Silencing of transient receptor potential canonical channel 4 inhibits endothelial progenitor cell angiogenesis by suppressing VEGF and SDF-1.

OBJECTIVES: Endothelial progenitor cells (EPCs) play a crucial role in acquired angiogenesis and endothelial injury repair. Transient receptor potential canonical channel 4 (TRPC4), a key component of store-operated calcium channels, is essential for EPC function. While the role of TRPCs has been clarified in vascular diseases, the relationship between TRPC4 and EPC function, along with the underlying molecular mechanisms, remains unclear and requires further elucidation. METHODS: EPCs were isolated from canine bone marrow and identified by morphology and flow cytometry. TRPC4 was transfected into EPCs using lentivirus or negative control, and its expression was assessed using real-time polymerase chain reaction (RT-PCR). Proliferation, migration, and tube formation were evaluated using Cell Counting Kit-8 (CCK-8), Transwell, and Matrigel assays, respectively. Levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: TRPC4 mRNA expression was significantly reduced in TRPC4-short hairpin RNA (shRNA) transfected EPCs compared to the normal control (NC)-shRNA groups. Migration and tube formation were significantly decreased after TRPC4 silencing, while proliferation showed no difference. Additionally, levels of SDF-1 and VEGF in EPCs were markedly reduced following TRPC4 silencing. CONCLUSION: TRPC4 plays a crucial role in regulating angiogenesis in EPCs. Silencing of TRPC4 can lead to decreased angiogenesis by inhibiting VEGF and SDF-1 expression, suggesting that TRPC4 knockdown might be a novel therapeutic strategy for vascular diseases.

Notoginsenoside Fc, a novel renoprotective agent, ameliorates glomerular endothelial cells pyroptosis and mitochondrial dysfunction in diabetic nephropathy through regulating HMGCS2 pathway.

BACKGROUND: Diabetic nephropathy (DN) is the primary cause of end-stage renal disease (ESRD), and the therapeutic strategies for DN are limited. Notoginsenoside Fc (Fc), a novel saponin isolated from Panax Notoginseng (PNG), has been reported to alleviate vascular injury in diabetic rats. However, the protective effects of Fc on DN remain unclear. PURPOSE: To investigate the beneficial effects and mechanisms of Fc on DN. METHODS: Db/db mice were treated with 2.5, 5 and 10 mg·kg-1·d-1 of Fc for 8 weeks. High glucose (HG) induced mouse glomerular endothelial cells (GECs) were treated with 2.5, 5 and 10 µM of Fc for 24 h. RESULTS: Our data found that Fc ameliorated urinary microalbumin level, kidney dysfunction and histopathological damage in diabetic mice. Moreover, Fc alleviated the accumulation of oxidative stress, the collapse of mitochondrial membrane potential and the expression of mitochondrial fission proteins, such as Drp-1 and Fis1, while increased the expression of mitochondrial fusion protein Mfn2. Fc also decreased pyroptosis-related proteins levels, such as TXNIP, NLRP3, cleaved caspase-1, and GSDMD-NT, indicating that Fc ameliorated GECs pyroptosis. In addition, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) expression was increased in diabetic group, which was partially abrogated by Fc. Our data further proved that knockdown of HMGCS2 could restrain HG-induced GECs mitochondrial dysfunction and pyroptosis. These results indicated that the inhibitory effects of Fc on mitochondrial damage and pyroptosis were associated with the suppression of HMGCS2. CONCLUSION: Taken together, this study clearly demonstrated that Fc ameliorated GECs pyroptosis and mitochondrial dysfunction partly through regulating HMGCS2 pathway, which might provide a novel drug candidate for DN.

Combination of Astragalus membranaceus and Panax notoginseng as Main Components in the Treatment of Diabetic Nephropathy: A Systematic Review and Meta-Analysis.

