Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3.

OBJECTIVE: The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs. METHODS: Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining. RESULTS: The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol. CONCLUSION: Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

Loganin alleviates macrophage infiltration and activation by inhibiting the MCP-1/CCR2 axis in diabetic nephropathy.

BACKGROUND/AIMS: The theory of inflammation is one of the important theories in the pathogenesis of diabetic nephropathy (DN). We herein aimed to explore whether loganin affected macrophage infiltration and activation upon diabetic nephropathy (DN) by a spontaneous DN mice and a co-culture system of glomerular mesangial cells (GMCs) and macrophage cells (RAW264.7) which was induced by advanced glycation end products (AGEs). METHODS AND KEY FINDINGS: Loganin showed remarkable capacity on protecting renal from damage by mitigating diabetic symptoms, improving the histomorphology of the kidney, decreasing the expression of extracellular matrix such as FN, COL-IV and TGF-ß, reversing the production of IL-12 and IL-10 and decreasing the number of infiltrating macrophages in the kidney. Moreover, loganin showed markedly effects by suppressing iNOS and CD16/32 expressions (M1 markers), increasing Arg-1 and CD206 expressions (M2 markers), which were the phenotypic transformation of macrophage. These effects may be attributed to the inhibition of the receptor for AGEs (RAGE) /monocyte chemotactic protein-1 (MCP-1)/CC chemokine receptor 2 (CCR2) signaling pathway, with significantly down-regulated expressions of RAGE, MCP-1 and CCR2 by loganin. Loganin further decreased MCP-1 secretion when RAGE was silenced, which means other target was involved in regulating the MCP-1 expression. While loganin combinated with the inhibitor of CCR2 exerted stronger anti-inhibition effects of iNOS expression, suggesting that CCR2 was the target of loganin in regulating the activation of macrophages. SIGNIFICANCE: Loganin could ameliorate DN kidney damage by inhibiting macrophage infiltration and activation via the MCP-1/CCR2 signaling pathway in DN.

Synergistic protection of tetramethylpyrazine phosphate and borneol on brain microvascular endothelium cells injured by hypoxia.

The combination of tetramethylpyrazine phosphate (TMPP) and borneol (BO) protects against cerebral ischemia. However, the mechanism for their synergistic effect is unclear. In this study, an oxygen-glucose deprivation (OGD) injured brain model was induced in microvascular endothelium cells (BMECs). TMPP and BO concentrations were optimized according to an MTT assay. Cells were divided into five groups: control, model, TMPP, BO, and TMPP+BO. Subsequently, oxidative stress was evaluated based on the levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS). Intracellular calcium ([Ca2+]i) was detected using a laser confocal microscope. Cellular apoptosis was examined via Hoechst 33342 staining, flow cytometry, and expression of p53, B-cell lymphoma 2 (BCL-2), BCL-2-like protein 4 (BAX), and caspase-3 mRNA. Angiogenesis was evaluated based on expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), fibroblast growth factor receptor 1 (FGFR1), Vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2. Results showed that 5.0 µM TMPP and 0.5 µM BO were optimal. Monotherapy significantly enhanced CAT, BCL-2, and VEGF, and also reduced [Ca2+]i, apoptosis, and BAX. TMPP increased SOD, GSH-Px, and bFGF, and reduced MDA, ROS, p53, and caspase-3 levels. BO reduced VEGFR1 expression. TMPP+BO combination exhibited synergistic effects in decreasing apoptosis, and modulating expression of BCL-2, BAX, and VEGFR1. These results indicate that protection of OGD-injured BMECs by TMPP+BO combination involves anti-oxidation, apoptosis inhibition, and angiogenesis. Moreover, their synergistic mechanism was mainly related to the regulation of apoptosis and angiogenesis.

Effects of continuous haemofiltration on serum enzyme concentrations, endotoxemia, homeostasis and survival in dogs with severe heat stroke.

AIM: To examine the effectiveness of continuous haemofiltration as a treatment for severe heat stroke in dogs. METHODS: Dogs were randomly allocated to a control or continuous haemofiltration group (both n=8). Heat stroke was induced by placing anaesthetised dogs in a high temperature cabin simulator. Upon confirmation of heat stroke (rectal temperature>42 °C, mean arterial pressure (MAP) decrease>25 mmHg), dogs were removed from the chamber and continuous haemofiltration was initiated and continued for 3h for dogs in the continuous haemofiltration group. Dogs in the control group were observed at room temperature. RESULTS: Rectal temperature, haemodynamics, pH, blood gases and electrolyte concentrations rapidly returned to baseline in the continuous haemofiltration group, but not the control group. After 3h, rectal temperature was 36.68±0.51 °C in the continuous haemofiltration group and 39.83±1.10 °C in the control group (P<0.05). Continuous haemofiltration prevented endotoxin and all serum enzyme concentrations from increasing and caused malondialdehyde concentrations to decrease. After 3h, endotoxin concentrations were 0.14±0.02 EU ml(-1) in the continuous haemofiltration group and 0.23±0.05 EU ml(-1) in the control group (P=0.003), while malondialdehyde concentrations were 4.86±0.61 mmol l(-1) in the continuous haemofiltration group and 8.63±0.66 mmol l(-1) in the control group (P<0.001). Five dogs died in the control group within 3h, whereas no dogs died in the continuous haemofiltration group. CONCLUSIONS: Continuous haemofiltration rapidly reduced body temperature, normalised haemodynamics and electrolytes, improved serum enzyme concentrations and increased survival in dogs with heat stroke. Continuous haemofiltration may be an effective treatment for heat stroke.

