Role Of Substance P In Improving Impaired Wounds In Streptozotocin-induced Diabetic Rats As Well As Its Application

Diabetes mellitus is a disorder characterized by hyperglycaemia due to an absolute or relative deficiency of insulin and/or insulin resistance, and affects 1–2% of the population worldwide. Diabetic patients are prone to long-term complications, such as retinopathy, cataract, atherosclerosis, neuropathy, nephropathy and refractory wound. Delayed wound healing is a troublesome complication of diabetes mellitus. The treatment of the impaired diabetic wounds presents a formidable challenge to clinical workers, even to the whole medical fields. Results from recent investigations concerning the potential cellular and molecular mechanisms responsible for diabetic wound healing deficiency are preliminary in nature. It is well known that the hyperglycaemic state is the most prominent characteristic of diabetes mellitus, yet the precise mechanism still remains uncertain. Undoubtedly, the primary causal factor for the development of most diabetic complications is hyperglycemia. On the other hand, many factors are involved in the mechanisms of impaired diabetic wounds. In recent years, some studies have demonstrated that the primary sensory nerves and sensory neuropeptide released from the primary sensory nerves play very important roles in healing of impaired diabetic wound. Substance P (SP) can not only improve the cell proliferation, migration and differentiation, but also modulate the synthesis of some important growth factors and itself in granulation tissue cells. The purpose of our study is to evaluate the role of SP and explore the way to improve its curative effect in wound repair. Does exogenous SP facilitate wound healing in diabetic models? What are the effect and the mechanism of SP? How to improve therapeutic effect of SP? Methods: After the streptozotocin-induced diabetic rat model was established, the content of SP in diabetic rat's skin was measured by means of ELISA and the cutaneous sensory nerve distribution examined by immunohistochemistry with antibodies against the special sensory nerve marked SP. At the same time, H&E stain and transmission electron microscope (TEM) were used to estimate the pathological changes of the skin especially the sensory nerves. Once it was determined that the number of sensory nerve and content of SP reduced and that pathological changes occurred in streptozotocin-induced diabetic rats skin, we could confirm that this kind of model was suitable for studying impaired diabetic wounds. Then, we used a well-validated excisional wound healing model to determine the effect of exogenous SP on the healing response in streptozotocin-induced diabetic rats. Closure time of the wounds and the percentage of the healed wounds were recorded during different time points after diabetic skin wound. Synchronously, the content of hydroxyproline was measured and the collagen typeⅠand collagen typeⅢestimated by immunohistochemiscal staining. In order to improve therapeutic effect of SP in impaired wounds, a sustained-release multivesicular lipid-based SP was formulated using DepoFoam technology, a novel vehicle for controlled delivery of peptide drugs. Relatively, encapsulation efficiency and drug release assays in vitro in normal sodium (NS) were measured by radioimmunity method, while particle size distribution and spherical particle morphology were observed and measured by TEM and optical microscope. A sustained therapeutic effect of Depo-SP was performed in vivo in the same impaired diabetic wounds. Finally, a hydroxylated analogue of SP was synthesized by the solid-phase method. The effect of this analogue on the diabetic wound healing was tested. In situ hybridization was used to explore the mechanism of SP improving diabetic wound healing process, and the immunohistochemiscal fluorescence staining was used to investigate the expression of NK1R and NK2R(two kinds of receptors of substance P)attempt to explore the receptor pathway of SP. Results: The level of blood glucose was elevated over 16.7 mmol/L a week after rats were treated with 60 mg/kg streptozotocin i.p. The density of SP+ nerve fibers diminished in the skin of diabetic rats and the content of SP decreased obviously after four weeks. In diabetic skin, epidermis layer reduced in thickness, with inhibited activity of fibroblast proliferation. Sensory nerve dropsy and degeneration as well as Schwann cell degeneration could be observed by TEM. In diabetic wound tissue, the content of SP lowered greatly during the first few days after wound infliction. The counts of SP+ and NK1R+ cells decreased, while the number of NK2R+ cells increased. Exogenous SP (10-7M) shortened the healing time (14 days) of diabetic wounds compared to untreated diabetic wounds (healing time for 21 days). Meanwhile, the percentage of the healed wounds was decreased but the content of hydroxyproline within wounds increased. The same changes with thecollagen typeⅠand collagen type Ⅲ. Exogenous SP strengthened the expression of SP mRNA in reparative cytes in diabetic wounds. Depo-SP was prepared by orthogonal design. The particle size ranged 0.75～27.75 μm and phase-transition temperature (PTT) was 34℃,as indicated stable thermodynamic quality of the Depo-SP. Local application of the Depo-SP PBS solution could obtain the same therapeutic effect. Finally, solid-phase method was employed to synthesize the hydroxylated analogue of SP that was a kind of white powder with the peptide purity of 98.5% (measured by HPLC), molecular weight of 756.93 (measured by TLC) and sequence of Gln-Phe-Phe(4OH)-Gly-Leu-Met-NH2. When this dynamic peptide was used in diabetic wounds (10-6M, PBS solution, pH 6.0), the peptide improved the impaired wound healing at certain degree, the closure time of the wounds was shortened for 2-3 days or so. Conclusions 1. The decrease of SP in the diabetic rat skin is closely related to pathological changes of the skin. 2. The functional integrity of sensory nerves is the most important prerequisite in regulating the metabolism of SP in diabetic rat skin. 3. Local application of exogenous SP promoting the expression of SP mRNA in reparative cytes in diabetic wounds, as suggests that the peptides released by the peripheral sensory nerve terminals are the main factors in regulating the synthesis of endogenous SP in reparative cytes in diabetic wound healing process. 4. During the diabetic wound healing, the expression of NK2R (a special receptor of SP) was more significant than that of NK1R (another receptor of SP), as may suggest that NK2 is the more important signal transduction pathway than NK1 during course of SP promoting wound healing. 5. We have successfully prepared a Depo-SP by Depo-Foam MVL technology, the local application of Depo-SP in diabetic wounds is characterized by slow and stable release, and obviously decreased application frequency but is analogous with the normal SP solution upon therapeutic effect. 6. Since SP is so easy to be degraded by neutral endopeptidase, more frequent application in local skin leads to pain and discomfort, we have designed a hydroxylated analogue of [(4OH)Phe8]SP-6 according to it's molecular characterization and dynamicfragment. The novel peptide presents some dynamic activities in improving impaired diabetic wounds.