The Experimental Study On IGFBP-4Protecting Hindlimb Ischemic Injury

Hindlimb ischemia is the most common type of peripheral arterial disease (PAD),which can develop into serious limb ischemia requiring amputation. The quality of life inaffected patients is rated even lower than patients with chronic heart disease and cancerpatients. Current clinical treatment of hindlimb ischemia involve prescription drugs andsurgery, although there are limitations with each method. The pathological process ofhindlimb ischemia include vascular occlusion of the hindlimb muscle resulting in ahypoxic change, which induces production of oxygen free radicals, resulting in theoccurrence of DNA damage.Studies have shown that the Wnt signaling pathway plays an important role in therepair process of hindlimb ischemic injury. We also know that there is a very closerelationship between the activation of canonical Wnt signaling pathway and DNA damage,which prompted us to examine whether the inhibition of canonical Wnt pathway results ina reduction of DNA damage, which can play a possible role in protecting hindlimbischemia injury. Recent studies suggest that insulin-like growth factor binding protein4(IGFBP-4) is a strong inhibitor of canonical Wnt signaling. This study investigates therelationship between IGFBP-4and its role in hindlimb ischemic injury and repair, andprovide preliminary ideas on its possible clinical treatment. Major aspects of this studyinclude the following:Objective:Explore the protective effects of IGFBP-4in hindlimb ischemic disease,understanding the specific mechanism of action and provide a theoretical basis for possibleclinical treatment.Method:1.In vitro experiments using hydrogen peroxide to induce cell DNA damage; examinethe effect of IGFBP-4in protecting against DNA damage; using lithium chloride(canonical Wnt pathway agonist) stimulation and knockdown of β-catenin (canonical Wntpathway co-activator) to verify whether IGFBP-4reduces DNA damage through inhibitionof canonical Wnt signaling pathway.2.Mice experimental model through ligation of the femoral artery to induce hindlimbischemia; injection of purified IGFBP-4protein or PBS control at the site of ischemia; theuse of Laser Doppler and histological sectioning to observe the protective effect ofIGFBP-4on the hindlimb ischemia; western blotting to verify inhibition of Wnt canonicalpathway results in a reduction of DNA damage.Results:In in vitro studies, hydrogen peroxide treatment induced DNA damage andincreased the expression of γ-H2AX in Human umbilical vein endothelial cells (HUVEC);IGFBP-4reduced the expression of β-catenin, inhibiting the Wnt canonical signalingpathway; IGFBP-4pre-treatment in HUVEC reduced hydrogen peroxide induced DNAdamage, whereas Lithium chloride treatment increased β-catenin expression andaggravated DNA damage.Similarly in mice hindlimb ischemia model, Laser Doppler and and histology resultsshowed mice injected with IGFBP-4had significantly lower ischemic injury compared to control mice. Western blotting also showed mice injected with IGFBP-4had lowerβ-catenin expression in the ischemic tissue, as well as lower γ-H2AX expression andreduced DNA damage.Conclusion:1. IGFBP-4can protect the hydrogen peroxide induced DNA damage;2. IGFBP-4acts by inhibiting Wnt canonical signaling pathway in reducing DNAdamage in vitro;3. IGFBP-4can protect lower limb ischemia induced DNA damage;4. In vivo, IGFBP-4acts by inhibiting Wnt canonical signaling pathway in reducingDNA damage.