Hsin-Ju Chiang

Protective effect of melatonin-supported adipose-derived mesenchymal stem cells against small bowel ischemia-reperfusion injury in rat

We tested the hypothesis that combined melatonin and autologous adipose‐derived mesenchymal stem cells (ADMSC) was superior to either alone against small bowel ischemia‐reperfusion (SBIR) injury induced by superior mesenteric artery clamping for 30 min followed by reperfusion for 72 hr. Male adult Sprague Dawley rats (n = 50) were equally categorized into sham‐operated controls SC, SBIR, SBIR‐ADMSC (1.0 × 106 intravenous and 1.0 × 106 intrajejunal injection), SBIR‐melatonin (intraperitoneal 20 mg/kg at 30 min after SI ischemia and 50 mg/kg at 6 and 18 hr after SI reperfusion), and SBIR‐ADMSC‐melatonin groups. The results demonstrated that the circulating levels of TNF‐α, MPO, LyG6+ cells, CD68+ cells, WBC count, and gut permeability were highest in SBIR and lowest in SC, significantly higher in SBIR‐ADMSC group and further increased in SBIR‐melatonin group than in the combined therapy group (all P < 0.001). The ischemic mucosal damage score, the protein expressions of inflammation (TNF‐α, NF‐κB, MMP‐9, MPO, and iNOS), oxidative stress (NOX‐1, NOX‐2, and oxidized protein), apoptosis (APAF‐1, mitochondrial Bax, cleaved caspase‐3 and PARP), mitochondrial damage (cytosolic cytochrome C) and DNA damage (γ‐H2AX) markers, as well as cellular expressions of proliferation (PCNA), apoptosis (caspase‐3, TUNEL assay), and DNA damage (γ‐H2AX) showed an identical pattern, whereas mitochondrial cytochrome C exhibited an opposite pattern compared to that of inflammation among all groups (all P < 0.001). Besides, antioxidant expressions at protein (NQO‐1, GR, and GPx) and cellular (HO‐1) levels progressively increased from SC to the combined treatment group (all P < 0.001). In conclusion, combined melatonin‐ADMSC treatment offered additive beneficial effect against SBIR injury.

Melatonin treatment further improves adipose-derived mesenchymal stem cell therapy for acute interstitial cystitis in rat.

This study tests the hypothesis that combined melatonin and adipose‐derived mesenchymal stem cell (ADMSC, 1.2 × 106 given intravenously) treatment offer superior protection against cyclophosphamide (CYP 150 mg/kg)‐induced acute interstitial cystitis (AIC) in rats. Male adult Sprague‐Dawley rats were treated as follows: sham controls, AIC alone, AIC + melatonin, AIC + ADMSC, and AIC + melatonin +ADMSC. When melatonin was used, it was given as follows: 20 mg/kg at 30 min after CYP and 50 mg/kg at 6 and 18 hr after CYP. Twenty‐four‐hour urine volume, urine albumin level, and severity of hematuria were highest in AIC rats and lowest in the controls; likewise urine volume was higher in AIC + melatonin rats than in AIC + ADMSC and AIC + melatonin + ADMSC treated rats; in all cases, P < 0.001. The numbers of CD14+, CD74+, CD68+, MIP+, Cox‐2+, substance P+, cells and protein expression of IL‐6, IL‐12, RANTES, TNF‐α, NF‐κB, MMP‐9, iNOS (i.e. inflammatory biomarkers), glycosaminoglycan level, expression of oxidized protein, and protein expression of reactive oxygen species (NOX‐1, NOX‐2, NOX‐4) in the bladder tissue exhibited an identical pattern compared with that of hematuria among the five groups (all P < 0.0001). The integrity of epithelial layer and area of collagen deposition displayed an opposite pattern compared to that of hematuria among all groups (P < 0.0001). The cellular expressions of antioxidants (GR, GPx, HO‐1, NQO 1) showed a significant progressive increase form controls to AIC + melatonin + ADMSC (all P < 0.0001). Combined regimen of melatonin and ADMSC was superior to either alone in protecting against CYP‐induced AIC.

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia-reperfusion injury in rats through suppression of mitochondrial permeability transition.

