Background: We tested the hypothesis that febuxostat (Feb) therapy effectively protected cardiorenal syndrome (CRS) rats via repressing the xanthine-oxidase (XO)-caused oxidative stress. Methods and Results: Cellular levels of apoptosis/oxidative stress/mitochondrial-membrane potential were higher in p-Cresol treated-NRK-52E cells than in control group that were reversed by Feb treatment or silencing XO gene (all P<0.001). Pilot study demonstrated that: XO activity was significantly increased in CRS than in SC group; a significant negative correlation between XO activity and left ventricular ejection fraction (LVEF) (%); a significant positive correlation between XO activity and BNP/BUN/creatinine/proteinuria levels (all P<0.01). Male-adult SD-rats were classified into groups 1(sham-control)/2 (CRS)/3 [CRS+Feb (10mg/kg/day)]/4 [CRS+Feb (30mg/kg/day)]. By day-63, the survival rate was significantly lower in group 2 than in other groups (P=0.029), and circulatory levels of FGF23/BNP/XO-activity BUN/creatinine/proteinuria and renal-artery resistance were highest in group 2/lowest in group 1/significantly lower in group 4 than in group 3, whereas the LVEF exhibited an opposite pattern of XO among the groups (all P<0.0001). Cellular levels of fibrosis/XO/H2DCFDA/CD68/CHAC1, and protein expressions of oxidative-stress (NOX-2/NOX-4/XO)/inflammatory (NF-κB/IL-1β)/fibrotic (Smad3/TFG-β)/apoptotic (CHAC1/2)/mitochondrial-damaged (p-DRP1) biomarkers in kidney/heart tissues displayed a similar pattern of XO (all P<0.0001). Conclusion: Feb therapy improved cardiorenal function and prognostic outcome in CRS rats.

We examined whether combined exogenous mitochondria (ExMito) and cellular prion protein overexpression (Ove-PrPC) in adipose-derived mesenchymal stem cell (Ove-PrPC in ADMSCs) therapy is superior to a single therapy for protecting the brain against intracranial hemorrhage (ICH) in rats. In vitro, compared with the control group, ExMito transfusion into recipient cells (i.e., N2a cells) significantly increased under hypoxic conditions (P < 0.001) and augmented ρ0 cell proliferation and cell-cycle activation (P < 0.001). PrPC−OE in ADMSCs exhibited higher resistance to H2O2-induced cell senescence and mitochondrial and DNA damage compared to ADMSCs (P < 0.001). Rats were categorized into group 1 (sham-control), 2 (ICH), 3 [ICH + ExMito (350 μg) by intracranial injection at 3 h after ICH], 4 [ICH + PrPC−OE in ADMSCs (6.0 × 105 cells) and intracranial injection and 1.2 × 106 cells by intravenous injection)], and 5 (ICH + combined ExMito + PrPC−OE in ADMSCs). By day 28, the brain infarct volume, brain infarct area, inflammatory cell infiltration, and biomarkers for DNA and mitochondrial damage were highest in group 2, lowest in group 1, and significantly lower in group 5 than in groups 3 and 4. NeuN cells exhibited the opposite pattern for brain infarct volume, and neurological function (corner test) significantly improved in groups 3 and 4, with further improvement in group 5 compared with that in group 2 (P < 0.0001). Combined ExMito + PrPC−OE ADMSCs therapy was superior to either therapy alone in mitigating the ICH-induced brain damage.

Background and Aims: Ischemic stroke (IS) remains the third leading cause of death, and the treatment of acute ischemic stroke (AIS) is still a formidable challenge to clinicians. This study tested the hypothesis that combined silencing Mnf2 gene in adipose-derived mesenchymal stem cells (ADMSCssil-Mnf2) and melatonin (Mel) therapy was superior to monotherapy on attenuating the brain infarct volume (BIV) and improving neurological function in AIS rats. Results: In vitro and in vivo studies were conducted. In vitro results showed that as compared with the controls (i.e., ADMSCs/N2a cells), the cellular/protein levels of oxidative stress/reactive oxygen species (ROS)/mitochondrial and DNA damaged/apoptotic/cell stress signaling (tumor necrosis factor [TNF] receptor associated factor 6/ apoptosis signal regulating kinase/MKK4/7/JUN/ERK1/2/c-Jun) biomarkers were significantly increased in these cells treated by H2O2 that were significantly reversed by ADMSCssil-Mnf2 or Mel and further significantly reversed by combined therapy (all p < 0.0001). Animals were categorized into groups 1 (sham-operated control)/2 (AIS)/3 (AIS + Mel)/4 (AIS + ADMSCssil-Mnf2)/5 (AIS + Mel-ADMSCssil-Mnf2) and euthanized by day 28 after AIS. By day 28, the BIV and the brain infarct area (BIA) were lowest in group 1/highest in group 2/significantly lower in group 5 than in groups 3 and 4/significantly increased in group 4 than in group 3, whereas the neurological function displayed an opposite manner of BIV (all p < 0.0001). The protein expressions of oxidative stress/mitochondrial damaged/apoptotic/inflammatory/cell stress signaling biomarkers displayed an identical pattern, whereas the protein expressions of mitochondrial biogenesis/antioxidants and cellular level of neuronal cells exhibited an opposite manner of BIV among the groups (all p < 0.0001). Innovation and Conclusion: ADMSCssil-Mnf2 and Mel combined therapy offered synergic effects on attenuating the BIV/BIA and preserving neurological function in rodents after AIS mainly through suppressing oxidative stress/ROS/inflammatory signalings and upregulating antioxidants. Combined ADMSCssil-Mnf2 and Mel therapy offered additional benefits on protecting the brain against AIS in rodents. Antioxid. Redox Signal. 43, 427-447.