Objective: This meta-analysis evaluated the curative effect of the compatibility of Astragalus membranaceus and Panax notoginseng (ARPN) as main components on diabetic nephropathy. Methods: We used various Chinese and English databases, including the Cochrane Library, PubMed, Embase, Web of Science, the China National Knowledge Infrastructure (CNKI), China Biology Medicine Disc (SinoMed), VIP, and Wanfang, to search for randomized controlled trials on the compatibility of Astragalus membranaceus and Panax notoginseng as main components. After data extraction, meta-analysis was performed with Review Manager 5.4.0 and Stata 15, and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework was used to evaluate the quality of the evidence. Result: A total of 17 studies involving 1342 patients with diabetic nephropathy were included. Compared with the control group, ARPN can significantly improve the clinical effective rate of diabetic nephropathy (OR 5.12, 95% CI 3.42 to 7.66, P < 0.00001), and the curative effect of reducing UAER (MD -26.67, 95% CI -31.30 to -22.04, P < 0.00001) and 24 h urinary protein (SMD -0.58, 95% CI -0.75 to -0.41, P < 0.00001) is also significantly better than that of the control group, and it can also improve the renal function(Scr: MD -13.78, 95% CI -25.39 to -2.17, P=0.02; BUN: MD -0.74, 95% CI -1.27 to -0.20, P=0.007). In addition, it can also reduce glycosylated hemoglobin (SMD -1.30, 95% CI -2.33 to -0.27, P=0.01) and blood lipid(TC: SMD -0.62, 95% CI -0.95 to -0.29, P=0.0002; TG: SMD -0.47, 95% CI -0.75 to -0.19, P=0.0009; LDL: SMD -0.43, 95% CI -0.68 to -0.18, P=0.0008), and improve the TCM syndrome score (MD -4.87, 95% CI -6.17 to -3.57, P < 0.00001). Subgroup analysis suggested that the treatment plan of the control group could be the sources of heterogeneity. All the included studies had no obvious adverse effects. Conclusions: The compatibility of Radix Astragali and Radix notoginseng as the main components can effectively improve the renal function of patients with diabetic nephropathy and delay the progress of diabetic nephropathy. However, the results of this study need further research to be confirmed because of the uncertainty of the evidence and the suboptimal risk bias.

CD206+CD68+ mono-macrophages and serum soluble CD206 level are increased in antineutrophil cytoplasmic antibodies associated glomerulonephritis.

BACKGROUND: Antineutrophil Cytoplasmic Antibodies (ANCA) associated glomerulonephritis (AGN) is a group of autoimmune diseases and mono-macrophages are involved in its glomerular injuries. In this study, we aim to investigate the role of CD206+ mono-macrophages in AGN. METHODS: 27 AGN patients (14 active AGN, 13 remissive AGN) together with healthy controls (n = 9), disease controls (n = 6) and kidney function adjusted controls (n = 9) from Department of Nephrology, Ruijin hospital were recruited. Flow cytometry was used to study proportion of CD206+ cells in peripheral blood. Immunohistochemistry for CD206 staining was performed and CD206 expression was scored in different kidney regions. Serum soluble CD206 (sCD206) was measured by enzyme-linked immunosorbent assay (ELISA). We also generated murine myeloperoxidase (MPO) (muMPO) ANCA by immunizing Mpo-/- mice. Mouse bone marrow-derived macrophages (BMDMs) from wild C57BL/6 mice and peripheral blood mononuclear cell (PBMC) derived macrophages from healthy donors were treated with MPO ANCA with or without its inhibitor AZD5904 to investigate the effects of MPO-ANCA on CD206 expression. RESULTS: The proportion of peripheral CD206+CD68+ cells in active AGN patients were significantly higher than that in remissive patients (p < 0.001), healthy controls (p < 0.001) and kidney function adjusted controls (p < 0.001). Serum sCD206 level in active AGN patients was higher than that in healthy controls (p < 0.05) and remissive patients (p < 0.01). Immunohistochemistry showed CD206 was highly expressed in different kidney regions including fibrinoid necrosis or crescent formation, glomeruli, periglomerular and tubulointerstitial compartment in active AGN patients in comparison with disease controls. Further studies showed MPO ANCA could induce CD206 expression in BMDMs and PBMC derived macrophages and such effects could be reversed by its inhibitor AZD5904. CONCLUSION: ANCA could induce CD206 expression on mono-macrophages and CD206+ mono-macrophages are activated in AGN. CD206 might be involved in the pathogenesis of AAV and may be a potential target for the disease.

Notoginsenoside Fc ameliorates renal tubular injury and mitochondrial damage in acetaminophen-induced acute kidney injury partly by regulating SIRT3/SOD2 pathway.

Introduction: Mitochondria dysfunction is one of the primary causes of tubular injury in acute kidney injury (AKI). Notoginsenoside Fc (Fc), a new saponin isolated from Panax notoginseng, exhibited numerous pharmacological actions. However, the beneficial effects of Fc on renal tubular impairment and mitochondrial dysfunction in AKI have not been fully studied. Methods: In this study, we established acetaminophen (APAP)-induced AKI model in mice to examine the therapeutic impacts of Fc on AKI. Results: Our results showed that Fc could decrease the levels of the serum creatinine (Scr), blood urea nitrogen (BUN) and Cystatin C in mice with AKI. Fc also ameliorated renal histopathology, renal tubular cells apoptosis and restored expression of apoptosis-related proteins such as Bax, Bcl-2 and caspase3 (C-caspase3). Additionally, Fc increased the protein expression of SIRT3 and SOD2 in kidneys from mice with AKI. In vitro studies further showed Fc reduced the apoptosis of HK-2 cells exposure to APAP, attenuated the loss of mitochondrial membrane potential and decreased the formation of mitochondrial superoxide. Fc also partly restored the protein expression of Bax, Bcl-2, C-Caspase3, SIRT3, and SOD2 in HK-2 cells exposure to APAP. Conclusion: In summary, Fc might reduce renal tubular injury and mitochondrial dysfunction in AKI partly through the regulation of SIRT3/SOD2 pathway.

