Study On The Role Of Janus Kinase 2 In Diabetic Erectile Dysfunction With Conditional Gene Knockout Mice

Part Ⅰ The effect of JAK2 knockout on erectile function in diabetic miceObjective: The purpose of this study was to explore the effect of JAK2 knockout on erectile function in diabetic mice.Methods: Forty 8-week-old male Cre^+/+-Jak2^fl/fl mice were divided into four groups: control,JAK2 knockout,diabetes,and diabetes with JAK2 knockout.Diabetes was induced by intraperitoneal injection of 60mg/kg/d streptozotocin for 5 consecutive days.3 months later,Jak2 knockout was induced by intraperitoneal injection of 20mg/kg/d tamoxifen dissolved in corn oil for 5 consecutive days in the diabetes with Jak2 deficiency group.After 1 month,erectile function was measured by electrical stimulation of the cavernous nerve and the ratio of maximal intracavernosal pressure（ICP）to mean systemic arterial blood pressure（MAP）was calculated.Penis samples were harvested to detect the expression and activity of relevant proteins with Western blot,immunofluorescence,immunohistochemistry,etc..Results: In mice without diabetes,knockout of JAK2 did not influence erectile function.Diabetic mice had impaired erectile function and elevated JAK2 phosphorylation（P<0.05）.JAK2 knockout could improve erectile function in diabetic mice（P<0.05）.In diabetic mice,the phosphorylation of endothelial nitric oxide synthase（eNOS）and the expression of NO-cyclic guanosine monophosphate（cGMP）pathway were reduced,whereas the expressions of Rho A-ROCK pathway,NADPH oxidases（NOXs）,malonaldehyde（MDA）,and the transforming growth factor beta 1（TGFβ1）-Smad2/3-Collagen IV pathway were all increased（P<0.05）.Caspase3 activity was also enhanced by diabetes（P<0.05）.However,JAK2 knockout could reverse these changes to some extent（P<0.05）.Conclusions: Diabetes could impair erectile function partially via activating JAK2.JAK2 knockout improved diabetic erectile dysfunction,and the underlying mechanism might involve ameliorated oxidative stress,upregulation of NO-c GMP pathway,enhanced diastolic function of smooth muscle,apoptosis reduction and cavernous fibrosis remission..Part Ⅱ The effect of JAK2 knockout on cavernous smooth muscle cells under high glucose culture conditionObjective: The purpose of this study wass to explore the effect of JAK2 knockout on cavernous smooth muscle cells（CSMCs）under high glucose condition.Methods: Primary CSMCs were extracted from an 8-week-old male Cre^+/+-Jak2^fl/fl mouse and divided into four groups: control,JAK2 knockout,high glucose and high glucose with JAK2 knockout.Cells in the former two groups were cultured in DMEM with low glucose concentration（5mmol/L）,whereas cells in the latter two groups were cultured in DMEM with high glucose concentration（30mmol/L）.JAK2 knockout was induced by the addition of 5×10^-7mol/L 4-OH-tamoxifen.1 week later,relevant proteins were detected with Western blot.The levels of reactive oxygen species（ROS）and Ca²⁺ were also measured.Results: High glucose could stimulate the phosphorylation of JAK2 in CSMCs（P<0.05）.In addition,the expression of three major NADPH oxidases including NOX1,NOX2 and NOX4,ROS level,Caspase3 activity and Ca²⁺ concentration were also increased by high glucose（P<0.05）.JAK2 knockout could reverse these changes to some extent（P<0.05）.However,JAK2 knockout had limited influence on CSMCs cultured in in DMEM with low glucose concentration.Conclusions: High glucose could impair CSMCs via activating JAK2.JAK2 knockout ameliorated apoptosis,oxidative stress and reduce Ca²⁺ concentration in CSMCs under high glucose condition.Part Ⅲ The effect of JAK2 knockout on endothelial cells under high glucose culture conditionObjective: The purpose of this study was to explore the effect of JAK2 knockout on endothelial cells（ECs）under high glucose condition.Methods: Primary ECs were extracted from the aorta of an 8-week-old male Cre^+/+-Jak2^fl/fl mouse and divided into four groups: control,JAK2 knockout,high glucose and high glucose with JAK2 knockout.Cells in the former two groups were cultured in EGM-2 with low glucose concentration（5mmol/L）,whereas cells in the latter two groups were cultured in EGM-2 with high glucose concentration（30mmol/L）.JAK2 knockout was induced by the addition of 5×10-7mol/L 4-OH-tamoxifen.1 week later,relevant proteins were detected with Western blot.The levels of reactive oxygen species（ROS）and NO were also measured.Results: High glucose could stimulate the phosphorylation of JAK2 in ECs（P<0.05）.In addition,the expression of three major NADPH oxidases including NOX1,NOX2 and NOX4,ROS level and Caspase3 activity were also increased by high glucose（P<0.05）.High glucose reduced the phosphorylation of e NOS and the concentration of NO（P<0.05）.JAK2 knockout could reverse these changes to some extent（P<0.05）.However,JAK2 knockout had limited influence on ECs cultured in EGM-2 with low glucose concentration.Conclusions: High glucose could impair ECs via activating JAK2.JAK2 knockout elevated NO level and ameliorated apoptosis and oxidative stress in ECs under high glucose condition.