Pei-Lin Shao

Adipose-Derived Mesenchymal Stem Cell Protects Kidneys against Ischemia-Reperfusion Injury through Suppressing Oxidative Stress and Inflammatory Reaction

BackgroundReactive oxygen species are important mediators exerting toxic effects on various organs during ischemia-reperfusion (IR) injury. We hypothesized that adipose-derived mesenchymal stem cells (ADMSCs) protect the kidney against oxidative stress and inflammatory stimuli in rat during renal IR injury.MethodsAdult male Sprague-Dawley (SD) rats (n = 24) were equally randomized into group 1 (sham control), group 2 (IR plus culture medium only), and group 3 (IR plus immediate intra-renal administration of 1.0 × 106 autologous ADMSCs, followed by intravenous ADMSCs at 6 h and 24 h after IR). The duration of ischemia was 1 h, followed by 72 hours of reperfusion before the animals were sacrificed.ResultsSerum creatinine and blood urea nitrogen levels and the degree of histological abnormalities were markedly lower in group 3 than in group 2 (all p < 0.03). The mRNA expressions of inflammatory, oxidative stress, and apoptotic biomarkers were lower, whereas the anti-inflammatory, anti-oxidative, and anti-apoptotic biomarkers were higher in group 3 than in group 2 (all p < 0.03). Immunofluorescent staining showed a higher number of CD31+, von Willebrand Factor+, and heme oxygenase (HO)-1+ cells in group 3 than in group 2 (all p < 0.05). Western blot showed notably higher NAD(P)H quinone oxidoreductase 1 and HO-1 activities, two indicators of anti-oxidative capacity, in group 3 than those in group 2 (all p < 0.04). Immunohistochemical staining showed higher glutathione peroxidase and glutathione reductase activities in group 3 than in group 2 (all p < 0.02)ConclusionADMSC therapy minimized kidney damage after IR injury through suppressing oxidative stress and inflammatory response.

Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia–reperfusion injury in rats

Objectives:Mesenchymal stem cells have previously been shown to offer significant therapeutic benefit in ischemic organ injuries. This study aimed at investigating the therapeutic role of adipose tissue-derived mesenchymal stem cells in hepatic ischemia–reperfusion injury and the underlying mechanisms. Design:Adult male Fisher rats (n = 30) were equally divided into three groups (group 1: Sham-operated normal controls; group 2: Ischemia-reperfusion injury with intravenous fresh culture medium; group 3: Ischemia-reperfusion injury with intravenous adipose tissue-derived mesenchymal stem cells). Ischemia-reperfusion injury was induced by occluding the vascular supplies of left lobe liver for 60 minutes followed by reperfusion for 72 hrs. Adipose tissue-derived mesenchymal stem cells (1.2 × 106) were administered through tail vein immediately after reperfusion and at 6 hrs and 24 hrs after reperfusion in group 3. All animals were sacrificed 72 hrs after reperfusion. Setting:Animal laboratory at a medical institute. Measurements and Main Results:Histologic features, plasma aspartate aminotransferase, hepatic cytokine profile, oxidative stress, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling were analyzed. Seventy-two hrs after reperfusion, plasma aspartate aminotransferase, hepatic oxidative stress, messenger RNA expressions of tumor necrosis factor-a, transforming growth factor-b, interleukin-1b, interleukin-6, endothelin-1, matrix metalloproteinase-9, plasminogen activator inhibitor-1, Bax and caspase-3, protein expression of intercellular adhesion molecule as well as the number of apoptotic nuclei were significantly increased in group 2 compared with group 3, whereas messenger RNA expressions of endothelial nitric oxide synthase, Bcl-2, interleukin-10, protein expressions of reduced nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1, and heme oxygenase-1 were lower in group 2 than group 3. Conclusions:The results showed that systemic adipose tissue-derived mesenchymal stem cell administration significantly preserved hepatocyte integrity and suppressed inflammatory responses, oxidative stress, and apoptosis in a rodent model of hepatic ischemia–reperfusion injury. (Crit Care Med 2012; 40:–1290)

Extracorporeal Shock Wave Therapy Reverses Ischemia-Related Left Ventricular Dysfunction and Remodeling: Molecular-Cellular and Functional Assessment