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia–reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione–melatonin treatment, 4.0 × 105 each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham‐control (SC), IR (60‐min left‐lobe ischemia + 72‐hr reperfusion), IR‐Mel (melatonin at 30 min/6/8 hr after reperfusion), IR‐Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR‐Mel‐Mito. Following menadione treatment in vitro, oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (cleaved caspase‐3/PARP), DNA damage (γ‐H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR‐Mito group than that in groups IR‐Mel and IR‐Mel‐Mito, and higher in IR‐Mel group than that in IR‐Mel‐Mito group after 72‐hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF‐α/NF‐κB/IL‐1β/MMP‐9), oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (caspase‐3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO+/CD68+/CD14+ cells), and DNA damage (γ‐H2AX+ cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin‐supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.

Sitagliptin protects rat kidneys from acute ischemia-reperfusion injury via upregulation of GLP-1 and GLP-1 receptors

Aim:Sitagliptin, an oral glucose-lowering agent, has been found to produce cardiovascular protection possibly via anti-inflammatory and anti-atherosclerotic activities of glucagon-like peptide-1 receptor (GLP-1). The aim of this study was to investigate whether sitagliptin protected the kidney function from acute ischemia-reperfusion (IR) injury in rats.Methods:Adult male SD rats were categorized into 4 groups: sham control, IR injury, IR+sitagliptin (300 mg/kg) and IR+sitagliptin (600 mg/kg). Acute renal IR injury of both kidneys was induced by clamping the renal pedicles for 1 h. The drug was orally administered at 1, 24 and 48 h after acute IR. Blood samples and 24-h urine were collected before and at 72 h after acute IR. Then the rats were sacrificed, and the kidneys were harvested for biochemical and immunohistochemical studies.Results:Acute IR procedure markedly increased serum levels of creatinine and BUN and the ratio of urine protein to creatinine. The kidney injury score, inflammatory biomarkers (MMP-9, TNF-α and NF-κB) levels and CD68+ cells in IR kidneys were considerably increased. The expression of oxidized protein, reactive oxygen species (NOX-1, NOX-2) and apoptosis proteins (Bax, caspase-3, PARP) in IR kidneys was also significantly upregulated. All these pathological changes were suppressed by sitagliptin in a dose-dependent manner. Furthermore, the serum GLP-1 level, and the expression of GLP-1 receptor, anti-oxidant biomarkers (HO-1 and NQO-1 cells, as well as SOD-1, NQO-1 and HO-1 proteins), and angiogenesis markers (SDF-1α+ and CXCR4+ cells) in IR kidneys were significantly increased, and further upregulated by sitagliptin.Conclusion:Sitagliptin dose-dependently protects rat kidneys from acute IR injury via upregulation of serum GLP-1 and GLP-1 receptor expression in kidneys.

The cardioprotective effect of melatonin and exendin-4 treatment in a rat model of cardiorenal syndrome

We investigated the cardioprotective effect of melatonin (Mel) and exendin‐4 (Ex4) treatment in a rat model of cardiorenal syndrome (CRS). Adult male SD rats (n=48) were randomly and equally divided into sham control (SC), dilated cardiomyopathy (DCM) (doxorubicin 7 mg/kg i.p. every five days/4 doses), CRS (defined as DCM+CKD) only, CRS‐Mel (20 mg/kg/d), CRS‐Ex4 (10 μg/kg/d), and CRS‐Mel‐Ex4 groups. In vitro results showed protein expressions of oxidative stress (NOX‐1/NOX‐2/oxidized protein), DNA/mitochondrial damage (γ‐H2AX/cytosolic cytochrome c), apoptosis (cleaved caspase‐3/PARP), and senescence (β‐galactosidase cells) biomarkers were upregulated, whereas mitochondrial ATP level was decreased in doxorubicin/p‐cresol‐treated H9c2 cells that were revised by Mel and Ex4 treatments (all P<.001). By day 60, LVEF was highest in the SC and lowest in the CRS, significantly lower in the DCM than in other treatment groups, lower in the CRS‐Mel and CRS‐Ex4 than in the CRS‐Mel‐Ex4, and lower in the CRS‐Mel than in the CRS‐Ex4, whereas LV chamber size and histopathology score showed a pattern opposite to that of LVEF among all groups (all P<.001). Plasma creatinine level was highest in the CRS and lowest in the SC and progressively decreased from the CRS‐Mel, CRS‐Ex4, CRS‐Mel‐Ex4 to DCM (P<.0001). Protein expressions of inflammation (TNF‐α/NF‐κB/MMP‐2/MMP‐9/IL‐1β), apoptosis/DNA damage (Bax/c‐caspase‐3/c‐PARP/γ‐H2AX), fibrosis (Smad3/TGF‐β), oxidative stress (NOX‐1/NOX‐2/NOX‐4/oxidized protein), cardiac hypertrophy/pressure overload (BNP/β‐MHC), and cardiac integrity (Cx43/α‐MHC) biomarkers in LV myocardium showed an opposite pattern compared to that of LVEF among all groups (all P<.001). Fibrotic area, DNA damage (γ‐H2AX+/53BP1+CD90+/XRCC1+CD90+), and inflammation (CD14+/CD68+) biomarkers in LV myocardium displayed a pattern opposite to that of LVEF among all groups (all P<.001). Combined melatonin and exendin‐4 treatment suppressed CRS‐induced deterioration of LVEF and LV remodeling.

