Astragalus Polysaccharides On The Effect And Mechanism Of Oxidative Damage Caused By The Treatment Of Skin Ulcers

In recent years, the relationship between oxidative stress and skin damages attracts people's more and more interests, it becomes a hot issue in surgery research, such as photoallergic dermatitis, vitiligo and various acute and/or chronic ulceration etc., The oxygen radicals have more and more importance in wound healing process, in one hand they can work as signal molecular, clearance infection of microorganism; in the other hand, excess of oxygen radical causes tissue injuries, which are not benefit to tissue repair. The effective antioxidant systems in organism keep the balance between oxidant and antioxidant. Destroy of equilibrium causes oxidative stress if the antioxidant system cannot eliminate oxygen radicals, or over expression of free radicals, it causes tissue injuries and disadvantageous to wound healing. Thus, antioxidant therapy is one of path for related skin diseases. This thesis studied the effective polysaccharide monomer of one traditional Chinese Medicine, called Huangqi (Astragalus polysaccharide, APS), which has effect of purging toxin and improving tissue regeneration. There are two major parts in this thesis, the first part is in vivo study, we studied the role of APS and their antioxidant mechanism by using a chronic oxidative ulcerated animal model which induced by Epirubicin. The second part is in vitro study, a model of oxidative injury in rat skin fibroblasts were replicated by H₂O₂, we demonstrated the mechanism of APS against oxidative damages, explored the relationship among APS, oxidative damages, lysosomes and mitochindria, we also investigated the early intervention and targets of APS in apoptosis induced by oxidative stress.1 In vivo Studies1.1 Replication of oxidative stress-induced dermal ulceration induced by Epirubicin in mice.Epirubicin (EPI) was interdermally injected of 2 mg/mL, 0.15 mL per mouse to back skin to replicate the chronic and oxidative stress induced dermal ulceration. Healing days and wound area were measured, HE and Massson stain used to evaluate histological changes, red Sirius stain for collagen reconstitution, finally, biochemical methods used to measure MDA level and SOD activity.Out data showed that there were edema and formation of ulceration 3 to 5 days after EPI injection, maximum ulcer area was observed 11 to 13 days after injection; then the ulcer began contracted with new hair-growth at the border of wound; on 28th to 30th days, scar formatted with new hair-growth on the healed wound surface.Histological studies showed that 13 days after EPI injection; collagen was damaged and reduced with disordered structure, less fibroblasts and inflammatory infiltration and edema, especially at the border of wound. 27 days after EPI injection, capillaries and fibers regenerated with re-epithelization, collagen reconstituted with high degree of maturity, but disorder structured. The results of oxidative injuries shown that compared to Control mice, 13 days after EPI injection, MDA level was increased(p < 0.01) while SOD activity was decreased markedly(p < 0.01); 27 days after EPI injection, MDA still stay at high level(p < 0.01)SOD activity was low(p < 0.01).These results demonstrated that EPI successfully replicated the chronic oxidative ulcer model, which can be used to study the therapeutic effect and mechanism of traditional Chinese medicine.1.2 Effect and mechanism of APS on chronic dermal ulcerThe same animal model was used to evaluate the effect and mechanism of APS treatments on skin ulcer. Healing days and wound area were measured, HE and Massson stain used to evaluate histological changes, Red Sirius stain for observation collagen reconstitution, biochemical methods used to measure MDA , LDH level and SOD activity, immunohistochemical methods for Cathepsin D and Bax expression.We studied the action of APS at high dose(APSH, 0.8 mg/mL) and low dose(APSL, 0.4 mg/mL), with positive control treatments of antioxidant Trolox(3 mg/mL)and growth factor rhEGF(2000 IU/mL); compared their effect on dermal ulceration, explored their mechanism.Our data revealed that mice in APSH and APSL treatment groups crusted earlier than those in Model group, they exhibited an improvement on wound healing, their healing days are 23.25±4.80 days and 24.44±3.50 days, less than rhEGF group(26.29±2.50 days),Model group(29.75±3.58 days)and Trolox group(29.57±3.03 days). APS reduced the wounded area in EPI-induced skin ulcer 13 days after injection, which had the same effect as rhEGF treatment, whileas Trolox treatment was not effective as APS.13 days after injection, APS treatment improved the histological changes of tissue injuries, granulation tissues regenerated, with more fibroblasts and less inflammatory cells, promoted the collagen deposition and epidermis repairing. However, Trolox showed less effect on reducing inflammatory infiltration and improving ulcer healing. 