This study presents an examination of whether the double overexpression of ZNF746 and cellular prion protein (PrPC) genes in rat adipose-derived mesenchymal stromal cells (ADMSCs) (ie, MSCDGe-OVE) offered enhanced protection to the livers of rats against ischemia‒reperfusion (IR) injury. The in vitro results revealed that compared with those of rat ADMSCs, cell activities (viability/proliferation/growth/cell cycle process) were significantly upregulated by the overexpression of either gene in rat ADMSCs and were further significantly increased by MSCDGe-OVE, whereas the expression of biomarkers of oxidative stress/ROS/apoptosis/fibrosis/autophagy decreased with increasing cell viability among the groups (all P < 0.001). Male adult SD rats (n = 50) were equally categorized into groups 1 (sham-operated-control), 2 (IR), 3 (IR-MSCOVE-PrPC), 4 (IR-MSCOVE- ZNF746), and 5 (IR-MSCDGe-OVE), and livers were harvested by day 3. By day 3, the number of circulatory inflammatory/immune cells, protein expression of oxidative stress/apoptotic/fibrotic/mitochondrial damage/autophagic biomarkers, and cellular levels of DNA damage/fibrosis/inflammation in the liver parenchyma were lowest in group 1, highest in group 2 and significantly lower in groups 3/4 than in group 5 (all P < 0.0001). Liver fibrosis detected by ultrasound and the liver injury score displayed identical patterns of circulatory levels of immune cells among the groups (all P < 0.0001). Upstream and downstream inflammatory and cell-stress signaling pathways were identified as playing crucial roles in acute liver IR injury. In conclusion, MSCDGe-OVE enhanced cell proliferation and growth and ameliorated IR-induced liver damage.

BACKGROUND:This study investigated whether the combined treatment of melatonin and tofacitinib offers enhanced protection against dextran sulfate sodium-induced acute colitis (AC) in rats. Using CCD-18Co fibroblasts and a rat colitis model, we assessed the anti-inflammatory, anti-apoptotic, and immunomodulatory effects of the combination therapy. METHODS:CCD-18Co cells were grouped as A1 (CCD-18Co), A2 (CCD-18Co + lipopolysaccharide (LPS)), A3 (CCD-18Co + LPS + Melatonin), A4 (CCD-18Co + LPS + Tofacitinib), or A5 (CCD-18Co + LPS + melatonin + tofacitinib). Sprague-Dawley rats were categorized into groups 1 (normal control), 2 (AC), 3 (AC + melatonin), 4 (AC + tofacitinib), and 5 (AC + melatonin + tofacitinib), and the colons were harvested 14 days after AC induction. KEY FINDINGS:Cell viability at time points of 24, 48, and 72 h was the highest in A1, lowest in A2, and progressively increased from A3 to A5 (all P < 0.0001). The protein expression levels of inflammatory, DNA-damaged, and autophagic (ratio of LC3-BII to LC3-BI) biomarkers displayed identical patterns of apoptosis among the groups (all P < 0.0001). Additionally, the blood stool, colon leakage, and colon injury scores were the lowest in group 1, highest in group 2, and significantly progressively reduced from groups 3 to 5 (all P < 0.0001). The protein expression of the Janus kinase family-signal transducer and activator of transcriptions/cell-stress signaling, inflammation, oxidative stress, DNA-damaged, apoptotic biomarkers, and cellular expression of immune and inflammatory factors exhibited an identical pattern of colon injury scores among the groups. CONCLUSIONS:Combined melatonin-tofacitinib treatment effectively protected the colon against dextran sulfate sodium-induced damage, mainly through the suppression of Janus kinase family-signal transducer and activator of transcriptions/cell-stress signaling, inflammation, and oxidative stress.

We tested how the coordination between cyclin D1/cyclin-dependent kinase (CDK) and the cellular prion protein (PrPC) activates mitogenic/cell proliferation signaling to improve neurological outcomes in acute ischemic stroke (AIS) rats. Compared with those in adipose-derived mesenchymal stem cells (ADMSCs) and the N2a cell line, the cell viability, cell proliferation, cell-stress signaling, and wound healing rates were significantly increased upon overexpression of PrPC (PrPC-OE) in ADMSCs (all P<0.001). The cell viability, proliferation. mitochondrial mass, and protein expression of mitogenic signaling markers (cyclin D1, cyclin E1, CDK2, and CDK4) were significantly increased upon PrPC-OE in ADMSCs compared to ADMSCs that were subjected to a significant reversal of PrPC-OE by treatment with promazine (a PrPC formation inhibitor) (all P<0.001). After 3 h of serum-free/hypoxic conditions, the protein expression levels of cyclin D1/CDK, p-Akt and mitogenic signaling markers were significantly increased upon PrPC-OE in ADMSCs compared with ADMSCs that were treated with palbociclib (a cyclin D1/CDK inhibitor). Adult male Sprague-Dawley rats (n=40) were grouped into Groups 1 (AC), 2 (AIS), 3 (AIS + ADMSCs), and 4 (AIS + ADMSCs with PrPC-OE). By Day 28 after AIS induction, the neurological function and numbers of NeuN+ cells and myelin basic protein (MBP)+ cells were lowest in Group 2, highest in Group 1 and significantly increased in Group 4 compared with Group 3, whereas the cellular levels of fibrosis and inflammation markers and protein levels of markers of apoptosis, mitochondrial and DNA damage and autophagy exhibited the opposite pattern to neurological function, and protein expression levels of cell-stress signaling proteins (PI3K, Akt, and m-TOR) and PrPC progressively increased from Groups 1 to 4 (all P<0.0001). In conclusion, activated cyclin D1/CDK coordinated with PrPC to improve neurological function in the AIS setting.