An optimal treatment for patients with diffuse obstructive arterial disease unsuitable for catheter-based or surgical intervention is still pending. This study tested the hypothesis that extracorporeal shock wave (ECSW) therapy may be a therapeutic alternative under such clinical situation. Myocardial ischemia was induced in male mini-pigs through applying an ameroid constrictor over mid-left anterior descending artery (LAD). Twelve mini-pigs were equally randomized into group 1 (Constrictor over LAD only) and group 2 (Constrictor over LAD plus ECSW [800 impulses at 0.09 mJ/mm2] once 3 months after the procedure). Results showed that the parameters measured by echocardiography did not differ between two groups on days 0 and 90. However, echocardiography and left ventricular (LV) angiography showed higher LV ejection fraction and lower LV end-systolic dimension and volume in group 2 on day 180 (p<0.035). Besides, mRNA and protein expressions of CXCR4 and SDF-1α were increased in group 2 (p<0.04). Immunofluorescence staining also showed higher number of vWF-, CD31-, SDF-1α-, and CXCR4-positive cells in group 2 (all p<0.04). Moreover, immunohistochemical staining showed notably higher vessel density but lower mean fibrosis area, number of CD40-positive cells and apoptotic nuclei in group 2 (all p<0.045). Mitochondrial protein expression of oxidative stress was lower, whereas cytochrome-C was higher in group 2 (all p<0.03). Furthermore, mRNA expressions of MMP-9, Bax and caspase-3 were lower, whereas Bcl-2, eNOS, VEGF and PGC-1α were higher in group 2 (all p<0.01). In conclusion, ECSW therapy effectively reversed ischemia-elicited LV dysfunction and remodeling through enhancing angiogenesis and attenuating inflammation and oxidative stress.

Benefit of combined extracorporeal shock wave and bone marrow-derived endothelial progenitor cells in protection against critical limb ischemia in rats*

Objectives:We hypothesized that combined treatment with extracorporeal shock wave and bone marrow-derived endothelial progenitor cells might exert enhanced protection against critical limb ischemia in rats. Methods:Male Sprague-Dawley rats (n = 9 for laser Doppler study and n = 6 for laboratory examinations in each group) were divided into group 1 (sham control), group 2 (critical limb ischemia treated with culture medium), group 3 (critical limb ischemia treated with intramuscular bone marrow-derived endothelial progenitor cells [2.0 × 106 cells]), group 4 (critical limb ischemia treated with extracorporeal shock wave [280 impulses at 0.1 mJ/mm2]), and group 5 (combined bone marrow-derived endothelial progenitor cell-extracorporeal shock wave) after critical limb ischemia induction. Results:By day 21, laser Doppler showed substantially lower ratios of ischemic/normal blood flow in group 2 compared with other groups (p < .001). The protein expressions of mitochondrial cytochrome c, stromal cell-derived factor-1, C-X-C chemokine receptor type 4, vascular endothelial growth factor, and endothelial nitric oxide synthase were remarkably higher in group 5 than in groups 2 to 4, and notably higher in groups 3 and 4 than in group 2 (all p < .01). The messenger RNA expressions of proinflammatory and apoptotic biomarkers and oxidative stress were reduced in group 5 compared with groups 2 to 4, and notably lower in groups 3 and 4 than in group 2 (all p < .01). The messenger RNA expressions of anti-inflammatory and antiapoptotic biomarkers were lower in group 2 than in other groups (all p < .01). Immunofluorescent staining showed higher numbers of CD31+ stromal cell-derived factor-1+, chemokine receptor type 4+, and von Willebrand factor+ cells, and vessels in the ischemic area in group 5 than in groups 2 to 4, and in groups 3 and 4 than in group 2 (all p < .04). Conclusion:Combined treatment with bone marrow-derived endothelial progenitor cells and extracorporeal shock wave is superior to either bone marrow-derived endothelial progenitor cells or extracorporeal shock wave alone in improving ischemia in rodent critical limb ischemia.

Intravenous administration of xenogenic adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke

We tested the hypothesis that combined xenogenic (from mini-pig) adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy could reduce brain-infarct zone (BIZ) and enhance neurological recovery in rat after acute ischemic stroke (AIS) induced by 50-min left middle cerebral artery occlusion. Adult-male Sprague-Dawley rats (n = 60) were divided equally into group 1 (sham-control), group 2 (AIS), group 3 [AIS-ADMSC (1.2×106 cells)], group 4 [AIS-exosome (100μg)], and group 5 (AIS-exosome-ADMSC). All therapies were provided intravenously at 3h after AIS procedure. BIZ determined by histopathology (by day-60) and brain MRI (by day-28) were highest in group 2, lowest in group 1, higher in groups 3 and 4 than in group 5, but they showed no difference between groups 3 and 4 (all p < 0.0001). By day-28, sensorimotor functional results exhibited an opposite pattern to BIZ among the five groups (p < 0.005). Protein expressions of inflammatory (inducible nitric oxide synthase/tumor necrosis factor-α/nuclear factor-κB/interleukin-1β/matrix metalloproteinase-9/plasminogen activator inhibitor-1/RANTES), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/ Poly-ADP-ribose polymerase), and fibrotic (Smad3/transforming growth factor-β) biomarkers, and cellular expressions of brain-damaged (γ-H2AX+/ XRCC1-CD90+/p53BP1-CD90+), inflammatory (CD11+/CD68+/glial fibrillary acid protein+) and brain-edema (aquaporin-4+) markers showed a similar pattern of BIZ among the groups (all n < 0.0001). In conclusion, xenogenic ADMSC/ADMSC-derived exosome therapy was safe and offered the additional benefit of reducing BIZ and improving neurological function in rat AIS.