Combined melatonin and exendin-4 therapy preserves renal ultrastructural integrity after ischemia-reperfusion injury in the male rat.

We tested whether combined melatonin (Mel) and exendin‐4 (Ex4) treatment can better preserve glomerular structural integrity after ischemia–reperfusion (IR) injury compared with either alone. Adult male Sprague Dawley rats (n = 50) were equally divided into sham control (SC), IR, IR‐Ex4 (10 μg/kg subcutaneously 30 min after reperfusion and daily for 5 days), IR‐Mel (20 mg/kg intraperitoneally at 30 min postreperfusion and 50 mg/kg at 6 and 18 hr), and IR‐Ex4‐Mel were euthanized at day 14. Serum creatinine level and urine protein‐to‐creatinine ratio at days 3 and 14 were highest in IR group and lowest in SC, significantly higher in IR‐Ex4 and IR‐Mel groups than in IR‐Ex4‐Mel group (all P < 0.001) without significant difference between IR‐Ex4 and IR‐Mel groups. Changes in podocyte injury score (PIS) and kidney injury score were highest in IR group and lowest in SC, significantly higher in IR‐Ex4 and IR‐Mel groups than in IR‐Ex4‐Mel, and significantly higher in IR‐Mel group than in IR‐Ex4 group (all P < 0.001). Immunohistochemical microscopic findings of the expressions of FSP‐1 and WT‐1 (two glomerular damage indicators) and KIM‐1 and snail (two renal tubular‐damaged indicators) showed an identical pattern, whereas the expressions of ZO‐1, p‐cadherin, podocin, dystroglycan, fibronectin, and synaptopodin (six indices of glomerular integrity) demonstrated an opposite pattern compared to that of PIS among five groups (all P < 0.001). Protein expressions of inflammatory (TNF‐α/NF‐κB/MMP‐9) and oxidative stress (NOX‐1, NOX‐2, oxidized protein) biomarkers exhibited an identical pattern to that of PIS among five groups (all P < 0.001). Combined melatonin–exednin‐4 therapy further protected glomerulus from IR injury.

Combined Therapy With Adipose-Derived Mesenchymal Stem Cells and Ciprofloxacin Against Acute Urogenital Organ Damage in Rat Sepsis Syndrome Induced by Intrapelvic Injection of Cecal Bacteria

We hypothesized that combined treatment with autologous adipose‐derived mesenchymal stem cell (ADMSC) and ciprofloxacin is superior to ciprofloxacin only in reducing sepsis‐induced urogenital organ damage and mortality in rat sepsis syndrome (SS) caused by intrapelvic injection of cecal bacteria (1.0 × 104 cells per milliliter; total, 5.0 ml). Male Sprague‐Dawley rats (n = 60) equally divided into group 1 (sham‐control), group 2 (SS), group 3 (SS‐ADMSC [5.0 × 105 intravenously at 0.5, 6, and 18 hours after sepsis induction]), group 4 (SS‐ciprofloxacin [3.0 mg/kg, b.i.d.] for 5 days), and group 5 (SS‐ADMSC‐ciprofloxacin) were sacrificed by day 5. Mortality rate and creatinine level were highest in group 2 and lowest in group 1 and significantly higher in groups 3 and 4 than those in group 5, but there was no difference between groups 3 and 4 (all p < .005). The kidney injury score, inflammatory biomarker expressions at protein (tumor necrosis factor‐1α, nuclear factor‐κB, matrix metallopeptidase‐9, regulated on activation, normal T‐cell expressed and secreted, interleukin‐1β) and cellular (CD14+, migratory inhibitor factor positive, CD68+) levels in kidneys and urinary bladder were lowest in group 1 and highest in group 2, higher in group 4 than in groups 3 and 5, and higher in group 3 than in group 5 (all p < .001). Protein expressions of apoptosis (Bax, cleaved caspase 3 and poly[ADP‐ribose] polymerase 1, p21 protein [Cdc42/Rac]‐activated kinase 2) and oxidative stress (oxidized protein, NADPH oxidase (NOX)‐1, NOX‐2) in these organs showed an identical pattern compared with that of inflammation in all groups (all p < .001). In conclusion, ADMSC‐assisted ciprofloxacin therapy offered an additional benefit by reducing acute urogenital organ damage in rat.