This study tested whether combined ceftriaxone and adipose-derived mesenchymal stem cells (ADMSCs) would defend the spinal cord against acute spinal infection (ASI) in rodent. Adult-Male-SD rats were grouped into groups 1 (SC)/2 (ASI)/3 (ASI + ceftriaxone from days 2 to 28 after ASI induction)/4 (ASI + allogenic ADMSCs from day 2 for a total of 3 doses/3 consecutive intervals by intravenous injection)/5 (ASI + combined ceftriaxone and ADMSC) and spinal cord tissues were harvested by day 28. Circulatory levels of TNF-α/IL-6 at days 7 and 28, and these two parameters in spinal fluid at day 28 were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3/4, and significantly lower in group 3 than in group 4 (all p < 0.0001). The day-28 bacterial colony formation unit (CFU) in vertebral bone and circulatory WBC counts at the time points of days 7/14/28, and the protein expressions of upstream (TRL-2/TLR-4/MYD88/TRAF6/IKKα/IKKβ /IKBβ/p-NF-κB) and downstream (IL-1β/IL-6/TNF-α/IFN-γ/iNOS) inflammatory signalings displayed a similar pattern of inflammatory biomarkers in spinal fluid among the groups (all p < 0.0001). By day 28, the bone injury score/bone marrow density/ratio of bone volume (BV) to the bone tissue volume (TV)/ratio of bone surface (BS) to BV/ratio of BS to bone TV/trabecular number exhibited an opposite, whereas the trabecular space exhibited an alike pattern of inflammatory biomarkers among the groups (all p < 0.0001). Combined ceftriaxone and ADMSCs therapy offered an additional benefit on protecting the vertebral bone/spinal cord against ASI damage.

Background: This study investigated whether Nanog-overexpressing adipose-derived mesenchymal stem cells (NanogOE-ADMSCs) are superior to unmodified ADMSCs in improving the left ventricular ejection fraction (LEVF) in acute myocardial infarction (AMI) patients. Methods: We utilized silencing and overexpression of Nanog gene in ADMSCs and performed a wound healing assay/transwell migration assay/MTT cell viability assay/left coronary artery ligation for AMI induction. Additionally, we categorized the cells into three classes [i.e., (ADMSCs and NanogOE-ADMSCs); A1 (ADMSCs)/A2 (ADMSCs + CoCl2)/A3 (NanogOE-ADMSCs + CoCl2)/A4 (siRNA-Nanog-ADMSCs) + CoCl2); B1 (ADMSCs)/B2 (ADMSCs + H2O2)/B3 (NanogOE-ADMSCs + H2O2)/B4 (siRNA-Nanog gene in ADMSCs + H2O2)], and the rats (n=50) were evenly divided into Groups 1 (sham-operated control)/2 (AMI)/3 (AMI+ADMSCs)/4 (AMI+NanogOE-ADMSCs)/5 (AMI+siRNA-Nanog-ADMSCs). The hearts were harvested on Day 35. Results: In vitro experiments revealed significantly higher ATP, relative mitochondrial DNA/Nonog gene expression, mitochondrial cytochrome C+ cell, angiogenesis and exosome-specific marker (Alix/CD81/CD63/CD9) levels in NanogOE-ADMSCs than in ADMSCs. The cell viability, wound healing, and migration were highest in A1, lowest in A4, and significantly greater in A3 than in A2, whereas early/late apoptosis and intracellular and mitochondrial ROS displayed the opposite pattern of cell viability among the groups (all P<0.001). Additionally, the proteins expressions of phosphorylation (p) of the PI3K/Akt/mTOR, p-JAK2/p-STAT3, and Ras/Raf/MEK1/2/ERK1/2 signaling pathways were highest in A3, lowest in A4 and significantly greater in A1 than in A2 (all P<0.001). The levels of cell cycle proteins and mitochondrial electron transport train (ETC) complex I/II/III/IV components exhibited identical patterns as PI3K/Akt/mTOR among the groups B1 to B4 (all P<0.001). On Day 35, the LVEF was highest in Group 1, lowest in Group 2, significantly greater in Group 4 than in Groups 3 and 5, and significantly greater in Group 3 than in Group 5, with the opposite pattern for the LV remodeling index, infarct and fibrosis areas, and LV chamber size (all P < 0.0001). The p-AK/p-STAT3, p-PI3K/p-Akt/p-mTOR, and Ras/Raf/MEK1/2/ERK1/2 protein levels displayed the same pattern as the LVEF among the groups (all P < 0.001). Conclusion: NanogOE-ADMSCs rescued LVEF by upregulating JAK/STAT3-mediated cell proliferation/cell stress pathways and accelerating the cell cycle.

