Efficacy and Safety of Acupuncture for Restless Legs Syndrome in Patients with End-Stage Renal Disease: A Randomized-Controlled Trial at Hospital-Based Hemodialysis Center.

Background: Restless legs syndrome (RLS) is frequent in patients with hemodialysis (HD) and occurs predominantly in its most severe forms. The study was conducted to evaluate the efficacy and safety of acupuncture for RLS in patients with end-stage renal disease (ESRD) at hospital-based HD center. Methods: This single-blind, randomized controlled trial was performed on patients with HD and RLS who were randomly assigned to the experimental group and control group. Data were collected using the International Restless Legs Syndrome Rating Scale (IRLSRS), Insomnia Severity Index (ISI), and heart rate variability (HRV) records at baseline, after the therapeutic course (12 times/4 weeks), and 1-week follow-up. Result: A total of 47 patients were evaluated with IRLSRS score from 11 to 30 in this study. There were 41 patients enrolled in the study based on inclusion/exclusion criteria and allocated randomly into two groups. A total of 35 participants completed the trial, including 18 subjects in the experimental group and 17 subjects in the control group. The comparison of IRLSRS and ISI showed a significant reduction between two groups after acupuncture treatment (p = 0.002, p = 0.003). The ISI after 1-week follow-up also revealed significant decrease (p = 0.003). This HRV results showed that high frequency (HF%) increased significantly (p = 0.021) and low frequency (LF%) decreased significantly in the acupuncture group (p = 0.021). The generalized estimating equation showed that the IRLSRS improved by 2.902 points (p < 0.001) in the acupuncture group compared with the control group and by 1.340 points (p = 0.003) after 1-week follow-up. There were no adverse effects observed during HD in this study. Discussion: The authors conclude that acupuncture could effectively improve the symptoms of RLS significantly. The results from this study provide clinical evidence on the efficacy and safety of acupuncture to treat the patients with RLS at the HD center.

Fibroblast Growth Factor 19 Alters Bile Acids to Induce Dysbiosis in Mice With Alcohol-Induced Liver Disease.

BACKGROUND & AIMS: Excessive alcohol consumption can lead to alcohol-associated liver disease, a spectrum of conditions ranging from steatosis to fibrosis and cirrhosis. Bile acids regulate metabolic pathways by binding to cellular and nuclear receptors, and they also interact with the gut microbiome to control microbial overgrowth. Fibroblast growth factor 19 (FGF-19) is an ileum-derived hormone induced and released in response to bile acid activation of the nuclear receptor farnesoid X receptor. FGF-19 signaling is dysregulated with ethanol consumption and is increased in patients with alcoholic hepatitis. Here, we examined the effects of FGF-19 in a mouse model of chronic + binge ethanol feeding. METHODS: After injection of adeno-associated virus-green fluorescent protein or AAV-FGF-19, female C57BL/6J mice were pair-fed a Lieber DeCarli liquid diet (5% v/v) or control diet for 10 days and were given a bolus gavage of 5% ethanol or maltose control to represent a binge drinking episode. Tissues were collected for analysis 9 hours after the binge. RESULTS: Chronic + binge ethanol feeding induced steatosis regardless of FGF-19 expression. Interestingly, FGF-19 and ethanol resulted in significantly increased liver inflammation, as measured by Il6, Tgfß, and Tnfα, compared with ethanol alone. Both ethanol and FGF-19 decreased bile acid synthesis, and FGF-19 significantly reduced secondary bile acids, leading to overgrowth of specific pathogenic bacteria including Enterococcus faecalis, Escherichia coli, and Clostridium perfringens. CONCLUSIONS: Dysregulation of FGF-19 and consequent changes in bile acid synthesis and composition during alcohol consumption may be a contributing factor to alcohol-induced liver disease and dysbiosis.

Sex Dimorphic Effects of Bile Acid Metabolism in Liver Cancer in Mice.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a male-dominant disease, but targeted sex hormone therapies have not been successful. Bile acids are a potential liver carcinogen and are biomolecules with hormone-like effects. A few studies highlight their potential sex dimorphism in physiology and disease. We hypothesized that bile acids could be a potential molecular signature that explains sex disparity in HCC. METHODS & RESULTS: We used the farnesoid X receptor knockout (FxrKO) mouse model to study bile acid-dependent HCC. Temporal tracking of circulating bile acids determined more than 80% of FxrKO females developed spontaneous cholemia (ie, serum total bile acids ≥40 µmol/L) as early as 8 weeks old. Opposingly, FxrKO males were highly resistant to cholemia, with ∼23% incidence even when 26 weeks old. However, FxrKO males demonstrated higher levels of deoxycholate than females. Compared with males, FxrKO females had more severe cholestatic liver injury and further aberrancies in bile acid metabolism. Yet, FxrKO females expressed more detoxification transcripts and had greater renal excretion of bile acids. Intervention with CYP7A1 (rate limiting enzyme for bile acid biosynthesis) deficiency or taurine supplementation either completely or partially normalized bile acid levels and liver injury in FxrKO females. Despite higher cholemia prevalence in FxrKO females, their tumor burden was less compared with FxrKO males. An exception to this sex-dimorphic pattern was found in a subset of male and female FxrKO mice born with congenital cholemia due to portosystemic shunt, where both sexes had comparable robust HCC. CONCLUSIONS: Our study highlights bile acids as sex-dimorphic metabolites in HCC except in the case of portosystemic shunt.