Extracorporeal shock wave treatment attenuated left ventricular dysfunction and remodeling in mini-pig with cardiorenal syndrome

This study tested the hypothesis that extracorporeal shock wave (ECSW) treatment can improve ischemia-induced left ventricular (LV) dysfunction in mini-pig with co-existing chronic kidney disease (CKD). LV ischemia in mini-pigs was induced by applying an ameroid constrictor over mid-left anterior descending artery (LAD), while model of CKD was established by right nephrectomy with partial ligation of left renal arterioles 2 weeks before LAD constriction. Thirty mini-pigs were randomly divided into group 1 (sham-control), group 2 (LV-ischemia), group 3 (LV-ischemia + CKD), Group 4 [LV-ischemia + ECSW (applied 1200 shots at 0.1 mJ/m2/equally to 4-ischemic regions by day-90 after LAD constriction], and group 5 (LV-ischemia-CKD + ECSW). By day-180 after CKD induction, echocardiography showed that LV ejection fraction (LVEF) was highest in group 1, lowest in group 3, significantly lower in group 2 than that in groups 4 and 5, and significantly lower in group 5 than that in group 4, whereas LV-end systolic and diastolic dimensions displayed an opposite pattern (all p<0.001). Protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved-caspase-3/cleaved-PARP/mitochondrial-Bax), fibrotic (TGF-β/Smad3), pressure/volume-overload (BNP/β-MHC), endothelial (CD31/vWF) and mitochondrial-integrity (PGC-1/mitochondrial-cytochrome-C) biomarkers exhibited a pattern identical to that of LVEF, whereas angiogenesis factors (VEGF/CXCR4/SDF-1α) showed significant progressive increase among all groups (all p<0.0001). Microscopic findings of CD31+cells/vWF+cells/small-vessel density/sarcomere-length showed an identical pattern, whereas collagen-deposition area/fibrotic area/apoptotic nuclei expressed an opposite pattern compared to that of LVEF among all groups (all p<0.0001). In conclusion, CKD aggravated ischemia-induced LV dysfunction and remodeling and molecular-cellular perturbations that were reversed by ECSW treatment.This study tested the hypothesis that extracorporeal shock wave (ECSW) treatment can improve ischemia-induced left ventricular (LV) dysfunction in mini-pig with co-existing chronic kidney disease (CKD). LV ischemia in mini-pigs was induced by applying an ameroid constrictor over mid-left anterior descending artery (LAD), while model of CKD was established by right nephrectomy with partial ligation of left renal arterioles 2 weeks before LAD constriction. Thirty mini-pigs were randomly divided into group 1 (sham-control), group 2 (LV-ischemia), group 3 (LV-ischemia + CKD), Group 4 [LV-ischemia + ECSW (applied 1200 shots at 0.1 mJ/m2/equally to 4-ischemic regions by day-90 after LAD constriction], and group 5 (LV-ischemia-CKD + ECSW). By day-180 after CKD induction, echocardiography showed that LV ejection fraction (LVEF) was highest in group 1, lowest in group 3, significantly lower in group 2 than that in groups 4 and 5, and significantly lower in group 5 than that in group 4, whereas LV-end systolic and diastolic dimensions displayed an opposite pattern (all p<0.001). Protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved-caspase-3/cleaved-PARP/mitochondrial-Bax), fibrotic (TGF-β/Smad3), pressure/volume-overload (BNP/β-MHC), endothelial (CD31/vWF) and mitochondrial-integrity (PGC-1/mitochondrial-cytochrome-C) biomarkers exhibited a pattern identical to that of LVEF, whereas angiogenesis factors (VEGF/CXCR4/SDF-1α) showed significant progressive increase among all groups (all p<0.0001). Microscopic findings of CD31+cells/vWF+cells/small-vessel density/sarcomere-length showed an identical pattern, whereas collagen-deposition area/fibrotic area/apoptotic nuclei expressed an opposite pattern compared to that of LVEF among all groups (all p<0.0001). In conclusion, CKD aggravated ischemia-induced LV dysfunction and remodeling and molecular-cellular perturbations that were reversed by ECSW treatment.