BACKGROUND:This study tested the hypothesis that PI3K/Akt/GSK3β and TGF-β/Smad2/3 signaling play essential roles in mediating the epithelial-mesenchymal transition (EMT) and fibrosis, resulting in the deterioration of renal function and parenchyma in chronic kidney disease (CKD) rats, which is reversed by early empagliflozin treatment. METHODS AND RESULTS:NRK-52E cells were divided into the A1 (NRK-52E), A2 (NRK-52E + 200 μM p-Cresol), A3 (NRK-52E + 200 μM p-Cresol + 50 μM empagliflozin), B1 (NRK-52E), B2 (NRK-52E + 5 ng/mL TGF-β1) and B3 (NRK-52E + 5 ng/mL TGF-β1 + 50 μM empagliflozin) groups. Compared with those in the A1 group, the expression levels of proteins related to the EMT (TGF-β1/p-Smad2/p-Smad3/α-SMA), extracellular matrix (MMP2/9) and EMT (IGF-1) activators were significantly higher in the A2 group, but these changes were significantly reversed in the A3 group, whereas the protein expression levels of antifibrotic markers (TIMP1/TIMP2) exhibited the opposite pattern to the EMT-related proteins among the groups (all p<0.001). The expression of these proteins, along with the other EMT markers (snail, fibronectin, and vimentin) related to cellular function/protein expression, also exhibited an identical pattern to the A1 to A3 groups among Groups B1 to B3 (all p<0.001). Adult male SD rats were categorized into Groups 1 (sham-operated control), 2 (CKD) and 3 (CKD + empagliflozin). On Day 56 after CKD induction, the renal artery resistive index (RARI) was significantly higher in Group 2 than in Groups 1 and 3 and significantly higher in Group 3 than in Group 1 (all p<0.0001). The expression of EMT (Snail/α-SMA/fibronectin/vimentin/TGF-β1/p-Smad2/3), apoptotic (cleaved caspase-3/cleaved-PARP), inflammatory (HIF-1α/IL-1β/TNF-α/MPO/MMP-2/MMP-9), and cell stress signaling (p-PI3K/p-Akt/GSK-3β) proteins and the cellular kidney injury score, expression of fibrosis and EMT markers (Snail/vimentin)/glomerular-hypercellularity/fibrocellular crescent formation displayed an identical pattern, whereas the cellular expression of podocyte components (podocin/synaptopodin/ZO-1) displayed the opposite pattern to the RARIs among the groups (all p<0.0001). CONCLUSIONS:Empagliflozin protected kidney function and architecture mainly by suppressing fibrosis, cellular oxidative stress signaling, the EMT and inflammation.

Peritoneal fibrosis (PF) is a major complication of long-term peritoneal dialysis, leading to ultrafiltration failure and technique dropout, highlighting the urgent need for therapies that can preserve peritoneal membrane function and longevity. The present study evaluated the effectiveness of dulaglutide in preserving the functional integrity and durability of the peritoneum while inhibiting PF. In vitro Met-5A cells showed significant upregulation of inflammatory, oxidative stress, intracellular and mitochondrial reactive oxygen species (ROS), fibrotic, intracellular cytoskeletal, apoptotic and epithelial-mesenchymal transition (EMT) biomarkers, and dipeptidyl peptidase 4 (DPP4), following stimulation with a uremic toxin (p-Cresol), PF inducer [chlorhexidine gluconate (CG)] or endotoxin [lipopolysaccharide (LPS)]. Notably, these effects were significantly suppressed by dulaglutide or TGF-β/DPP4 double silencing. Furthermore, cell viability and glucagon-like peptide 1 (GLP-1) expression displayed an opposite pattern to ROS among the groups. Sprague-Dawley rats were divided into the following groups: i) Sham control (SC); ii) chronic kidney disease (CKD); iii) CKD + CG (mimicking renal failure and PF); and iv) CKD + CG + dulaglutide, and were euthanized by day 42. At this time point, the highest levels of peritoneal protein expression levels of oxidative stress (NOX-1, NOX-2 and DPP4), inflammation (NF-κB and TNF-α), angiogenesis (CD31 and von Willebrand factor) and EMT (TGF-β, Snail, β-catenin, vimentin, phosphorylated-Smad3, α-smooth muscle actin, collagen I, N-cadherin and fibronectin) factors; and cellular expression levels of fibrosis and inflammation markers, were observed in the CKD + CG group, the lowest were detected in the SC group, and the levels were significantly reduced in the CKD + CG + dulaglutide group compared with those in the CKD group. Furthermore, the expression levels of antioxidant proteins (nuclear factor erythroid 2-related factor 2, NAD(P)H quinone oxidoreductase 1 and GLP-1 receptor) exhibited an opposite trend to ROS-associated proteins among the groups. Additional Sprague-Dawley rats were categorized into the following groups: i) SC; ii) LPS-induced peritonitis; iii) LPS-induced peritonitis + dulaglutide, and were euthanized by day 5 after peritonitis induction. At this time point, flow cytometry revealed significantly increased levels of inflammatory cells (CD11b/c+, myeloperoxidase+ and Ly6G+ cells) in the circulation and abdominal fluid, and increased peritoneal permeability in the LPS-induced peritonitis group compared with those in the SC group; these levels were significantly reversed in the LPS-induced peritonitis + dulaglutide group. In conclusion, dulaglutide may effectively maintain peritoneal integrity primarily by suppressing inflammation, oxidative stress, EMT and fibrosis.

