Effect of anaphylatoxin C3a, C5a on the tubular epithelial-myofibroblast transdifferentiation in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-β1 (TGF-β)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT.</p><p><b>METHODS</b>HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-β1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-β1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-β1 receptor antagonist (TGF-β1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-β1.</p><p><b>RESULTS</b>HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-β1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-β1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-β1RA (P < 0.05).</p><p><b>CONCLUSION</b>C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-β1/CTGF signaling pathway in vitro.</p>

Detection and significance of phosphatidylinositol 3-kinase in adipose tissue of polycystic ovary syndrome patients with insulin resistance / 中华妇产科杂志

Objective To investigate the expression of phosphatidylinositol 3-kinase(PI-3K)in adipose tissue of polycystic ovary syndrome patients(PCOS),and explore molecular mechanisms of insulin resistance(IR)in PCOS.Methods Samples from patients with PCOS with IR(n=19),PCOS without IR (n=10)and controls(n=15)were collected.Serum fasting insulin(FIN)and fasting plasma glucose (FPG)were measure& Insulin resistance index was calculated using homeostasis model assessment (HOMA)to analyze the relationship between these markers and IR.Western blot technique was used to detect the PI-3K p85 subunit.Gene expression of PI-3K p85 subunit was detected by reverse transcription polymerase chain reaction(RT-PCR)method.Kinase activity was detected by immunoprecipitation,thin- layer chromatography and gamma scintillation counting.Results(1)The levels of FIN[(25.2?3.8) mU/L]and HOMA-IR(1.6?0.3)in PCOS with IR were significantly higher than those in PCOS without IR[(13.4 +3.8)mU/L,0.9?0.3]and controls[(9.5 +2.6)mU/L,0.5?0.3;all P＜0.05).(2) There was no significant difference in the protein(0.65?0.10)and gene expression(0.92?0.12)of PI-3 K p85 subunit in PCOS with IR compared with PCOS without IR(0.72?0.10,1.01?0.10)and control groups(0.73?0.14,1.00?0.12;P＞0.05).(3)PI-3K activity in PCOS with IR(81%)and PCOS without IR(89%)was significantly decreased(P＜0.01,P＜0.05)and negatively correlated with HOMA- IR(r=-0.69,P＜0.01;r=-0.62,P＜0.05).Conclusions No significant difference in the protein and gene expression of PI-3K p85 subunit in PCOS with IR is found.The decreased PI-3K activity may lead to IR of PCOS.