Panax notoginseng Saponin Promotes Bone Regeneration in Distraction Osteogenesis via the TGF-ß1 Signaling Pathway.

Distraction osteogenesis (DO) is an efficient strategy that is employed for the treatment of large bone defects in craniomaxillofacial surgery. Despite its utility, however, DO is associated with a prolonged consolidation phase and a high complication rate that hinder its more widespread utilization. Panax notoginseng saponin (PNS) is a traditional Chinese medicine that is frequently administered for the treatment of a range of conditions. Herein, we explored the ability of PNS treatment to influence osteogenic differentiation using both rabbit bone marrow mesenchymal cells (BMSCs) and a model of mandibular DO. BMSC proliferation was assessed via CCK-8 assay, while osteogenic differentiation was monitored through ALP and alizarin red S staining. A PCR approach was used to evaluate the expression of genes associated with osteogenesis (ALP, Runx2, and OCN) and genes linked to the TGF pathway (TßR-II, SMAD2, SMAD3, and PPM1A). For in vivo experiments, treated BMSCs were locally injected into the DO gap, with PNS being injected into treated rabbits every other day throughout the experimental period. The quality of the regenerative process was assessed via scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray imaging, and hematoxylin and eosin (H&E) staining. These analyses revealed that PNS was able to promote BMSC osteogenesis and mandibular generation, driving the upregulation of osteogenesis-related genes at the mRNA levels through the modulation of the TGF-ß1/Smad pathway. Consistently, the overexpression or silencing of TßR-II in PNS-treated BMSCs was sufficient to modulate their osteogenic potential. Analyses of in vivo mandibular DO outcomes revealed significantly augmented new bone growth in the PNS-treated group relative to control animals, with maximal osteogenesis in the group overexpressing rabbit TßR-II. Together, these results highlight the PNS as a promising and cost-effective therapeutic tool with the potential to enhance bone regeneration in clinical contexts through the modulation of the TGF-ß1/Smad pathway.

The RNA Methyltransferase METTL3 Promotes Endothelial Progenitor Cell Angiogenesis in Mandibular Distraction Osteogenesis via the PI3K/AKT Pathway.

Distraction osteogenesis (DO) is used to treat large bone defects in the field of oral and maxillofacial surgery. Successful DO-mediated bone regeneration is dependent upon angiogenesis, and endothelial progenitor cells (EPCs) are key mediators of angiogenic processes. The N6-methyladenosine (m6A) methyltransferase has been identified as an important regulator of diverse biological processes, but its role in EPC-mediated angiogenesis during DO remains to be clarified. In the present study, we found that the level of m6A modification was significantly elevated during the process of DO and that it was also increased in the context of EPC angiogenesis under hypoxic conditions, which was characterized by increased METTL3 levels. After knocking down METTL3 in EPCs, m6A RNA methylation, proliferation, tube formation, migration, and chicken embryo chorioallantoic membrane (CAM) angiogenic activity were inhibited, whereas the opposite was observed upon the overexpression of METTL3. Mechanistically, METTL3 silencing reduced the levels of VEGF and PI3Kp110 as well as the phosphorylation of AKT, whereas METTL3 overexpression reduced these levels. SC79-mediated AKT phosphorylation was also able to restore the angiogenic capabilities of METTL3-deficient EPCs in vitro and ex vivo. In vivo, METTL3-overexpressing EPCs were additionally transplanted into the DO callus, significantly enhancing bone regeneration as evidenced by improved radiological and histological manifestations in a canine mandibular DO model after consolidation over a 4-week period. Overall, these results indicate that METTL3 accelerates bone regeneration during DO by enhancing EPC angiogenesis via the PI3K/AKT pathway.

Panax notoginseng saponins promote endothelial progenitor cell angiogenesis via the Wnt/ß-catenin pathway.