BACKGROUND:Atherogenic dyslipidemia remains an essential predictor of cardiovascular disease. This study tested whether combined dapagliflozin and alirocumab therapy could exert a synergistic effect on reducing dyslipidemia by inhibiting CD36 expression. MATERIALS AND RESULTS:In vitro experiments revealed that a stepwise increase in glyceryl trioleate (TG) or oxidized low-density lipoprotein (ox-LDL) concentrations led to increased lipid-droplet formation (LDF) in macrophages. Silencing receptor CD36, but not the LDL receptor, as well as dapagliflozin/alirocumab therapy, significantly attenuated LDF in macrophages. Lipid droplets were transferred from TG or ox-LDL-treated macrophages to untreated macrophages using tunneling nanotubes. Combined dapagliflozin/alirocumab therapy was superior to monotherapy in upregulating lipoprotein lipase activity (LPLa) and suppressing the expression levels of receptor CD36, angiopoietin-4 (ANGPT4), LDF, proinflammatory cytokines, oxidative-stress markers, and Peroxisome proliferator-activated receptor gamma (PPARγ) in macrophages and hepatocytes. This was achieved by downregulating Tyrosine-protein kinase Fyn (Fyn)/proto-oncogene tyrosine-protein kinase Src (Src)/Mitogen-Activated Protein Kinase (MAPK)-family signaling. In vivo, db/db mice were categorized into groups 1 [ db/db + high-fat diet (HFD)]/2 ( db/db + HFD + dapagliflozin)/3 ( db/db + HFD + alirocumab)/4 ( db/db + HFD + combined dapagliflozin/alirocumab). After 12 weeks of HFD feeding, circulatory levels of angiopoietin-like protein 4, TG, total cholesterol, and LDL, as well as the number of aortic atheromas, inflammatory cell count, aortic tension, and circulatory proinflammatory cytokine levels were all significantly higher in group 1, while these were significantly reversed in groups 2/3, and further significantly reversed in group 4. Meanwhile, body weight, blood sugar, HB1AC, and LPLa exhibited an opposite pattern among the groups (all P < 0.0001). CONCLUSION:Receptor CD36 plays a critical role in LDF/atherogenic dyslipidemia, which were synergistically suppressed by combined dapagliflozin-alirocumab therapy.

Background: This study tested the hypothesis that extracorporeal shockwave therapy (ECSWT) effectively rescues critical limb ischemia (CLI) in mice through the upregulation of GPR120, which protects against inflammation and angiogenesis to restore blood flow in the ischemic area. Methods and results: Compared with the control, ECSWT-induced GPR120-mediated anti-inflammatory effects significantly suppressed the expression of inflammatory signaling biomarkers (TAK1/MAPK family/NF-κB/IL-1β/IL-6/TNF-α/MCP-1) in HUVECs, and these effects were abolished by silencing GPR120 or by the GPR120 antagonist AH7614 (all P < 0.001). C57BL/6 mice (n = 40) were equally categorized into Groups 1 (sham-operated control), 2 (CLI), 3 (CLI + ECSWT), and 4 (CLI + ECSWT + AH7614). By Days 7, 14, and 28 just prior to harvesting the quadriceps muscle, the laser Doppler results showed that the ratio of ischemia to normal blood flow (INBF) in the CLI area was highest in Group 1, lowest in Group 2, and significantly greater in Group 3 than in Group 4 (all P < 0.0001). Endothelial cell markers (CD31/vWF) and GPR120 + cells exhibited identical patterns of INBF among the groups, whereas angiogenesis biomarkers (CXCR4/SDF-1/VEGF/VEGFR2) were significantly and progressively upregulated from Groups 1 to 4 (all P < 0.0001). The protein levels of inflammation (MMP-9, IL-6, and TNF-α) and oxidative stress (NOX-1 and NOX-2) and the cellular levels of inflammation (CD68+)/DNA damage (γ-H2AX+) displayed opposite patterns, whereas the small vessel density in the CLI area displayed an identical pattern of INBF among the groups (all P < 0.0001). Conclusions: ECSWT rescued CLI by increasing GPR120-mediated suppression of inflammation and enhancing angiogenesis via activation of VEGFR2.

