Effects and potential mechanisms of bradykinin B2 receptor on growth and biological function of human extravillous trophoblast cells / 中华围产医学杂志

Objective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells).Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized.HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group),pcDNA3.1-B2R (B2R expression plasmid group),siRNA negative control and B2R-specific siRNA,respectively.Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R,matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells.Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle,respectively.Cell migration assay and cell invasion assay were used to detect cell migration and invasion,respectively.Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells.All data were analyzed with t test.Results (1) Compared with the blank plasmid group,expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28,t=7.226,P=0.002) and protein levels (1.34 ± 0.07 vs 1.00± 0.05,t=3.727,P=0.006).And the expression of B2R in HTR 8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17,t=3.667,P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05,t=4.097,P=0.006) comparing with the siRNA negative control group.(2) Compared with the blank plasmid group,HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50 ±0.03 vs 1.34± 0.04) promoting G0/G1 to S phase transition;compared with the siRNA negative control group,B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06 ± 0.04 vs 1.20± 0.02) and arrested the cell cycle at G0/G 1 phase (all P＜0.05).(3) Compared with the blank plasmid group,B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm],the number of cells penetrating matrigel gel (360.70 ±12.33 vs 268.70 ±14.45) and the number of cells having tube-like structures (28.20 ± 2.47 vs 14.00± 1.67),while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance:(56.00±3.51) vs (87.00±1.53) μ m,number of cells penetrating matrigel gel:143.30± 12.91 vs 252.30± 17.07;number of tube-like structures:6.25±1.49 vs 15.75 ±2.02;all P＜0.05].(4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level,and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group,and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P＜0.05).Conclusions B2R might enhance the activity,migration,invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A.

The association of BDKRB2 gene polymorphism with osteoarthritis risk / 医学研究生学报

Bradykinin is a kind of inflammation mediums and plays an important role in the development of inflammation .Bra-dykinin B2 receptor(BDKRB2) mediates most bradykinins-associated inflammation,and widely exists in various tissues, including joint tissue.Recent studies increasingly suggest that BDKRB 2 gene polymorphisms is certainly associated with the onset of osteoarthritis ( OA) .This paper reviews the influence of BDKRB 2 gene polymorphism on the susceptibility , severity and related products expression of OA, which facilitates a better understanding of OA and providing new targets and theoretical basis for the OA treatment.

The role of bradykinin in lung ischemia-reperfusion injury in a rat lung transplantation model

ABSTRACT PURPOSE: To investigate the role of bradykinin in a rat lung transplantation (LTx) model and preliminarily discuss the relationship between bradykinin and CD26/DPP-4. METHODS: Rats were randomly divided into four groups: Control (CON), Sham, low potassium dextranglucose (LPD), and AB192 (n=15/group). Orthotopic single LTx was performed in the LPD and AB192 groups. The donor lungs were flush-perfused and preserved with low potassium dextranglucose (LPD) or LPD+CD26/DPP-4 catalytic inhibitor (AB192). LTx was performed after 18 h cold ischemia time and harvested two days post-LTx. Blood gas analysis (PO2), wet/dry weight ratio (W/D), myeloperoxidase activity (MPO), and lipid peroxidation (MDA) were analyzed at 48 hr after transplantation. Immunohistochemical (IHC) analysis was performed in the same sample and validated by Western-Blot. RESULTS: Compared to the LPD group, the AB192 group showed higher PO2, lower W/D ratio, and decreased MPO and MDA. IHC studies showed strong bradykinin β2 receptor (B2R) staining in the LPD group, especially in inflammatory cells, alveolar macrophages, and respiratory epithelial cells. Expression of B2R by Western-Blot was significantly different between the AB192 and LPD groups. CONCLUSION: Bradykinin may be a competitive substrate of DPP-4, and decreased bradykinin levels may enhance protective effects against ischemia/reperfusion injury during LTx.

Interference of human tissue kailikrein on renal interstitial fibrosis in rats with 5/6 nephrectomy / 中华肾脏病杂志