BACKGROUND: Distraction osteogenesis (DO) is an effective treatment in craniomaxillofacial surgery. However, the issue of sufficient blood supply at the regeneration tissue has limited its wide application. Panax notoginseng saponins (PNS) is a Traditional Chinese Medicine that is commonly used to treat a range of angiogenic diseases. However, the mechanisms whereby PNS alters angiogenesis in endothelial progenitor cells (EPCs) have yet to be clarified. METHODS: EPCs were identified by immunofluorescence, confirmed by their uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1. EPCs were treated with different concentrations of PNS, and the effects of PNS on cell proliferation were measured on the optimal concentration of PNS determined. The effects of PNS on angiogenesis and migration, angiogenic cytokines mRNA expression and the proteins of the Wnt pathway were investigated. Then knocked down ß-catenin in EPCs and treated with the optimum concentrational PNS, their angiogenic potential was evaluated in tube formation and migration assays. In addition, the expression of cytokines associated with angiogenesis and Wnt/ß-catenin was then assessed via WB and RT-qPCR. RESULTS: We were able to determine the optimal concentration of PNS in the promotion of cell proliferation, tube formation, and migration to be 6.25 mg/L. PNS treatment increased the mRNA levels of VEGF, bFGF, VE-Cadherin, WNT3a, LRP5, ß-catenin, and TCF4. After knocked down ß-catenin expression, we found that PNS could sufficient to partially reverse the suppression of EPC angiogenesis. CONCLUSIONS: Overall, 6.25 mg/L PNS can promote EPC angiogenesis via Wnt/ß-catenin signaling pathway activation.

Evaluating renal outcome of ANCA-associated renal vasculitis: comparative study of two histopathological scoring systems.

OBJECTIVES: Renal risk score (RRS) and chronicity score (CS) are both newly proposed tools to predict end stage renal disease (ESRD) which could be applicable in antineutrophil cytoplasmic antibody (ANCA)-associated renal vasculitis patients. Their predictive value has not been fully studied and compared. METHODS: 252 patients with newly biopsy-proven ANCA-associated renal vasculitis were retrospectively studied at the Department of Nephrology, Ruijin Hospital, China. Patients were evaluated with RRS and CS for clinical factors, pathological lesions and outcome. Their predictive value of renal survival was also compared. RESULTS: The median RRS score point at diagnosis was 6 (interquartile range [IQR] 0-9) and CS score point was 4 (IQR 3-7). In accordance with severity of RRS category and CS grade, percentage of hypertensive patients, dialysis dependency, and level of proteinuria increased accordingly. Significant differences were found regarding dialysis dependency within RRS and CS groups (p<0.001 and p<0.01 respectively). The addition of RRS or CS scoring scheme to the base model of dialysis dependency significantly improved discrimination. The C statistic, integrated discrimination improvement and net reclassification improvement were significantly increased by adding either RRS/CS or both. Furthermore, RRS had better ROC. CONCLUSIONS: Among ANCA associated renal vasculitis patients, RRS and CS achieved similar discrimination, but the discrimination of RRS was superior.

Inhibitory mechanism of quercetin against the formation of 5-(hydroxymethyl)-2-furaldehyde in buckwheat flour bread by ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry.

Ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS) was employed to investigate the inhibitory mechanism of quercetin against the formation of 5-(hydroxymethyl)-2-furaldehyde (HMF) in buckwheat flour bread. The HMF and HMF precursors (3,4-dideoxyglucosone-3-ene (3,4-DGE), 3-deoxyglucosone (3-DG), or fructofuranosyl cation dehydration products (FCDPs)) adducts of quercetin were detected in buckwheat flour bread, with the trapping of these compounds by quercetin to form corresponding adducts with HMF or its precursors in 1:1, 1:2, 1:3, and 1:4 ratios (where "1" refers to quercetin in all cases). The structures of these adducts were elucidated by UPLC-HRMS/MS. Carbohydrate module labeling (CAMOLA) techniques and the labelled quercetin in model reactions were utilized to further confirm the inhibitory mechanism. Effects of baking temperature and time on the HMF inhibition rate were investigated in wheat flour bread, and a maximum inhibition rate of 86.0% was obtained with the baking of wheat flour bread (with the added quercetin concentration of 1.90mg/g) at 160°C for 30min.

[Effect of phillyrin on the anti-obesity in nutritive obesity mice].

OBJECTIVE: To study on the anti-obesity effect of phillyrin in nutritive obesity mice. METHOD: The alimentary obesity model was established by hyperalimentation. The wet weight of fat, fat index, number of fat cells per unit visual field, Lee's index, jejunum microvillus area, serum triglyceride and cholesterol were selected to observe the anti-obesity effect of phillyrin. RESULT: Phillyrin could lower wet weight of fat (P < 0.01), fat index (P < 0.05 or P < 0.01), diameter of fat cell and Lee's index (P < 0.05), decrease the jejunum microvillus area, lower the level of serum triglyceride and cholesterol. CONCLUSION: Phillyrin has anti-obesity effect in nutritive obesity mice.