This study revealed that adipose-derived mesenchymal stem cell-facilitated ciprofloxacin therapy effectively protected the kidney parenchyma and functional integrity against acute pyelonephritis damage in rodents. In vitro studies revealed that adipose-derived mesenchymal stem cell-derived media significantly suppressed the number of bacterial colony formation units (P < 0.0001). Additionally, the combination of adipose-derived mesenchymal stem cell-ciprofloxacin was superior to either treatment alone on suppressing lipopolysaccharide-induced inflammatory reactions in macrophages and peripheral blood-derived mononuclear cells and attenuated lipopolysaccharide-induced apoptosis/DNA damage in uroepithelial cells (Simian virus Hydrologic Unit Code 1) (all P < 0.0001). Sprague-Dawley rats were categorized into groups 1 (sham-control)/2 (acute pyelonephritis)/3 (acute pyelonephritis-ciprofloxacin)/4 (acute pyelonephritis- adipose-derived mesenchymal stem cell)/5 (acute pyelonephritis-adipose-derived mesenchymal stem cell-ciprofloxacin), and kidneys were harvested by day 5 after acute pyelonephritis induction. The in vivo results revealed that the day-5 mortality rate and creatinine levels at days 2 and 5 were significantly greater in group 2 than in groups 1 and 5 (P = 0.01), whereas the kidney injury score and inflammatory cell infiltration in the kidney were highest in group 2, lowest in group 1, and significantly greater in groups 3 and 4 than in group 5; however, there was no difference between groups 3 and 4 (all P < 0.0001). The upstream inflammatory signaling (toll-like receptor-4, myeloid differentiation primary response 88, tumor necrosis factor receptor associated factor 6 and nuclear factor-kappa B) and downstream inflammatory signaling (tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6) biomarkers exhibited identical patterns of kidney injury scores among the groups (all P < 0.0001). The results of the present study showed that adipose-derived mesenchymal stem cell-facilitated ciprofloxacin reduced inflammatory signaling-induced kidney parenchymal damage and acute pyelonephritis-induced mortality and preserved kidney function.

Background: Diabetic kidney disease (DKD) is one of the most significant public health burdens worldwide. This study explored the renal protections of combined adipose-derived mesenchymal stem cells (ADMSCs) and empagliflozin (EMPA) in DKD rats. Methods: Adult-male-SD rats were equally allocated into group 1 (sham-operated-control), group 2 (DKD), group 3 (DKD + EMPA/20 mg/kg/day since day-14 after CKD-induction), group 4 [DKD + ADMSCs (6.0 x 10(5)/ intrarenal-arterial-injection/post-day-28, followed by 1.2 x 10(6)/intravenous injection post-days 35 and 42 after CKD-induction, i.e., defined as repeated administration)] and group 5 (DKD + ADMSCs + EMPA) and kidney was harvested post-day-60 CKD-induction. Results: The result showed that the blood sugar and circulatory levels of BUN/creatinine and the ratio of urine protein/creatinine at day 60 were greatly increased in group 2 as compared the SC (i.e., group 1), significantly increased in groups 3 and 4 than in groups 5, but these parameters showed the similar manner in groups 3 and 4, except for blood sugar that was significantly lower in group 3 than in group 4 (all p < 0.0001). The protein levels of inflammation (NF-kappa B/FNF-alpha/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized protein/p22-phox)/apoptosis (cleaved-caspase-3/cleaved-PARP/mitochondrial-Bax)/fibrosis (TGF-beta/Smad 3)/mitochondrial/DNA-damaged (p-DRP1/gamma-H2AX) biomarkers revealed a similar manner of creatinine level among the groups (all p < 0.0001). Kidney injury score/fibrotic area/oxidative-stress score (8-OHdG) and cellular levels of kidneydamaged biomarkers (KIM-1/gamma-H2AX) showed a unanimous manner. In contrast, the cellular expressions of podocyte coponents (ZO-1/synaptopodin) revealed an antithetical manner of creatinine among the groups (all p < 0.0001). Conclusion: Combined ADMSCs-EMPA was superior to just one therapy for protecting kidney function and ultra structural integrity in DKD rodents.

BACKGROUND:This study investigates whether phloretin, a brain-edema inhibitor, can enhance the therapeutic effects of human-derived platelet-rich plasma (hPRP) in reducing brain hemorrhagic volume (BHV) and preserving neurological function in rodents following acute traumatic brain damage (TBD) METHODS:Forty rats were divided into five groups: sham-control, TBD, TBD + phloretin (80 mg/kg/dose intraperitoneally at 30 minutes and on days 2/3 post-TBD), TBD + hPRP (80μL by left intra-carotid-artery injection at 3 hours post-TBD), and TBD + phloretin + hPRP. Cerebral tissues were harvested on day 28 post-TBD for analysis. RESULTS:Brain MRI on day 28 showed the lowest BHV in the sham-control group and the highest in the TBD group. BHV was significantly lower in the phloretin + hPRP group compared to the phloretin or hPRP alone groups, which had similar BHV. Neurological function followed an inverse pattern to BHV. By day 28, protein levels of upstream (HGMB1, TLR-2, TLR-4, MyD88, Mal, TRAM, TRIF, TRAF6, IKK-α, IKK-ß, p-NF-κB) and downstream (IL-1ß, TNF-α, iNOS) inflammation signalings, apoptosis (caspase3, PARP), and fibrosis (Smad3, TGF-ß) biomarkers, as well as flow cytometric assessment of inflammatory cells (CD11b/c+, Ly6G+, PMO+) and early (AN-V+/PI-) and late (AN-V+/PI+) mononuclear-cell apoptosis, displayed patterns similar to BHV. The number of inflammatory (CD68+, MMP9+) and brain-swelling/myelin-damaged (AQP4+, GFAP+) mediators also followed this pattern, while neuronal-myelin (Doublecortin+, NeuN, nestin) mediators showed an inverse relationship with BHV (all p<0.0001). CONCLUSION:Combined phloretin and hPRP therapy is superior to either treatment alone in protecting the brain against TBD, primarily by suppressing inflammatory signaling and brain-swelling biomarkers.