Prion Protein Overexpression in Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Effectively Protected Rodent Kidney Against Ischemia-Reperfusion Injury Via Enhancing ATP/Mitochondrial Biogenesis-Role of ADMSC Rejuvenation and Proliferation.

BACKGROUND: We tested the hypothesis that overexpression of cellular-prion-protein in adipose-derived mesenchymal stem cells (PrPCOE-ADMSCs) effectively protected the kidney against ischemia-reperfusion (IR) injury in rat. METHODS: Part I of cell culture was categorized into A1(ADMSCs)/A2(ADMSCs+p-Cresol)/A3(PrPCOE in ADMSCs)/A4 (PrPCOE in ADMSCs+p-Cresol). Part II of cell culture was divided into B1(ADMSCs)/B2[ADMSCs+lipopolysaccharide (LPS)]/B3(PrPCOE in ADMSCs)/B4(PrPCOE in ADMSCs+LPS). Sprague-Dawley (SD) rats (n = 50) were equally categorized into groups 1 (sham-operated-control)/2 (IR)/3 (IR+ADMSCs/6.0 × 105 equally divided into bilateral-renal arteries and 6.0 × 105 intravenous administration by 1 h after IR)/4 [IR+PrPCOE-ADMSCs (identical dosage administered as group 3)]/5 [IR+silencing PRNP -ADMSCs (identical dosage administered as group 3)], and kidneys were harvested post-day 3 IR injury. RESULTS: Part I results demonstrated that the cell viability at 24/48/72 h, BrdU uptake/number of mitDNA/APT concentration/mitochondrial-cytochrome-C+ cells and the protein expressions of ki67/PrPC at 72 h-cell culturing were significantly higher in PrPCOE-ADMSCs than in ADMSCs (all P < 0.001). The protein expressions of oxidative-stress (NOX-1/NOX2/NOX4/oxidized protein)/mitochondrial-damaged (p22-phox/cytosolic-cytochrome-C)/inflammatory (p-NF-κB/IL-1ß/TNF-α/IL-6)/apoptotic (cleaved caspase-3/cleaved-PARP) biomarkers were lowest in A1/A3 and significantly higher in A2 than in A4 (all P < 0.001). Part II result showed that the protein expressions of inflammatory (p-NF-κB/IL-1ß/TNF-α/IL-6)/apoptotic (cleaved caspase-3/cleaved-PARP) biomarkers exhibited an identical pattern of part I among the groups (all P < 0.001). The protein expressions of inflammatory (p-NF-κB/IL-1ß/TNF-α/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized-protein)/mitochondrial-damaged (cytosolic-cytochrome-C/p22-phox)/apoptotic (cleaved caspase-3/cleaved-PARP/mitochondrial-Bx)/autophagic (beclin-1/ratio of LC3B-II/LC3B-I)/fibrotic (Smad3/TGF-ß) biomarkers and kidney-injury-score/creatinine level were lowest in group 1, highest in group 2, significantly higher in group 5 than in groups 3/4 (all P < 0.0001). CONCLUSION: PrPCOE in ADMSCs rejuvenated these cells and played a cardinal role on protecting the kidney against IR injury.

Addition of adipose tissue-derived mesenchymal stem cells improves empagliflozin therapy for alleviating hyperglycemia--induced neuropathy.

BACKGROUND: We examined the impact of adipose-derived mesenchymal stem cell (ADMSC)-facilitated empagliflozin (EMPA) therapy for alleviating hyperglycemic induced neuropathy [i.e., diabetic neuropathy (DN)]. METHODS: Study constituted N2a cell culture and rats to be classified into groups 1 (sham-operated-control)/2 (DN)/3 (DN + empagliflozin/20 mg/kg/daily orally for 6 weeks since post-day-7 DN induction)/4 (DN + ADMSCs/1.2 × 106 cells by vein transfusion at time intervals of 1/3/5 weeks after DN induction)/5 (DN + empagliflozin + ADMSCs) and sacrificed by day-42 after DN induction. RESULTS: In vitro results showed that, compared to N2a cells, the cellular levels of senescence/DNA-damage and protein expressions of oxidative-stress (OS), apoptotic, autophagic and inflammatory biomarkers were significantly higher in N2a + glucose (25 mM) but were significantly reversed in N2a + glucose + ADMSCs, whereas the cellular levels of mitochondrial cytochrome C and protein levels of anti-oxidants displayed an opposite pattern of OS (all P<0.001). The above-mentioned parameters (i.e., OS/apoptosis/fibrosis/autophagy/DNA-damage) were lowest in N2a cells, highest in N2a + glucose and significantly higher in N2a + glucose + EMPA (50 µM) than in N2a + glucose + EMPA (150 µM) (all P<0.001). By days 7/14/21/28/35/42 after DN induction, the values of thermal paw-withdrawal-latency (TPWL)/mechanical-paw-withdrawal-threshold were highest in group 1 and significantly progressively increased from groups 2/4/3/5 (all P<0.0001). The cellular levels of unmyelinated C- and myelinated A-δ fibers, and protein levels of OS/apoptotic/DNA-damaged/fibrotic/autophagic/inflammatory/pain-facilitated/voltage-gated sodium channel biomarkers in L4-L5 levels of dorsal-root-ganglia exhibited an contradictory manner of TPWL among the groups (all P<0.0001). CONCLUSIONS: Combination of EMPA and ADMSC therapy was superior to either alone for improving outcomes of DN.