Combination of cyclosporine and erythropoietin improves brain infarct size and neurological function in rats after ischemic stroke

BackgroundThis study tested the superiority of combined cyclosporine A (CsA)-erythropoietin (EPO) therapy compared with either one in limiting brain infarction area (BIA) and preserving neurological function in rat after ischemic stroke (IS).MethodsFifty adult-male SD rats were equally divided into sham control (group 1), IS plus intra-peritoneal physiological saline (at 0.5/24/48 h after IS) (group 2), IS plus CsA (20.0 mg/kg at 0.5/24h, intra-peritoneal) (group 3), IS plus EPO (5,000IU/kg at 0.5/24/48h, subcutaneous) (group 4), combined CsA and EPO (same route and dosage as groups 3 and 4) treatment (group 5) after occlusion of distal left internal carotid artery.ResultsBIA on day 21 after acute IS was higher in group 2 than in other groups and lowest in group 5 (all p < 0.01). The sensorimotor functional test showed higher frequency of left turning in group 2 than in other groups and lowest in group 5 (all p < 0.05). mRNA and protein expressions of apoptotic markers and number of apoptotic nuclei on TUNEL were higher in group 2 than in other groups and lowest in group 1 and 5, whereas the anti-apoptotic markers exhibited an opposite trend (all p < 0.05). The expressions of inflammatory and oxidized protein were higher in group 2 than in other groups and lowest in group 1 and 5, whereas anti-inflammatory markers showed reversed changes in group 1 and other groups (all p < 0.05). The number of aquaporin-4+ and glial fibrillary acid protein+ stained cells were higher in group 2 as compared to other groups and lowest in groups 1 and 5 (all p < 0.01).Conclusioncombined treatment with CsA and EPO was superior to either one alone in protecting rat brain from ischemic damage after IS.

Shock Wave Therapy Effectively Attenuates Inflammation in Rat Carotid Artery following Endothelial Denudation by Balloon Catheter

Background: This study investigates the effectiveness of extracorporeal shock wave (ECSW) in ameliorating inflammatory mediator expression and neointimal formation in a rat model of vascular injury. Methods and Results: Male Sprague-Dawley rats with left carotid artery (LCA) injury induced by balloon dilatation (BD; group 1) were compared with group 2 [LCA injury plus ECSW-181 (defined as 181 total shocks given in LCA at 0.011 mJ/mm2) on day 2 post-LCA injury], and group 3 (normal controls). The rats in each group were further divided into 3 subgroups (n = 6, each) that were sacrificed on postoperative day 3, 7 and 14, respectively. The results demonstrated that, compared to groups 2 and 3, group 1 had significantly increased cellular expression of CD40, interleukin-18, and connexin 43 at each analyzed time point (all p < 0.001). Additionally, LCCA macrophage (CD68) recruitment was substantially increased in group 1 compared to groups 2 and 3 (all p < 0.001). Furthermore, LCA neointimal proliferation and media thickness were markedly higher in group 1 than in groups 2 and 3 on days 7 and 14 post-BD (all p < 0.001). Conclusions: ECSW markedly attenuates inflammatory responses, proliferation of neointima and smooth muscle cells in a rat vascular injury model.

Continuing Exposure to Low-Dose Nonylphenol Aggravates Adenine-Induced Chronic Renal Dysfunction and Role of Rosuvastatin Therapy

BackgroundNonylphenol (NP), an environmental organic compound, has been demonstrated to enhance reactive-oxygen species (ROS) synthesis. Chronic exposure to low-dose adenine (AD) has been reported to induce chronic kidney disease (CKD).MethodsIn this study, we tested the hypothesis that chronic exposure to NP will aggravate AD-induced CKD through increasing generations of inflammation, ROS, and apoptosis that could be attenuated by rosuvastatin. Fifty male Wistar rats were equally divided into group 1 (control), group 2 (AD in fodder at a concentration of 0.25%), group 3 (NP: 2 mg/kg/day), group 4 (combined AD & NP), and group 5 (AD-NP + rosuvastatin: 20 mg/kg/day). Treatment was continued for 24 weeks for all animals before being sacrificed.ResultsBy the end of 24 weeks, serum blood urea nitrogen (BUN) and creatinine levels were increased in group 4 than in groups 1–3, but significantly reduced in group 5 as compared with group 4 (all p < 0.05). Histopathology scorings of renal-parenchymal and tubular damages were significantly higher in group 4 than in groups 1–3, but remarkably lower in group 5 compared with group 4 (all p < 0.01). Both gene and protein levels of inflammation, oxidative stress, ROS, and cellular apoptosis were remarkably higher in group 4 compared with groups 1–3, but lowered in group 5 than in group 4 (all p < 0.001). Conversely, both gene and protein levels of anti-oxidants, anti-inflammation and anti-apoptosis were markedly increased in group 5 compared with group 4 (all p < 0.001).ConclusionNP worsened AD-induced CKD that could be reversed by rosuvastatin therapy.

