| Part ⅠConstruction of transgenic rat model and the effect of hKLK1 on erectile function of diabetic rats Objective:To identify the existence and expression of hKLK1 gene in transgenic rats.In addition,we build diabetic rat model and evaluate the effect of hKLK1 on diabetic erectile dysfunction.Methods:The homozygous transgenic rats harboring hKLK1(TGR(hKLK1))were generous gift from Max-Delbruck-Center for Molecular Medicine in Germany.The construction process can be briefly described as the microinjection of the entire hKLK1 gene fragment into the oocyte of a female rat under the control of the heavy metal responsive mouse metallothionein promoter.The stable inherited homozygous TGR(hKLK1)were verified by southern blotting and multi-passage selections.Tails from the offspring were used for hKLK1 gene detection.Intraperitoneal injection of 60 mg/kg streptozotocin(STZ)was utilized to induce diabetic rat.Rats with fasting glucose over 16.7 mM were considered as diabetic rats.Forty rats were divided into five groups:wild type rats group(WTR),transgenic rats group(TGR),wild type diabetic rats group(WTDM),transgenic diabetic rats group(TGDM),transgenic diabetic rats with HOE 140 administration group(TGDMH).Twelve weeks later,cavernous nerve electrostimulation were used to record intracavernosal pressure(ICP)and mean artery blood pressure(MAP)and calculated peak ICP/MAP and the ratio of area under the ICP curve(AUC)to MAP to evaluate erectile function of all rats.Cavernous tissues were harvested and hKLKl gene was verified at genome DNA,mRNA and protein levels.Results:All transgenic rats contained hKLKl gene in their tails’ genome DNA.After 12 weeks of STZ administration,all diabetic rats had high fasting glucose and weight loss.The ratios of peak ICP/MAP and AUC/MAP significantly decreased in WTDM group when compared to the normal rats,but they were much improved in TGDM group.However,both two ratios in TGDMH group dropped down again when hKLK1-B2R signaling was blocked by HOE140.Re-identification showed hKLKl gene was only existed and expressed in penile tissues of transgenic rats.Conclusion:The transgenic rat model and diabetic rat model are reliable and the expression of hKLK1 gene can dramatically improve the erectile function of diabetic rats induced by STZ.Part ⅡThe influence and mechanisms of hKLK1 on corpus cavernous tissue in diabetic ratsObjective:To identify the mechanisms of the improvement of diabetic erectile dysfunction by hKLKl in corpus cavernous tissue.Methods:Immunohistochemical staining(IHC)was conducted to evaluate CD31 andα-SMA location and expression in paraffin slides of cavernous tissues.Proteins were extracted from frozen cavernous tissues and western blotting(WB)was used to detect total and phosphor protein expressions of PI3K,AKT and eNOS.Then the NOS activity,down stream release of NO,cGMP,and the level of PDE5,ion channels BKCa and ICa-L were measured.The expressions of RhoA-ROCK pathway were also analyzed.To evaluate oxidative stress level,we detected the expression of advanced glycation end products receptors(RAGE),subunits of NADPH oxidase,and the level of malondialdehyde(DMA)and superoxide dismutase(SOD).We measured cavernous Caspase3,cleaved Caspase3,Bax,Bcl-2 level by WB and apoptotic index(AI)by TUNEL staining to explore the influence of hKLKl on tissue apoptosis.To elucidate how hKLKl impacts cavernous tissue fibrosis,TGFβ1 location and level in penile tissue were detected by IHC and the ratio of smooth muscle to collagen were assayed by Masson straining.WB was utilized to show the protein level of TGFβ1,smad2/3,CTGF,collagen Ⅰ and collagen Ⅳ.Results:When compared to the normal rats,WTDM group showed lower endothelium and smooth muscle content.The protein level of PI3K,AKT,eNOS and the phosphorylation of eNOS(S1177)were significantly decreased while phosphorylation of eNOS(T495)was increased.The NOS activity,down stream release of NO and cGMP declined and the protein expressions of PDE5,BKCa were also dropped down.Conversely,the expressions of ICa-L,RhoA,ROCK1 and ROCK2 were obviously increased.Meanwhile,protein level of RAGE,subunits of NADPH oxidase(NOX2,phox-P67,phox-P47),and MDA were dramatically increased and SOD was decreased.The apoptosis related protein expressions of Caspase3,Cleaved Caspase3,the ratio of Bax/Bcl-2 and the AI by TUNEL in WTDM group were markedly went up.TGFβ1-smad2/3-CTGF-collagen Ⅳ signaling was activated in cavernous tissue of diabetic rat and Masson staining showed lower ratio of smooth muscle to collagen in WTDM group.Except for BKCa,all the parameters above were much improved in TGDM group while the beneficial effects by hKLK1 were abolished by administration of HOE140.Conclusion:hKLK1 can improve erectile function of diabetic rats through activation of PI3K-AKT-eNOS-NO-cGMP pathway,inhibition of RhoA-ROCK pathway,restriction of tissue oxidative stress,apoptosis and fibrosis in corpus cavernous tissue.Part ⅢThe influence of hKLK1 on endothelial cells and cavernous smooth muscle cells in ratsObjective:To explore the effects of hKLKl on endothelial cells and cavernous smooth muscle cells in rats.Methods:Primary culture and purification of cavernous smooth muscle cells and endothelial cells were conducted from rat penis and aorta,respectively.Detection ofα-SMA and desmin in smooth muscle cells or vWF and CD31 in endothelial cells were conducted by immunofluorescence for cell type identification.The detection of hKLK1 by immunofluorescence was used for verification of endothelial cells derived from transgenic rat.Co-culture system of these two cells was built and the substrate of hKLKl,low molecular weight kininogen(LMWK),or HOE 140,LY294002,L-NAME,the inhibitors of B2R,PI3K,eNOS,were added into the small well with endothelial cells inside.The cGMP level and intracellular calcium ion concentration were detected in cavernous smooth muscle cells.Then,cavernous endothelial cells and smooth muscle cells were cultured with glucose at concentration of 5 mM or 30 mM,and treated with LMWK or HOE 140 for 3 days.cGMP level was assayed by ELISA for each group.Western blotting was performed to detect protein expressions of RhoA,ROCK1,ROCK2,NOX2,Bax and Bcl-2 and evaluate the influence of hKLKl on ROS production and apoptosis in smooth muscle cells.Results:Smooth muscle cells and aortal endothelial cells were successfully isolated and purified from corpus cavernosum of wild type and transgenic rat.When treated with LMWK,cavernous smooth muscle cells presented higher cGMP level and lower intracellular calcium ion concentration and transgenic cell type showed larger changes.When HOE140,LY294002,L-NAME were added,both two parameters changes were significantly diminished.In the high glucose condition,the cGMP level was decreased and the expressions of RhoA,ROCK1,ROCK2,NOX2 and the ratio of Bax/Bcl-2 were raised up in smooth muscle cells.The ROS production and apoptotic cells portion also grew up in wild type cavernous smooth muscle cells.Fortunately,hKLK1 could partly restore these increased indices.However,HOE 140 administration inhibited these protective effects of hKLK1.Conclusion:hKLK1 can cleave LMWK and its productions were able to activate PI3K-AKT-eNOS pathway in endothelial cells,induce cGMP generation and decrease calcium ion concentration in cavernous smooth muscle cells by binding to bradykinin receptor at physiological condition.In addition,hKLKl can restore cGMP level,inhibit RhoA-ROCK signaling,protect excessive oxidative stress and apoptosis level in cavernous smooth muscle cells induced by high glucose administration. |