Interplay Between Inflammatory-immune and Interleukin-17 Signalings Plays a Cardinal Role on Liver Ischemia-reperfusion Injury-Synergic Effect of IL-17Ab, Tacrolimus and ADMSCs on Rescuing the Liver Damage.

BACKGROUND: This study tested the hypothesis that inflammatory and interleukin (IL)-17 signalings were essential for acute liver ischemia (1 h)-reperfusion (72 h) injury (IRI) that was effectively ameliorated by adipose-derived mesenchymal stem cells (ADMSCs) and tacrolimus. METHODS: Adult-male SD rats (n = 50) were equally categorized into groups 1 (sham-operated-control), 2 (IRI), 3 [IRI + IL-17-monoclonic antibody (Ab)], 4 (IRI + tacrolimus), 5 (IRI + ADMSCs) and 6 (IRI + tacrolimus-ADMSCs) and liver was harvested at 72 h. RESULTS: The main findings included: (1) circulatory levels: inflammatory cells, immune cells, and proinflammatory cytokines as well as liver-damage enzyme at the time point of 72 h were highest in group 2, lowest in group 1 and significantly lower in group 6 than in groups 3 to 5 (all p < 0.0001), but they did not differ among these three latter groups; (2) histopathology: the liver injury score, fibrosis, inflammatory and immune cell infiltration in liver immunity displayed an identical pattern of inflammatory cells among the groups (all p < 0.0001); and (3) protein levels: upstream and downstream inflammatory signalings, oxidative-stress, apoptotic and mitochondrial-damaged biomarkers exhibited an identical pattern of inflammatory cells among the groups (all p < 0.0001). CONCLUSION: Our results obtained from circulatory, pathology and molecular-cellular levels delineated that acute IRI was an intricate syndrome that elicited complex upstream and downstream inflammatory and immune signalings to damage liver parenchyma that greatly suppressed by combined tacrolimus and ADMSCs therapy.

Early and Dose-Dependent Xenogeneic Mesenchymal Stem Cell Therapy Improved Outcomes in Acute Respiratory Distress Syndrome Rodent Through Ameliorating Inflammation, Oxidative Stress, and Immune Reaction.

This study tested whether human umbilical cord-derived mesenchymal stem cells (HUCDMSCs) treatment effectively protected the rat lung against acute respiratory distress syndrome (ARDS) injury, and benefits of early and dose-dependent treatment. Rat pulmonary epithelial cell line L2 (PECL2) were categorized into G1 (PECL2), G2 (PECL2 + healthy rat lung-derived extraction/50 mg/ml co-cultured for 24 h), G3 (PECL2 + ARDS rat lung-derived extraction/50 mg/ml co-cultured for 24 h), and G4 (condition as G3 + HUCDMSCs/1 × 105/co-cultured for 24 h). The result showed that the protein expressions of inflammatory (HMGB-1/TLR-2/TLR-4/MAL/TRAM/MyD88/TRIF/TRAF6/IkB/NF-κB/IL-1ß/TNF-α), oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/ASK1/p-MKK4/p-MKK7/JNKs/JUN/cytosolic-cytochrome-C/cyclophilin-D/DRP1), and cell-apoptotic/fibrotic (cleaved-caspase 3/cleaved-PARP/TGF-ß/p-Smad3) biomarkers were significantly increased in G3 than in G1/G2 and were significantly reversed in G4 (all P < 0.001), but they were similar between G1/G2. Adult male rats (n = 42) were equally categorized into group 1 (normal control), group 2 (ARDS only), group 3 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 3 h after 48 h ARDS induction (i.e., early treatment)], group 4 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 24 h after 48 h ARDS induction (late treatment)], and group 5 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 3 h/24 h after-48 h ARDS induction (dose-dependent treatment)]. By day 5 after ARDS induction, the SaO2%/immune regulatory T cells were highest in group 1, lowest in group 2, significantly lower in group 4 than in groups 3/5, and significantly lower in group 3 than in group 5, whereas the circulatory/bronchioalveolar lavage fluid inflammatory cells (CD11b-c+/LyG6+/MPO+)/circulatory immune cells (CD3-C4+/CD3-CD8+)/lung-leakage-albumin level/lung injury score/lung protein expressions of inflammatory (HMGB-1/TLR-2/TLR-4/MAL/TRAM/MyD88/TRIF/TRAF6/IκB-ß/p-NF-κB/IL-1ß/TNF-α)/fibrotic (p-SMad3/TGF-ß), apoptosis (mitochondrial-Bax/cleaved-caspase-3)/oxidative-cell-stress (NOX-1/NOX-2/ASK1/p-MKK4/p-MKK7/p-JNKs/p-cJUN)/mitochondrial damaged (cyclophilin-D/DRP1/cytosolic-cytochrome-C) biomarkers displayed an opposite pattern of SaO2% among the groups (all P < 0.0001). Early administration was superior to and two-dose counterpart was even more superior to late HUCDMSCs treatment for protecting the lung against ARDS injury.