Background: This study tested whether combined dapagliflozin (DAPA) and roxadustat (ROX) therapy was superior to a singular therapy in protecting heart and kidney functions in rats with cardiorenal syndrome (CRS). Methods and results: An in vitro study demonstrated that the cell survival (PI3K/Akt/mTOR)/cell stress (ERK1/2, JNK/p-38) signaling was significantly activated by combination therapy with ROX-DAPA (all p<0.001). Additionally, these two signaling pathways further significantly upregulated the hypoxia-induced factor (HIF)-1α which, in turn, significantly upregulated Nrf2/ARE (HO-1/NQO-1) and angiogenesis/cell-growth factors (EPO/SDF-1α/VEGF/FGF/IGF-2) and downregulated hypoxia-inducible factor prolyl-4-hydroxylase-1 (all p<0.001). Adult-male SD rats were categorized into Groups 1 (sham-operated control)/2 (CRS)/3 (CRS+ROX)/4 (CRS+DAPA)/5 (CRS+ROX+DAPA). By Day 60 after rodent CRS induction, the levels of BUN/creatinine and the ratio of urine protein to creatinine were lowest in Group 1, highest in Group 2, and significantly lower in Group 5 than in Groups 3 and 4; however, they were similar in the latter two groups, whereas the left-ventricular-ejection-fraction exhibited the opposite trend of creatinine among the groups (all p<0.0001). The protein expression levels of cell-survival (p-PI3K/p-Akt-p-mTOR)/cell-stress (p-JNK/p-p38/p-ERK1/2)/Nrf2-ARE (HO-1/NQO-1/SIRT1/SIRT3) signaling factors and angiogenesis factors (HIF-1α/VEGF/SDF-1α/FGF/IGF-2/EPO) significantly and progressively increased from Groups 1–5 (all p<0.0001). Conclusion: Combined DAPA-ROX therapy has a synergistic effect on protecting heart and kidney functions against CRS-induced damage in rodents.

Background: This study tested whether combined shock wave (SW)-facilitated melatonin (Mel) delivered into endothelial progenitor cells (EPCs) (EPC (SW-Mel)) plus S-nitroso-N-acetyl-DL-penicillamine (SNAP) was superior to merely one modality alone for improving critical limb ischemia (CLI) in rats. Methods: SD rats (n = 50) were equally categorized into group 1 (sham-control), group 2 (CLI), group 3 (CLI + SNAP), group 4 (CLI + EPC (SW-Mel) ), and group 5 (CLI + EPC (SW-Mel)+ SNAP), and ischemia-involved quadriceps were harvested by day 14. Results: An in vitro study showed that at time points of 24/48/72 h, the cell viability/protein expression of endothelial nitric oxide synthase (eNOS)/and cellular expression of nitric oxide (NO) were highest in EPCs, lowest in EPCs + menadione, and much higher in EPC (SW-Mel) + Mena than in EPCs + Mena + Mel. Protein levels of oxidative-stress (NOX-1/NOX-2/oxidized protein)/early (AN-V+/PI-)/late + /PI- )/late (AN-V+/PI+) + /PI + ) apoptosis and total intracellular/mitochondrial reactive oxygen species ROS exhibited an antithetical trend of cell viability among the groups (all P<0.0001). Matrigel assay of angiogenesis/positively-stained NO cells showed that they were much higher in EPCs + SNAP than in EPCs only (all P<0.0001). Ex vivo angiogenesis/arterial relaxation of carotid-artery rings were highest in left-common-carotid-artery (LCCA) + SNAP, lowest in LCCA + Mena, and notably higher in LCCA than in LCCA + Mena + SNAP (all P<0.0001). Laser Doppler showed ischemic to normal-blood-flow (INBF) ratio was highest in group 1, lowest in group 2, and it progressively increased from groups 3 to 5 (all P<0.0001). The protein levels of oxidative-stress (NOX-1/NOX-4/oxidized protein)/ apoptotic [cleaved-caspase-3/cleaved apoptosis/mitochondrial-damage (cytosolic-cytochrome-C/p-DRP-1)]/fibrotic (Smad3/TGF-(3)/inflammatory (MMP-9/IL-1(3/TNF-alpha/NF-kappa B) biomarkers, exhibited an opposite trend, whereas the protein level of endothelial-cell surface markers (CD31/vWF/eNOS) and number of small vessels exhibited an identical pattern of INBF ratio among the groups (all P<0.0001). Conclusions: Combined EPCSW-Mel and SNAP therapy offered a synergic effect toward rescuing from CLI.

