 1 (SIRT1), has been reported to be involved in the regulation of various important biological processes, including inflammation, mitochondrial biogenesis, as well as cell senescence and consequent aging. The level of SIRT1 is decreased in both transcriptional and postranscriptional conditions during aging, accompanied by attenuated mitochondrial biogenesis, an important component of aging-related diseases. Over the last decade, extensive studies have demonstrated that SIRT1 can activate several transcriptional factors, such as peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) and hypoxia-inducible factor 1α (HIF-1α) resulting in ameliorated mitochondria biogenesis and extended life span. In this review, we focus on the molecular regulation of SIRT1 and its role in mitochondrial biogenesis during in the context of aging and aging-related diseases.

Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury

Hypoxia/reoxygenation (H/R)‐induced injury is the key factor associated with islet graft dysfunction. This study aims to examine the effect of mesenchymal stem cells (MSCs) on islet survival and insulin secretion under H/R conditions. Islets from rats were isolated, purified, cultured with or without MSCs, and exposed to hypoxia (O2 ≤ 1%) for 8 h and reoxygenation for 24 and 48 h, respectively. Islet function was evaluated by measuring basal and glucose‐stimulated insulin secretion (GSIS). Apoptotic islet cells were quantified using Annexin V‐FITC. Anti‐apoptotic effects were confirmed by mRNA expression analysis of hypoxia‐resistant molecules, HIF‐1α, HO‐1, and COX‐2, using semi‐quantitative retrieval polymerase chain reaction (RT‐PCR). Insulin expression in the implanted islets was detected by immunohistological analysis. The main results show that the stimulation index (SI) of GSIS was maintained at higher levels in islets co‐cultured with MSCs. The MSCs protected the islets from H/R‐induced injury by decreasing the apoptotic cell ratio and increasing HIF‐1α, HO‐1, and COX‐2 mRNA expression. Seven days after islet transplantation, insulin expression in the MSC‐islets group significantly differed from that of the islets‐alone group. We proposed that MSCs could promote anti‐apoptotic gene expression by enhancing their resistance to H/R‐induced apoptosis and dysfunction. This study provides an experimental basis for therapeutic strategies based on enhancing islet function. Copyright

Reference values of clinical chemistry and hematology parameters in rhesus monkeys (Macaca mulatta)

Chen Y, Qin S, Ding Y, Wei L, Zhang J, Li H, Bu H, Lu Y, Cheng J. Reference values of clinical chemistry and hematology parameters in rhesus monkeys (Macaca mulatta).
Xenotransplantation 2009; 16: 496–501.

Comparison of single high-dose streptozotocin with partial pancreatectomy combined with low-dose streptozotocin for diabetes induction in rhesus monkeys.

Monkeys with insulin-dependent diabetes are important experimental models for islet xenotransplantation. However, with regard to diabetes induction, total pancreatectomy is a difficult operation with a high complication rate, while streptozotocin (STZ) administration may cause serious toxic effects and individual difference in metabolism. We compared two strategies involving pancreatectomy and STZ to successfully and safely induce diabetes in rhesus monkeys. Thirteen rhesus monkeys were divided into two groups: single high-dose STZ administration (80, 100 and 120 mg/kg, n = 3 for each dose) (group 1) and partial pancreatectomy (70–75%) combined with low-dose STZ (15 mg/kg, n = 4) (group 2). Induction of diabetes was evaluated by blood glucose, insulin, C-peptide, intravenous glucose tolerance test (IVGTT) and arginine stimulation test (AST). Detection of hematological and serum biochemical parameters and biopsies of pancreas, liver and kidney were periodically performed. In our study, animals in both groups developed diabetes. Serum C-peptide levels in groups 1 and 2 decreased to 0.08 ± 0.07 and 0.35 ± 0.06 nmol/L, respectively. IVGTT and AST indicated severely impaired glucose tolerance. Immunohistochemistry demonstrated that rare insulin-positive cells remained in the pancreas. In terms of STZ toxicity, four monkeys died 8–14 days after STZ administration (3 with 120 mg/kg STZ and 1 with 100 mg/kg STZ). Group 1 animals developed liver and kidney injury evidenced by increased alanine aminotransferase, aspartate aminotransferase, total cholesterol, LDL, triglyceride and blood urea nitrogen for one month, and histological abnormality including hepatic steatosis, renal glomerulus and tubular injury. Nevertheless, moderate histological injuries were seen in animals with 80 mg/kg STZ, with subsequent recovery. In contrast, group 2 animals displayed normal biochemical parameters and histology, with generally less risk of postoperative complications. We conclude that injection of 80 mg/kg STZ could induce diabetes with moderate injuries. Partial pancreatectomy with low-dose STZ is a safer and more reproducible method for inducing diabetes in rhesus monkeys.