Repeated administration of adipose-derived mesenchymal stem cells added on beneficial effects of empagliflozin on protecting renal function in diabetic kidney disease rat.

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 × 105/intrarenal-arterial-injection/post-day-28, followed by 1.2 × 106/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-κB/FNF-α/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized protein/p22-phox)/apoptosis (cleaved-caspase-3/cleaved-PARP/mitochondrial-Bax)/fibrosis (TGF-ß/Smad 3)/mitochondrial/DNA-damaged (p-DRP1/γ-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 kidney-damaged biomarkers (KIM-1/γ-H2AX) showed a unanimous manner. In contrast, the cellular expressions of podocyte components (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.

Effect of in-hospital evolocumab therapy on lipoprotein(a) in patients with acute myocardial infarction: a retrospective cohort study and a propensity score matching analysis.

Elevated lipoprotein(a) is associated with an increased risk of atherosclerotic cardiovascular disease. Evolocumab, a proprotein convertase subtilisin/kexin type 9 inhibitor, has been shown to reduce lipoprotein(a). However, the effect of evolocumab on lipoprotein(a) in patients with acute myocardial infarction (AMI) is poorly studied. This study aims to investigate the change in lipoprotein(a) under evolocumab therapy in patients with AMI. Methods: This retrospective cohort analysis included a total of 467 AMI patients with LDL-C level >2.6 mmol/L upon admission, among whom 132 received in-hospital evolocumab (140 mg every 2 weeks) plus statin (20 mg atorvastatin or 10 mg rosuvastatin per day) and the remaining 335 received statin only. Lipid profiles at 1-month follow-up were compared between the two groups. A propensity score matching analysis was also conducted based on age, sex, and baseline lipoprotein(a) at a 1:1 ratio using a 0.02 caliper. Results: At the 1-month follow-up, the lipoprotein(a) level decreased from 27.0 (17.5, 50.6) mg/dL to 20.9 (9.4, 52.5) mg/dL in evolocumab plus statin group, but increased from 24.5 (13.2, 41.1) mg/dL to 27.9 (14.8, 58.6) mg/dL in statin only group. The propensity score matching analysis included 262 patients (131 in each group). In subgroup analysis of the propensity score matching cohort stratified by the baseline lipoprotein(a) at cutoff values of 20 and 50 mg/dL, the absolute change in lipoprotein(a) was -4.9 (-8.5, -1.3), -5.0 (-13.9, 1.9), -0.2 (-9.9, 16.9) mg/dL in three subgroups in evolocumab plus statin group, and 0.9 (-1.7, 5.5), 10.7 (4.6, 21.9), 12.2 (2.9, 35.6) mg/dL in three subgroups in statin only group. In comparison to statin only group, evolocumab plus statin group had lower lipoprotein(a) level at 1 month in all subgroups (P < 0.05). Conclusions: In-hospital initiation of evolocumab on a background statin therapy reduced lipoprotein(a) level at 1-month follow-up in patients with AMI. Evolocumab plus statin therapy inhibited the increase in lipoprotein(a) in statin only therapy, regardless of the baseline lipoprotein(a) level.

Tgr5-/- mice are protected from ethanol-induced metabolic alterations through enhanced leptin and Fgf21 signaling.