Melatonin attenuated brain death tissue extract-induced cardiac damage by suppressing DAMP signaling

We tested the hypothesis that melatonin prevents brain death (BD) tissue extract (BDEX)-induced cardiac damage by suppressing inflammatory damage-associated molecular pattern (DAMP) signaling in rats. Six hours after BD induction, levels of a DAMP component (HMGB1) and inflammatory markers (TLR-2, TLR-4, MYD88, IκB, NF-κB, IL-1β, IFN-γ, TNF-α and IL-6) were higher in brain tissue from BD animals than controls. Levels of HMGB1 and inflammatory markers were higher in BDEX-treated H9C2 cardiac myoblasts than in cells treated with healthy brain tissue extract. These increases were attenuated by melatonin but re-induced with luzindole (all P < 0.001). Additional male rats (n = 30) were divided into groups 1 (negative control), 2 (healthy brain tissue extract implanted in the left ventricular myocardium [LVM]), 3 (BDEX-LVM), 4 (BDEX-LVM + melatonin), and 5 (BDEX-LVM + melatonin + luzindole). Collagen deposition/fibrosis and LVM levels of MTR2, HMGB1, inflammatory markers, oxidative stress, apoptosis, mitochondrial damage and DNA damage were highest in group 3, lowest in groups 1 and 2, and higher in group 5 than in group 4. Heart function and LVM levels of MTR1 and anti-inflammatory, mitochondrial-integrity and anti-oxidative markers exhibited a pattern opposite that of the inflammatory markers in the five groups (all P < 0.0001). These results indicate melatonin inhibits BDEX-induced cardiac damage by suppressing the DAMP inflammatory axis.

Combined Therapy with SS31 and Mitochondria Mitigates Myocardial Ischemia-Reperfusion Injury in Rats

Myocardial ischemia-reperfusion (IR) injury contributes to adverse cardiac outcomes after myocardial ischemia, cardiac surgery, or circulatory arrest. In this study, we evaluated the ability of combined SS31-mitochondria (Mito) therapy to protect heart cells from myocardial IR injury. Adult male SD rats (n = 8/each group) were randomized: group 1 (sham-operated control), group 2 (IR, 30-min ischemia/72 h reperfusion), group 3 (IR-SS31 (2 mg intra-peritoneal injection at 30 min/24 h/48 h after IR)), group 4 (IR-mitochondria (2 mg/derived from donor liver/intra-venous administration/30 min after IR procedure)), and group 5 (IR-SS31-mitochondria). In H9C2 cells, SS31 suppressed menadione-induced oxidative-stress markers (NOX-1, NOX-2, oxidized protein) while it increased SIRT1/SIRT3 expression and ATP levels. In adult male rats 72 h after IR, left ventricular ejection fraction (LVEF) was highest in sham-operated control animals and lowest in the IR group. LVEF was also higher in IR rats treated with SS31-Mito than untreated IR rats or those treated with Mito or SS31 alone. Areas of fibrosis/collagen-deposition showed the opposite pattern. Likewise, levels of oxidative-stress markers (NOX-1, NOX-2, oxidized protein), inflammatory markers (MMP-9, CD11, IL-1β, TNF-α), apoptotic markers (mitochondrial-Bax, cleaved-caspase-3, PARP), fibrosis markers (p-Smad3, TGF-β), DNA-damage (γ-H2AX), sarcomere-length, and pressure/volume overload markers (BNP, β-MHC) all showed a pattern opposite that of LVEF. Conversely, anti-apoptotic (BMP-2, Smad1/5) and energy integrity (PGC-1α/mitochondrial cytochrome-C) markers exhibited a pattern identical to that of LVEF. This study demonstrates that the combined SS31-Mito therapy is superior to either therapy alone for protecting myocardium from IR injury and indicates that the responsible mechanisms involved increased SIRT1/SIRT3 expression, which suppresses inflammation and oxidative stress and protects mitochondrial integrity.