Induction of Diabetes in Rhesus Monkeys and Establishment of Insulin Administration Strategy

OBJECTIVES The diabetic rhesus monkey seems to be a useful model for preclinical investigations of islet transplantation and new drug treatments for type 1 diabetes mellitus (T1DM). Information is limited regarding a standard technique to induce and assess diabetes in rhesus monkeys as well as the strategy to apply insulin administration. Herein, we have established and characterized a model of diabetic rhesus macaques. METHODS Four monkeys were divided into 2 groups of 2 each: group 1, total pancreatectomy; and group 2, partial pancreatectomy (75%) with low-dose streptozotocin (STZ) administration. Pancreatic function was measured using intravenous glucose tolerance tests before the operation. Spiral computed tomography (CT) scans of the pancreas were obtained before and after pancreatectomy. Fasting blood glucose and postprandial blood glucose levels were monitored twice daily using blood samples from the fingers or toes. Various types and doses of insulin were administered twice daily. We performed regular assessments of hematological and serum biochemical parameters, insulin, and C-peptide. RESULTS Both total pancreatectomy and partial pancreatectomy (75%) with STZ administration induced T1DM in rhesus monkeys; there was interindividual variation in the STZ dose. Excluding C-peptide and insulin, the hematological and serum biochemical parameters did not differ significantly from normal values postoperatively. The various insulin treatment strategies are achieved stable blood glucose (BG) levels. CONCLUSIONS STZ injection after partial pancreatectomy may be a safe, reproducible method to induce T1DM. Porcine insulin administration was a safe, economical method to control BG levels in a diabetic rhesus monkey before islet transplantation.

Anti-thrombosis effect of diosgenyl saponins in vitro and in vivo.

Thrombosis in coronary or cerebral arteries is the major cause of morbidity and mortality worldwide. Diosgenin and total steroidal saponins extracted from the rhizome of Dioscorea zingiberensis C.H. Wright are demonstrated to have anti-thrombotic activity. However, few studies describe the anti-thrombotic activity of the diosgenyl saponin monomer. In the present study, a simple and convenient method for the preparation of a new disaccharide saponin, diosgenyl β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside (3), is described. We evaluated the anti-thrombotic effects of diosgenin and four diosgenyl saponins by measuring the bleeding time; the results showed that compound 3 exhibits outstanding efficiency in prolonging the bleeding time. Furthermore, we assessed whether compound 3 could alter platelet aggregation in vitro and in vivo. In addition, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), coagulation factors and protection rate in mice were measured to evaluate the anti-thrombotic effect of compound 3. The results show that compound 3 inhibited platelet aggregation, prolonged APTT, inhibited factor VIII activities in rats, and increased the protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that diosgenyl saponins, especially compound 3, had anti-thrombotic activity. It may execute anti-thrombotic activity through inhibiting factor VIII activities and platelet aggregation.

A Preliminary Study on the Feasibility of Gene Expression Profile of Rhesus Monkey Detected With Human Microarray

OBJECTIVES A pig-to-monkey transplant model was initiated to investigate the outcome of pig-to-human xenotransplantation. Though monkey is close to human in biology and physiology, the genetic differences between the two species remains unclear. This study sought to compare the gene expressions of three tissues from humans and rhesus monkey. METHODS RNA samples extracted from liver, spleen, and peripheral blood cells were hybridized onto Illumina gene expression microarray. Genes with detected signals greater than 1000 and diff-scores higher than 100 were selected as significant results. The data were analyzed with Illumina software. mRNA expression levels were confirmed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS Of the 47,293 transcripts tested on every gene chip, more than 6000 genes were expressed in three tissues. Total numbers of genes detected and the similarity ratios followed the same rule as liver < PBC < spleen. The 136 IRI-related genes, 192 immunological-related genes, and 131 cell cycle-related genes selected and analyzed showed gene expression concordance rates of 82.35%, 72.92%, and 77.10%, respectively. RT-PCR tests indicated similar mRNA expression levels of RTN4, interleukin (IL)-1beta, NF-kappaB1, IL-8, and G0S2 to the results on chips. CONCLUSIONS The detected mRNA expressions in human and monkey tissues showed an average consistency in 85.78%, indicating that a human microarray might provide a part of the information for monkey sample testing. Therefore, in pig-to-monkey transplant models, monkey microarray may be used to determine recipient gene expressions. The genetic difference between human and monkey must be taken into account in interpreting the experimental results.

Serum metabolic variables associated with impaired glucose tolerance induced by high-fat-high-cholesterol diet in Macaca mulatta

Dyslipidemia caused by ‘Western-diet pattern’ is a strong risk factor for the onset of diabetes. This study aimed to disclose the relationship between the serum metabolite changes induced by habitual intake of high-fat and high-cholesterol (HFHC) diet and the development of impaired glucose tolerance (IGT) and insulin resistance through animal models of Macaca mulatta. Sixteen M. mulatta (six months old) were fed a control diet or a HFHC diet for 18 months. The diet effect on serum metabolic profiles was investigated by longitudinal research. Islet function was assessed by intravenous glucose tolerance and hyperinsulinemic-euglycemic clamp test. Metabonomics were determined by 1 H proton nuclear magnetic resonance spectroscopy. Prolonged diet-dependent hyperlipidemia facilitated visceral fat accumulation in liver and skeletal muscle and disorder of glucose homeostasis in juvenile monkeys. Glucose disappearance rate (KGlu) and insulin response to the glucose challenge effects in HFHC monkeys were significantly lower than in control monkeys. Otherwise, serum trimethylamine-N-oxide (TMAO), lactate and leucine/isoleucine were significantly higher in HFHC monkeys. Sphingomyelin and choline were the most positively correlated with KGlu (R2 = 0.778), as well as negative correlation (R2 = 0.64) with total cholesterol. The HFHC diet induced visceral fat, abnormal lipid metabolism and IGT prior to weight gain and body fat content increase in juvenile monkeys. We suggest that increased serum metabolites, such as TMAO, lactate, branched-chain amino acids and decreased sphingomyelin and choline, may serve as possible predictors for the evaluation of IGT and insulin resistance risks in the prediabetic state.