BACKGROUND: Alcohol-associated liver disease (ALD) is caused by chronic use of alcohol and ranges from hepatic steatosis to fibrosis and cirrhosis. Bile acids are physiological detergents that also regulate hepatic glucose and lipid homeostasis by binding to several receptors. One such receptor, Takeda G protein-coupled receptor 5 (TGR5), may represent a therapeutic target for ALD. Here, we used a chronic 10-day + binge ethanol-feeding model in mice to study the role of TGR5 in alcohol-induced liver injury. METHODS: Female C57BL/6J wild-type mice and Tgr5-/- mice were pair-fed Lieber-DeCarli liquid diet with ethanol (5% v/v) or isocaloric control diet for 10 days followed by a gavage of 5% ethanol or isocaloric maltose control, respectively, to represent a binge-drinking episode. Tissues were harvested 9 hours following the binge, and metabolic phenotypes were characterized through examination of liver, adipose, and brain mechanistic pathways. RESULTS: Tgr5-/- mice were protected from alcohol-induced accumulation of hepatic triglycerides. Interestingly, liver and serum levels of Fgf21 were significantly increased during ethanol feeding in Tgr5-/- mice, as was phosphorylation of Stat3. Parallel to Fgf21 levels, increased leptin gene expression in white adipose tissue and increased leptin receptor in liver were detected in Tgr5-/- mice fed ethanol diet. Adipocyte lipase gene expression was significantly increased in Tgr5-/- mice regardless of diet, whereas adipose browning markers were also increased in ethanol-fed Tgr5-/- mice, indicating potential for enhanced white adipose metabolism. Lastly, hypothalamic mRNA targets of leptin, involved in the regulation of food intake, were significantly increased in Tgr5-/- mice fed ethanol diet. CONCLUSIONS: Tgr5-/- mice are protected from ethanol-induced liver damage and lipid accumulation. Alterations in lipid uptake and Fgf21 signaling, and enhanced metabolic activity of white adipose tissue, may mediate these effects.

My lifelong dedication to bile acid research.

It is a great honor to be invited to write a reflections article on my scientific journey and lifelong bile acid research for the Journal of Biological Chemistry, in which I am proud to have published 24 articles. I have also published 21 articles in the Journal of Lipid Research, another journal of the American Society of Biochemistry and Molecular Biology. I begin my reflections from my early education in Taiwan, my coming to America for graduate study, and continue with my postdoctoral training in cytochrome P450 research, and my lifelong bile acid research career at Northeast Ohio Medical University. I have witnessed and helped in the transformation of this rural not so visible medical school to a well-funded leader in liver research. Writing this reflections article on my long and rewarding journey in bile acid research brings back many good memories. I am proud of my scientific contributions and attribute my academic success to hard work, perseverance, good mentoring, and networking. I hope these reflections of my academic career would help inspire young investigators to pursue an academic career in biochemistry and metabolic diseases.

Bile acids as metabolic regulators: an update.

PURPOSE OF REVIEW: This review aims to provide a concise update on recent advances in understanding of the bile acid metabolism and signaling in health and diseases. RECENT FINDINGS: CYP2C70 has been identified as the murine cytochrome p450 enzyme that mediates the synthesis of muricholic acids to account for the major different bile acid composition between human and mice. Several studies have linked nutrient sensing bile acid signaling to the regulation of hepatic autophagy-lysosome activity, an integral pathway of the cellular adaptive response to starvation. Distinct bile acid-mediated signaling mechanisms have been shown to contribute to the complex metabolic changes post bariatric surgery, suggesting that pharmacological manipulation of the enterohepatic bile acid signaling could be a potential nonsurgical alternative to weight loss surgery. SUMMARY: Basic and clinical studies have continued to discover novel roles of the enterohepatic bile acid signaling in regulation of key metabolic pathways. Such knowledge forms the molecular basis needed for developing safe and effective bile acid-based therapeutics for treating metabolic and inflammatory diseases.

Jagged/Notch proteins promote endothelial-mesenchymal transition-mediated pulmonary arterial hypertension via upregulation of the expression of GATAs.

This study tested the hypothesis that Jagged2/Notches promoted the endothelial-mesenchymal transition (endMT)-mediated pulmonary arterial hypertension (PAH) (i.e. induction by monocrotaline [MCT]/63 mg/kg/subcutaneous injection) through increasing the expression of GATA-binding factors which were inhibited by propylthiouracil (PTU) (i.e. 0.1% in water for daily drinking since Day 5 after PAH induction) in rodent. As compared with the control (i.e. HUVECs), the protein expressions of GATAs (3/4/6) and endMT markers (Snail/Zeb1/N-cadherin/vimentin/fibronectin/α-SMA/p-Smad2) were significantly reduced, whereas the endothelial-phenotype markers (CD31/E-cadherin) were significantly increased in silenced JAG2 gene or in silenced GATA3 gene of HUVECs (all p < 0.001). As compared with the control, the protein expressions of intercellular signallings (GATAs [3/4/6], Jagged1/2, notch1/2 and Snail/Zeb1/N-cadherin/vimentin/fibronectin/α-SMA/p-Smad2) were significantly upregulated in TGF-ß/monocrotaline-treated HUVECs that were significantly reversed by PTU treatment (all p < 0.001). By Day 42, the results of animal study demonstrated that the right-ventricular systolic-blood-pressure (RVSBP), RV weight (RVW) and lung injury/fibrotic scores were significantly increased in MCT group than sham-control (SC) that were reversed in MCT + PTU groups, whereas arterial oxygen saturation (%) and vasorelaxation/nitric oxide production of PA exhibited an opposite pattern of RVW among the groups (all p < 0.0001). The protein expressions of hypertrophic (ß-MHC)/pressure-overload (BNP)/oxidative-stress (NOX-1/NOX-2) biomarkers in RV and the protein expressions of intercellular signalling (GATAs3/4/6, Jagged1/2, notch1/2) and endMT markers (Snail/Zeb1/N-cadherin/vimentin/fibronectin/TGF-ß/α-SMA/p-Smad2) in lung parenchyma displayed an identical pattern of RVW among the groups (all p < 0.0001). Jagged-Notch-GATAs signalling, endMT markers and RVSBP that were increased in PAH were suppressed by PTU.

