Role And Mechanism Of Immunoproteasome β5i In Lower Limb Ischemia Of Mice

Background:Atherosclerotic limb ischemia,a common disease in aging society nowadays,seriously threatens people’s health.Mild ischemia will cause intermittent claudication influencing life quality.Sever ischemia will lead to limb loss and even death.Both angiogenesis and inflammation play important role in the prognosis of patients with lower limb ischemia.Immunoproteasome produces the main effect in the regulation of the differentiation of T cells and the secretion of inflammatory factors.The role of β5i subunit,the main subunit of immunoproteasome,in the angiogenesis of ischemia of the lower extremity has not yet been elucidated.Objective:To investigate the effect of β5i activity regulation in lower limb ischemia of mouse.Materials and Methods:The model of lower limb ischemia was prepared using male ApoE-/-mice.The mice was divided into 4 groups:ApoE-/-group(ApoE-/-),ApoE-/-and operation group(ApoE-/--OP)),35i knockout in the ApoE-/-(DKO),β5i knockout in the ApoE-/-.and operation group(DKO-OP).Laser ultrasound Doppler was performed when determining the surgery success rate and monitoring postoperative blood perfusion.Western Blot was used to analyse the β5i level in lower limb ischemia tissue.H&E staining was used to observe the inflammation and vascular remodeling.The expression of VEGF in muscle was determined by Western Blot.Immunohistochemistry was used to determine collateral angiogenesis.Result:1.Femoral artery ligation is an effective method for the preparation of lower limb ischemia model in mice;2.The expression level and activity ofβ5i in the ApoE-/--OP group were higher than the ApoE-/-group;3.Laser Doppler monitoring showed that the recovery of ischemic blood perfusion in the lower limbs of the DKO-OP group was significantly increased compared to the ApoE-/--OP group.4.H&E staining revealed that the lower limb muscles of in the DKO-OP group showed less injury and more vessel remodeling than the ApoE-/--OP group.5.The expression of VEGF in DKO-OP group was significantly higher than that in ApoE-/--OP group by Western Blot examination.6.Immunohistochemistry showed that DKO-OP group had more angiogenesis in ischemic lower limb than ApoE-/--OP.group.Conclusion:This study showed that the immunoproteasome subunit β5i relieved the ischemic state of the lower limbs in mice by increasing the angiogenesis.Further studies showed that β5i promotes angiogenesis by regulating VEGF gene.Through this research,the pathophysiological mechanism of(35i involved in ischemia of the lower extremities was preliminarily identified,which providing a new vision for drug development to cure lower limb ischemia.