Abstract Background This phase II randomized controlled trial tested whether the intracarotid arterial administration (ICAA) of autologous CD34 + cells to patients within 14 ± 7 days after acute ischemic stroke (IS) could be safe and further improve short- and long-term outcomes. Methods Between January 2018 and March 2022, 28 consecutive patients were equally randomly allocated to the cell-treated group (CD34 + cells/3.0 × 107/patient) or the control group (receiving optimal medical therapy). CD34 + cells were transfused into the ipsilateral brain infarct zone of cell-treated patients via the ICAA in the catheterization room. Results The results demonstrated 100% safety and success rates for the procedure, and no long-term tumorigenesis was observed in cell-treated patients. In cell-treated patients, the angiogenesis capacity of circulating endothelial progenitor cells (EPCs)/Matrigel was significantly greater after treatment than before treatment with granulocyte colony-stimulating factor (all p < 0.001). Blood samples from the right internal jugular vein of the cell-treated patients presented significantly greater levels of the stromal cell-derived factor 1α/EPC at 5, 10 and 30 min compared with 0 min (all p < 0.005). The National Institute of Health Stroke Scale scores were similar upon presentation, but a greater response was observed by Days 30 and 90 in the cell-treated group than in the control group. Tc-99 m brain perfusion was significantly greater at 180 days in the cell-treated group than in the control group (p = 0.046). The combined long-term end points (defined as death/recurrent stroke/or severe disability) were notably lower in the control group compared with the cell-treated group (14.3% vs. 50.0%, p = 0.103). Conclusion Intracarotid transfusion of autologous CD34 + cells is safe and might improve long-term outcomes in patients with acute IS. Trial registration ISRCTN, ISRCTN15677760. Registered 23 April 2018- Retrospectively registered, https://doi.org/10.1186/ISRCTN15677760

Background: This study tested the hypothesis that combined therapy with probiotics and cytokine-induced killer (CIK) cells was superior to merely one on suppressing the peritoneal carcinomatosis and liver metastasis of colorectal cancer (CRC) cells in nude mice. Methods and Results: The in vitro study revealed that in HCT 116/SW620 CRC cell lines, cell viability, proliferation, colony formation, migratory ability, wound healing, and protein expression of PD-L1 and FAK were significantly and comparably suppressed and that apoptosis was significantly and comparably increased by probiotics and CIK cells, and these effects were further significantly enhanced by combined probiotics + CIK cell therapy (all p<0.001). Nude mice were categorized into Groups 1 (SC), 2 (HCT 116), 3 (HCT 116 + probiotics), 4 (HCT 116 + CIK cells), and 5 (HCT 116 + probiotics + CIK cells). CRC cells were intraperitoneally implanted into Groups 2 to 5, and the animals were euthanized by Day 28. The results demonstrated that the abdominal dissemination of CRC cells, tumor numbers, tumor weights, liver weights, liver necrosis areas and the expression of gamma-H2AX/PD-L1/FAK in harvested liver tumors were lowest in Group 1, highest in Group 2, and significantly and progressively decreased in Groups 3 to 5 (all p<0.0001). The protein expression levels of apoptotic and DNA damage biomarkers (Bax/c-caspase 3/c-PARP/gamma-H2AX), a metastatic biomarker (FAK) and three tumor proliferation and survival signaling biomarkers (JAK-STAT1, PI3K/Akt/m-TOR and Ras/Raf/MEK/ERK) exhibited identical patterns to that of a tumor immune escape biomarker (PD-L1) among the groups (all p<0.0001). Conclusion: The combination of probiotics and CIK cells was superior to either therapy alone in suppressing CRC cell growth, proliferation, liver metastasis and survival, mainly through downregulating cell proliferation and survival signaling pathways.

This study tested the hypothesis that empagliflozin (EMPA) therapy effectively protected renal and heart functions via downregulating reactive oxygen species (ROS) and activating AMPK signaling in cardiorenal syndrome (CRS) (induced by doxorubicin-5/6 nephrectomy) rats. In vitro result showed that underwent p-Cresol treatment, the H9C2/NRK-52E cell viabilities, were significantly suppressed, whereas cellular levels of ROS and early/late apoptosis of these cells were significantly increased that were significantly reversed by EMPA treatment (all p < 0.001). The protein levels of the cell-stress/oxidative signaling (p-PI3K/p-Akt/p-mTOR/NOXs/p-DRP1) were significantly activated, whereas the mitochondrial biogenesis signaling (p-AMPK/SIRT-1/TFAM/PGC-1α) was significantly repressed in these two cell lines treated by p-Cresol and all of these were significantly reversed by EMPA treatment (all p < 0.001). Male-adult-SD rats were categorized into groups 1 [sham-operated control (SC)]/2 [SC + high protein diet (HPD) since day 1 after CKD induction]/3 (CRS + HPD)/4 (CRS + HPD+EMPA/20 mg/kg/day) and heart/kidney were harvested by day 60. By day 63, the renal function parameters (creatinine/BUN/proteinuria)/renal artery restrictive index/cellular levels of ROS/inflammation were significantly increased in group 3 than in groups 1/2, whereas heart function exhibited an opposite pattern of ROS among the groups, and all of these parameters were significantly reversed by EMPA treatment (all p < 0.0001). The protein levels of inflammation/ oxidative-stress/cell-stress signalings were highest in group 2, lowest in group 1 and significantly lower in group 4 than in group 2, whereas the AMPK-mitochondrial biogenesis displayed an opposite manner of oxidative-stress among the groups (all p < 0.0001). EMPA treatment effectively protected the heart/kidney against CRS damage via suppressing ROS signaling and upregulating AMPK-mediated mitochondrial biogenesis.