Preactivated and Disaggregated Shape-changed Platelets Protected Against Acute Respiratory Distress Syndrome Complicated by Sepsis Through Inflammation Suppression.

Background: This study tested the hypothesis that preactivated and disaggregated shape-changed platelet (PreD-SCP) therapy attenuates lung injury from acute respiratory distress syndrome (ARDS) induced by 100% oxygen inhalation and complicated by sepsis through peritoneal administration of 1.5 mg/kg lipopolysaccharide (LPS). Methods: Adult male Sprague-Dawley rats, weighing 325 to 350 g, were randomized into group 1 (normal controls [NC]), group 2 (NC + PreD-SCP [3.0 × 108, intravenous administration]), group 3 (ARDS-LPS), and group 4 (ARDS-LPS + PreD-SCP), and sacrificed by 72 h after ARDS induction. Results: The lung injury score was significantly higher in group 3 than that in other groups, and significantly higher in group 4 than that in groups 1 and 2, whereas the numbers of alveolar sacs and oxygen saturation (%) showed a reversed pattern compared with that of lung injury score among the four groups (all P < 0.0001) without significant difference between groups 1 and 2. The expressions of proinflammatory cells (CD11+, CD14+, CD68+) and proteins (tumor necrosis factor [TNF]-&agr;, nuclear factor [NF]-&kgr;B, interleukin [IL]-1&bgr;&bgr;, matrix metalloproteinase [MMP]-9, inducible nitric oxide synthase, intercellular adhesion molecule-1) exhibited a pattern identical to the lung injury score. Circulating levels of white blood cell, IL-6, TNF-&agr;, myeloperoxidase and CCL5, and pulmonary protein expressions of oxidative stress (NOX-1/NOX-2, oxidized protein), apoptotic (Bax, cleaved caspase 3/poly (ADP-ribose) polymerase), fibrotic (Smad3, transforming growth factor [TGF]-&bgr;), and DNA damage (&ggr;-H2AX) biomarkers showed an identical pattern, whereas protein expressions of antifibrotic (Smad1/5, bone morphogenetic protein [BMP]-2) and anti-inflammatory (Bcl-2) biomarkers demonstrated an opposite pattern compared with the proinflammatory indices among the four groups (all P < 0.001). Conclusions: PreD-SCP therapy effectively improved lung injury in ARDS complicated by sepsis.

Enhanced protection against renal ischemia–reperfusion injury with combined melatonin and exendin-4 in a rodent model

We tested the hypothesis that combined treatment with melatonin, an anti-oxidant, and exendin-4, an anti-inflammatory agent, was superior to either alone for protecting the kidney from ischemia–reperfusion (IR) injury. Male adult Sprague-Dawley rats (n=40) were equally divided into group 1 (sham-operated control), group 2 (IR only, IR=1h/72h), group 3 (IR–exendin-4, 10 µg/kg at 30 min, 24 h, 48 h after IR procedure), group 4 (IR–melatonin, i.p. 50 mg at 30 min, then 20 mg at 6 and 18 h after IR procedure), and group 5 (combined IR–exendin-4–melatonin). All animals were sacrificed by 72 h after IR/sham procedure. The results showed that the kidney injury score, plasma creatinine, and blood urea nitrogen (BUN) levels were highest in group 2 and lowest in group 1, significantly higher in groups 3 and 4 than those in group 5 and significantly higher in group 3 than those in group 4 (all p < 0.001). The protein expressions of inflammatory (toll-like receptor 4, inducible nitric oxide synthase, interleukin-1β), apoptotic (mitochondrial Bax, cleaved caspase-3 and poly(ADP-ribose) polymerase, p53), podocyte integrity (E-cadherin, P-cadherin), and cell survival (phosphatidylinositol-3-kinase/AKT/mammalian target of rapamycin) biomarkers, as well the podocyte dysfunction biomarkers (Wnt1/Wnt4/β-catenin) displayed a pattern identical to that of creatinine level among the five groups (all p < 0.001). Microscopic findings demonstrated that podocyte dysfunction (Wnt1/Wnt4/β-catenin expression) and inflammatory (CD14 and F4/80-positively stained cells) biomarkers exhibited an identical pattern, whereas that of antioxidant (HO-1 + , NQO-1 + cells) biomarkers showed an opposite pattern compared to that of creatinine level among the five groups (all p < 0.001). Combined melatonin–exendin-4 therapy offered an additional benefit in protecting the kidney from acute IR injury.