Adipose-derived mesenchymal stem cells overexpressing prion improve outcomes via the NLRP3 inflammasome/DAMP signalling after spinal cord injury in rat.

Traumatic spinal cord injury (SCI) is a highly destructive disease in human neurological functions. Adipose-derived mesenchymal stem cells (ADMSCs) have tissue regenerations and anti-inflammations, especially with prion protein overexpression (PrPcOE ). Therefore, this study tested whether PrPcOE -ADMSCs therapy offered benefits in improving outcomes via regulating nod-like-receptor-protein-3 (NLRP3) inflammasome/DAMP signalling after acute SCI in rats. Compared with ADMSCs only, the capabilities of PrPcOE -ADMSCs were significantly enhanced in cellular viability, anti-oxidative stress and migration against H2 O2 and lipopolysaccharide damages. Similarly, PrPcOE -ADMSCs significantly inhibited the inflammatory patterns of Raw264.7 cells. The SD rats (n = 32) were categorized into group 1 (Sham-operated-control), group 2 (SCI), group 3 (SCI + ADMSCs) and group 4 (SCI + PrPcOE -ADMSCs). Compared with SCI group 2, both ADMSCs and PrPcOE -ADMSCs significantly improved neurological functions. Additionally, the circulatory inflammatory cytokines levels (TNF-α/IL-6) and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/TRIF/c-caspase8/FADD/p-NF-κB/NEK7/NRLP3/ASC/c-caspase1/IL-ß) and by Day 42 the protein expressions of DAMP-inflammatory signalling (HGMB1/TLR-4/MyD88/TRIF/TRAF6/p-NF-κB/TNF-α/IL-1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPcOE -ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.

Combined therapy with dapagliflozin and entresto offers an additional benefit on improving the heart function in rat after ischemia-reperfusion injury.

BACKGROUND: This study tested whether combined dapagliflozin and entresto treatment would be superior to either one alone for preserving the left-ventricular ejection-fraction (LVEF) in rat after ischemia-reperfusion (IR) injury. METHODS: Cell culture using H9C2 cells and IR injury in rat with dapagliflozin-entresto treatment were conducted in the present study. RESULTS: In vitro flow-cytometric result showed that the intracellular and mitochondrial reactive oxygen species and mitochondrial permeability transition pore, and protein levels of oxidative-stress/DNA-damaged markers [NADPH-oxidase-1 (NOX-1)/NOX-2/oxidized-protein/γ-H2A-histone-family member X (γ-H2AX)] were significantly higher in hydrogen peroxide (H2O2) (300µM)-treated H9C2 cells as compared with the controls that were significantly reversed in sacubitril/valsartan and dapagliflozin therapy in the same H2O2-treated condition, whereas the protein expressions of antioxidants [Sirtuin-1 (SIRT1)/SIRT3/superoxide dismutase/catalase/glutathione peroxidase) exhibited an opposite pattern among the groups (all p<0.001). Adult-male-Sprague-Dawley rat (n=40) were equally categorized into group 1 (sham-operated control), group 2 (IR), group 3 (IR+dapagliflozin/20mg/kg/orally at 3h and post-days 1/2/3 after IR), group 4 (IR+entresto/100mg/kg/orally at 3h and post-days 1/2/3 after IR) and group 5 (IR+dapagliflozin+entresto) and the hearts were harvested by day 3 after IR. The 3rd day's LVEF was highest in group 1, lowest in group 2 and significantly higher in group 5 than in groups 3/4, but it was similar between the latter two groups (p<0.001). The protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein), fibrotic (transforming-growth factor-ß/phosphorylated-Smad3), apoptotic [mitochondrial-Bax/cleaved-caspase-3/cleaved-poly (ADP-ribose) polymerase], mitochondria/DNA damaged (cytosolic-cytochrome-c/γ-H2AX), pressure-overload/heart-failure [brain natriuretic peptide (BNP)/ß-myosin heavy chain] and autophagic (ratio of meiotic cyclins CLB3-II/CLB3-I) biomarkers, and the upstream (high-mobility group box 1/Toll-like receptor-4/MyD88/phosphorylated-nuclear factor-κB and downstream [interleukin (IL)-1ß/IL-6/tumor necrosis factor-α] inflammatory signalings revealed an antithetical features of LVEF among the groups (all p<0.0001). The cellular levels of inflammatory (myeloperoxidase+/CD68+), pressure-overload/heart-failure (BNP+) and DNA-damage (γ-H2AX+) biomarkers as well as infarct area demonstrated an opposite pattern of LVEF among the groups (all p<0.0001). CONCLUSION: Incorporated entresto-dapagliflozin treatment was superior to either one alone on protecting the heart against IR injury.

Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis-Pharmacomodulation of Rosuvastatin.

This study tested the hypothesis that intrarenal arterial transfusion of oxidized low-density lipoprotein (ox-LDL) jeopardized the residual renal function and kidney architecture in rat chronic kidney disease ((CKD), i.e., induced by 5/6 nephrectomy) that was reversed by rosuvastatin. Cell culture was categorized into A1 (NRK-52E cells), A2 (NRK-52E + TGF-ß), A3 (NRK-52E + TGF-ß + ox-LDL) and A4 (NRK-52E + TGF-ß + ox-LD). The result of in vitro study showed that cell viability (at 24, 48 and 72 h), NRK-52E ox-LDL-uptake, protein expressions of epithelial−mesenchymal−transition (EMT) markers (i.e., p-Smad2/snail/α-SMA/FSP1) and cell migratory and wound healing capacities were significantly progressively increased from A1 to A4 (all p < 0.001). SD rats were categorized into group 1 (sham-operated control), group 2 (CKD), group 3 (CKD + ox-LDL/0.2 mg/rat at day 14 after CKD induction) and group 4 (CKD + ox-LDL-treated as group 3+ rosuvastatin/10 mg/kg/day by days 20 to 42 after CKD induction) and kidneys were harvested at day 42. The circulatory levels of BUN and creatinine, ratio of urine-protein to urine-creatinine and the protein expressions of the above-mentioned EMT, apoptotic (cleaved-caspase3/cleaved-PARP/mitochondrial-Bax) and oxidative-stress (NOX-1/NOX-2/oxidized-protein) markers were lowest in group 1, highest in group 3 and significantly higher in group 4 than in group 2 (all p < 0.0001). Histopathological findings demonstrated that the kidney injury score, fibrotic area and kidney injury molecule-1 (KIM-1) displayed an identical pattern, whereas the cellular expression of podocyte components (ZO-1/synaptopodin) exhibited an opposite pattern of EMT markers (all p < 0.0001). In conclusion, ox-LDL damaged the residual renal function and kidney ultrastructure in CKD mainly through augmenting oxidative stress, EMT and fibrosis that was remarkably reversed by rosuvastatin.

Synergic Effect of Combined Therapy of Hyperbaric Oxygen and Adipose-Derived Mesenchymal Stem Cells on Improving Locomotor Recovery After Acute Traumatic Spinal Cord Injury in Rat Mainly Through Downregulating Inflammatory and Cell-Stress Signalings.

This study tested whether combined hyperbaric oxygen (HBO) and allogenic adipose-derived mesenchymal stem cells (ADMSCs) would be superior to either one for improving the locomotor recovery in rat after acute traumatic spinal cord injury (TSCI) in rat. Adult-male Sprague-Dawley rats were equally categorized into group 1 (sham-operated control), group 2 (TSCI), group 3 (TSCI + HBO for 1.5 h/day for 14 consecutive days after TSCI), group 4 (TSCI + ADMSCs/1.2 × 106 cells by intravenous injection at 3 h and days 1/2 after TSCI), and group 5 (TSCI + HBO + ADMSCs), euthanized, and spinal cord tissue was harvested by day 49 after TSCI. The protein expressions of oxidative-stress (NOX-1/NOX-2), inflammatory-signaling (TLR-4/MyD88/IL-1ß/TNF-α/substance-p), cell-stress signaling (PI3K/p-AKT/p-mTOR), and the voltage-gated sodium channel (Nav1.3/1.8/1.9) biomarkers were highest in group 2, lowest in group 1, and significantly lower in group 5 than in groups 3/4 (all P <0.0001), but they did not differ between groups 3 and 4. The spinal cord damaged area, the cellular levels of inflammatory/DNA-damaged biomarkers (CD68+/GFAP+/γ-H2AX+ cells), mitogen-activated protein kinase family biomarkers (p-P38/p-JNK/p-ERK1/2), and cellular expressions of voltage-gated sodium channel (Nav.1.3, Nav.1.8, and Nav.1.9 in NF200+ cells) as well as the pain-facilitated cellular expressions (p-P38+/peripherin+ cells, p-JNK+/peripherin+ cells, p-ERK/NF200+ cells) exhibited an identical pattern of inflammation, whereas the locomotor recovery displayed an opposite pattern of inflammation among the groups (all P < 0.0001). Combined HBO-ADMSCs therapy offered additional benefits for preserving the neurological architecture and facilitated the locomotor recovery against acute TSCI.

Rejuvenated endothelial progenitor cells through overexpression of cellular prion protein effectively salvaged the critical limb ischemia in rats with preexisting chronic kidney disease.