Objective To investigate the interference and associated mechanism of hnman tissue kallikrein (HK) gene on renal interstitial fibrosis in rats with 5/6 nephrectomy. Methods Human kallikrein cDNA was packed in a recombinant adeno-associated virus(rAAV)-based plasmid vector. The rAAV-HK was produced by transfection in 293 cells. Twenty-four male Wistsr rats were divided into sham operation and operation groups. The rats with 5/6 nephrectomy were randomly divided into simple operation, control and experiment groups. The rats in experiment group received single dose rAAV-HK via the tail vein with 1×1011 pfu. Before nephrectomy and every month after surgery until the rats were sacrificed, the caudal arterial pressure was measured using tail cuff blood pressure determinator. Three months after HK gene delivery, the rats were sacrificed. The expression of HK in rats was assessed by RT-PCR, Western blot and enzyme-linked immunosorbent assay (ELISA). The pathological changes of renal interstitium were evaluated by Masson stainning, and the distribution of bradykinin B2 receptor (BKB2R) and angiotensin Ⅱ typel receptor (ATIR) was examined by immunohistochemistry. The expressions of BKB2R, AT1R, p-MAPK protein in renal tissue were detected by Western blot. Results Three months after HK gene delivery, the systolic blood pressure of experiment group was significantly decreased compared with the control group [(163±13) nun Hg vs (217±16) mm Hg, P<0.01](1 mm Hg=0.133 kPa). Compared with sham rats, the rats in simple operation group and control group had much more renal interstitial collagen deposition and more serious fibrosis performance, but renal interstitial collagen deposition and fibrosis were significantly ameliorated in the rats of experiment group. In addition, the tubulointerstitial injury index of HK transgenic rats was significantly lower than that of the rats in control group (1.33±0.73 vs 3.01±0.62, P<0.01). Up-regnlating expression of bradykinn B2 receptor protein and down-regulating expression of AT1 receptor and p-MAPK protein were found in renal tissues of experimental group after three months (P<0.05). Conclusion HK gene delivery significantly alleviates renal interstitial fibrosis in rats with 5/6 nephrectomy through regulating the expression of bradykinin B2 receptor, AT1 receptor and p-MAPK in renal tissue.

Down-regulation of Cardiac Bradykinin B2 Receptors and eNOs mRNA in Rats with Remnant Kidneys / 华中科技大学学报（医学）（英德文版）

Summary: The changes in the expression of cardiac bradykinin B2 receptors (BKB2Rs) and endogenous nitrix oxide synthase (eNOs) mRNA were studied in rats with remnant kidneys. Thirty-two rats were divided into sham-operated and experimental groups randomly (n=16 in each group). The remnant kidney model was established by 2-stage 5/6 nephrectomy. Blood pressure and serum Cr were measured before operation and 15, 30, 60, 120 days after 5/6 nephrectomy. Eight animals in each group were killed at the first month and 4th month after the operation. The expression of BKB2Rs and eNOs mRNAs was detected by using RT-real time PCR from isolated left ventricle, and their correlation was also analyzed. The results showed that blood pressure and serum Cr were increased significantly 15 days after 5/6 nephrectomy (both P<0.01), and the hypertension and azomia existed constantly till 120 days but had no significant fluctuation. Cardiac BKB2Rs and eNOs mRNA was declined time-dependently (both P<0.05). And there was a close positive correlation between cardiac BKB2Rs and eNOs mRNA (r=0.82, P<0.01). It was suggested that a significant chronic renal failure can be produced at least 15 days after 5/6 nephrotomy and can sustain more than 4 months. The expression of BKB2Rs and eNOs was down-regulated time-dependently in this model, and there was a significant correlation between them.

Influence of Bradykinin on Catecholamine Release from the Rat Adrenal Medulla

The present study was undertaken to investigate the effect of bradykinin on secretion of catecholamines (CA) evoked by stimulation of cholinergic receptors and membrane depolarization from the isolated perfused model of the rat adrenal glands, and to elucidate its mechanism of action. Bradykinin (3 X 10 (-8) M) alone produced a weak secretory response of the CA. however, the perfusion with bradykinin (3 X 10 (-8) M) into an adrenal vein of the rat adrenal gland for 90 min enhanced markedly the secretory responses of CA evoked by ACh (5.32 X 10 (-3) M), excess K+ (5.6 X 10 (-2) M, a membrane depolarizer), DMPP (10 (-4) M, a selective neuronal nicotinic agonist) and McN-A-343 (10 (-4) M, a selective M1-muscarinic agonist). Moreover, bradykinin (3 X 10 (-8) M) in to an adrenal vein for 90 min also augmented the CA release evoked by BAY-K-8644, an activator of the dihydropyridine L-type Ca2+ channels. However, in the presence of (N-Methyl-D-Phe7) -bradykinin trifluoroacetate salt (3 X 10 (-8) M), an antagonist of BK2-bradykinin receptor, bradykinin no longer enhanced the CA secretion evoked by Ach and high potassium whereas the pretreatment with Lys- (des-Arg9, Leu8) -bradykinin trifluoroacetate salt (3 X 10 (-8) M), an antagonist of BK1-bradykinin receptor did fail to affect them. Furthermore, the perfusion with bradykinin (3 X 10 (-6) M) into an adrenal vein of the rabbit adrenal gland for 90 min enhanced markedly the secretory responses of CA evoked by excess K+ (5.6 X 10 (-2) M). Collectively, these experimental results suggest that bradykinin enhances the CA secretion from the rat adrenal medulla evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) and membrane depolarization through the activation of B2-bradykinin receptors, not through B1-bradykinin receptors. This facilitatory effect of bradykinin seems to be associated to the increased Ca2+ influx through the activation of the dihydropyridine L-type Ca2+ channels.