[Influence of electroacupuncture plus intragastric administration of extract of hypericum perforatum L on ethology and brain microcirculation in depression rats].

OBJECTIVE: To observe the effect of combined administration of intragastric perfusion of extract of Hypericum Perforatum L (HP-L) and electroacupuncture (EA) of "Baihui" (GV 20) and "Yamen" (GV 15) on behavior and brain microcirculation in depression rats. METHODS: Female SD rats were randomized into control, model, lower-dose of HP-L (lower-dose in short, 10 mg/kg), lower-dose+ EA, higher-dose (20 mg/kg) and higher-dose+ EA groups (n = 10/group). Depression model was established by lonely raising and chronic unpredictable mild stress (tail cramping, water-deprivation, fasting, electrical shock stimulation, etc. ) for 21 days. EA (2 Hz, 1 mA) was applied to "Baihui"(GV 20) and "Yamen"(GV 15) for 20 min, once daily for 14 days. Changes of ethology including glucose-consumption during 1 h, crossing and rearing scores of open-field test during 3 min (for assessing the rats' locomoto)and laser Doppler flowmetry values of cortical regional cerebral bloodflow (r CBF) were detected, and Morris water maze test (for assessing the rats' learning-memory ability) was conducted. RESULTS: In comparison with the control group, the sucrose consumption, crossing and rearing scores of open-field test, the average swimming velocity (ASV). the ratios of path length and swimming duration near the hidden-platform and the path length and swimming duration far from the platform of Morris water maze test during 70 seconds, and the cortical r CBF value in the model group were decreased significantly (P < 0.01), while the total swimming distance and escape latency in the model group increased apparently (P < 0.01). Compared to the model group, the average sucrose consumption, crossing and rearing scores of open-field test, the ASV, and the ratios of path length and swimming duration near the platform and those far from the platform in the lower-dose. lower-dose + EA, higher-dose and higher-dose + EA groups, and the cortical r CBF in the lower-dose + EA and higher-dose + EA groups were increased considerably (P < 0.05, P < 0.01). The total swimming distances and escape latencies of lower-dose, lower-dose + EA, higher-dose and higher-dose + EA groups were significantly shortened in comparison with the model group (P < 0.05, P < 0.01). The sucrose consumption and crossing score were significantly higher in the higher-dose + EA group than the lower-dose group (P < 0.05). The escape latency was significantly shorter in the higher-dose + EA group than in the lower-dose group (P < 0.05). No significant differences were found among the lower-dose, lower-dose + EA and higher-dose groups the sucrose consumption, crossing score and escape latency: among the lower-dose, lower-dose + EA, higher-dose and higher-dose + EA groups in the rearing score and ASV; among the lower-dose, higher-dose and model groups in the cortical r CBF (P > 0.05). CONCLUSION: EA can enhance the effect of extract of HP-L in increasing sucrose consumption, crossing score and cerebral blood flow, and in shortening escape latency in depression rats, which may contribute to their effect in improving depression. But HP-L itself has no effect on cortical microcirculation.

Beneficial effect of Cyclovirobuxine D on heart failure rats following myocardial infarction.

The effect of Cyclovirobuxine D, an active ingredient from Buxus microphylla, was investigated in the potential prevention of cardiac dysfunction in rats with congestive heart failure. Heart failure was induced by left coronary artery occlusion and verified using echocardiography. Cyclovirobuxine D was administered for 30 days (0.5, 1.0 and 2.0mg/kg, ig) and mortality, cardiac function, hemodynamics, microcirculation, histology and ultrastructure assessments were observed. Results from the present study suggest that Cyclovirobuxine D is beneficial for heart failure induced by myocardial infarction and supports the potential for Cyclovirobuxine D as a new therapy for heart failure.

[The material character of sand dust and analysis on the cause of formation of the sand-dust storm in northeast district in spring of 2002].

The paper analyzed the particle distribution, pollen combination of sand dust storm in northeast district in spring, 2002. It was found that there is abundant pollen, based on pollen combination sand source at new angle can be analyzed, and the distribution laws of the bowling sand and sand dust storm in season, in yearly, in geological periods and mankind historic periods can be explained. The blowing sand and sand dust storm often appeared in geological period and mankind historic period in northeast district, and mainly appeared in winter and in spring and they dropped fluctuationally in fifty years. The frequency of the sand dust storm increased since 2000. The frequency of blowing sand and sand dust storm was controlled by the strength of the monsoon wind in winter. Which the Enino and anti-Enino alternately occurs was influenced by the periodic change of the strength of monsoon wind in winter in Eastern Asia. The cold and dry climate combination in winter and spring was favorable of the occurrence of sand dust weather. The frequency of the sand dust weather will not obviously increase in several years.