BACKGROUND: This study tested the hypothesis that overexpression of cellular prion protein in endothelial progenitor cells (PrPcOE-EPCs), defined as "rejuvenated EPCs," was superior to EPCs for salvaging the critical limb ischemia (CLI) induced after 28-day chronic kidney disease (CKD) induction in rat. METHODS AND RESULTS: Cell viability and flow cytometric analyses of early/late apoptosis/total-intracellular ROS/cell cycle (sub-G1, G2/M phase) were significantly higher in EPCs + H2O2 than in EPCs that were significantly reversed in PrPcOE-EPCs + H2O2 (all p < 0.001). The protein expressions of inflammation (IL-1ß/IL-6/MMP-9/p-NF-κB) were significantly increased in EPC + TNF-α than in EPCs that were significantly reversed in PrPcOE-EPCs + TNF-α (all p < 0.001). Adult-male SD rats (n = 8/each group) were categorized into group 1 (sham-operated control), group 2 (CKD + CLI), group 3 [CKD + CLI + EPCs by intravenous (0.6 × 105)/intra-muscular (0.6 × 105) injections at 3 h after CLI induction], group 4 (CKD + CLI + PrPcOE-EPCs/dose-administration as group 3) and group 5 (CKD + CLI + siPrnp-EPCs/dose-administration as group 3). By day 14 after CLI induction, the ratio of ischemia to normal blood flow (INBF) in CLI area was highest in group 1/lowest in group 2/significantly higher in group 4 than in groups 3/5 and significantly higher in group 3 than in group 5 (all p < 0.0001). Histopathology demonstrated that the angiogenesis (number of small vessels/CD31 + cells) exhibited a similar trend, whereas the fibrosis/kidney injury score exhibited an opposite pattern of INBF among the groups (all p < 0.0001). The protein expressions of angiogenesis (SDF-1α/VEGF/CXCR4)/cell-stress signaling (p-PI3K/p-Akt/p-m-TOR) were significantly and progressively increased from groups 1-4 that were reversed in group 5 (all p < 0.0001). The protein expressions of fibrotic (p-Smad3/TGF-ß)/oxidative-stress (NOX-1/NOX-2/oxidized-protein)/apoptotic (mitochondrial-Bax/cleaved caspase3/cleaved PARP)/mitochondrial-damaged (cytosolic-cytochrome-C) biomarkers displayed an opposite pattern of INBF among the groups (all p < 0.0001). CONCLUSION: PrPcOE-EPCs were superior to EPCs only therapy for salvaging the CLI.

Exploring the pivotal variables of tongue diagnosis between patients with acute ischemic stroke and health participants.

Background and aim: Stroke is a major cause of disability worldwide, and ischemic stroke is the most common type of stroke. The prevention and treatment of ischemic stroke remain a challenge worldwide. Traditional Chinese medicine (TCM) is often sought to provide an alternative therapy for the prevention and rehabilitation intervention of ischemic stroke in Taiwan. Therefore, this study explored the pivotal variables of tongue diagnosis among acute ischemic stroke and healthy participants in middle and older age. Experimental procedure: This was a cross-sectional and case-controlled study. Data were collected from 99 patients with acute ischemic stroke and 286 healthy participants who received tongue diagnoses at Changhua Christian Hospital (CCH) from September 1, 2014, to December 31, 2016. Tongue features were extracted using the automatic tongue diagnosis system. Nine tongue features, including tongue shape, tongue color, fur thickness, fur color, saliva, tongue fissures, ecchymoses, teeth marks, and red spots were analyzed. Results and conclusion: Objective image analysis techniques were used to identify significant differences in the many tongue features between patients with acute ischemic stroke and individuals without stroke. According to the logistic regression analysis, pale tongue color (OR:5.501, p = 0.001), bluish tongue color (OR:4.249, p = 0.014), ecchymoses (OR:1.058, p < 0.001), and tongue deviation angle (OR:1.218, p < 0.001) were associated with significantly increased odds ratios for acute ischemic stroke. The research revealed that tongue feature abnormalities were significantly related to the occurrence of ischemic stroke.

Interactive Relationships between Intestinal Flora and Bile Acids.

The digestive tract is replete with complex and diverse microbial communities that are important for the regulation of multiple pathophysiological processes in humans and animals, particularly those involved in the maintenance of intestinal homeostasis, immunity, inflammation, and tumorigenesis. The diversity of bile acids is a result of the joint efforts of host and intestinal microflora. There is a bidirectional relationship between the microbial community of the intestinal tract and bile acids in that, while the microbial flora tightly modulates the metabolism and synthesis of bile acids, the bile acid pool and composition affect the diversity and the homeostasis of the intestinal flora. Homeostatic imbalances of bile acid and intestinal flora systems may lead to the development of a variety of diseases, such as inflammatory bowel disease (IBD), colorectal cancer (CRC), hepatocellular carcinoma (HCC), type 2 diabetes (T2DM), and polycystic ovary syndrome (PCOS). The interactions between bile acids and intestinal flora may be (in)directly involved in the pathogenesis of these diseases.