27 days after injection, collagen became reconstituted with high degree of maturity and well organized in animals of APSL and rhEGF groups, closed to Control group.13 days after injection, MDA level and LDH activity of wound tissue increased(p < 0.01)while LDH activity decreased markedly(p < 0.01), compared to those of normal mice. 27 days after injection, MDA level and LDH activity of treatments groups dropped compared to Model group with better effect of rhEGF and two APS treated groups(p < 0.01). Trolox also showed certain effect on MDA level(p < 0.05). SOD activity of treated groups augmented than those on 13 days, but no statistic difference compared to mice of Model groups on 27 days, among them, SOD activity of Trolox group return to normal level(p > 0.05).We furtherlly observed that the positive expression of Cat D enhanced significantly, there were statistic differenc compared to those of Control groups; while as APS treatments showed marked difference compared to those of Model, with better effect of APSL group closing to control mice. Bax expression also enhanced significantly in various treatments groups mice with statistical differences compared to Control group(p < 0.01 ). By comparison with Model group, Trolox exhibited no effect(p > 0.05)while the others treatments reduced Bax expression markedly (p < 0.05)with better effect of those of APSL mice, which closed to those of Control group.These data of animal experiment suggested that APS can improve the healing of chronic dermal ulcer which induced by EPI, reduce the healing days, enhance healing quality, reduce inflammatory infiltration, improve collagen reconstitution; moreover, APS can inhibit lipid peroxidation and LDH activity, enhance SOD activity, to exhibit effect of against oxidative damages, it can also inhibit apoptosis of cells in the wound area.2 In vitro studiesBased on the effect of APS on Chronic oxidative dermal ulceration and its mechanism, we replicated the an in vitro model of oxidative stressed-dermal fibroblasts, furthermore, explored the mechanism of APS against oxidative stress induced apoptosis at cellular and molecular level, to discover the more effective action targets of APS.2.1 Regulation of APS on apoptosis induced by oxidative stress in dermal fibroblsatsH₂O₂,0.5 mM, added to rat fibroblasts, incubated 30 min, then added fresh culture medium containing different concentration of APS, cultured 24 h,48 h and 72 h, measured cell survival by MTT methods; and/or incubate 1 h,3 h,6 h and 24 h, then observed the changes of apoptotic nuclei by Hoechst 33258 stain, morphology changes of mitochondria by probe Mitotracker Green FM confocal microscope scanning, Caspase-9,-3 activation by fluorometric determination.Our data showed that 24 hours after stress, H₂O₂ markedly reduced cell number(p < 0.01)to 35.23% of Control (100%); 48 hours after stress, the cell survival continued to decrease to 31.80% , with those of 35.80% 72 hours after stress. However, APS increased cell survival to 52.52% 24 h after stress with a concentration of 1μg/ml, while 48 h 72 h APS showed effect of enhancing cell survival with concentration from 0.5μg/ml to 2μg/ml, better effect of 1μg/ml, thus, 1μg/ml was used for the followed mechanism studies.H₂O₂ produced cells with exhibited folded nuclei at 3 h, condensed and/or fragmented nuclei at 6 h, chromatin condensation and fragment apoptotic bodies 24 h after stress, while treated with APS significantly reduced percentage of apoptotic cells.1 hour after stress, tubular mitochondrial network was destroyed and formated of punctiform, 3 hours after stress shwed a typical changes, these fragmented mitochondria moved from the cell periphery to the nuclei, this situation lasted to 24 hours after stress. APS reversed this situation, some mitochondria was still with tubular network and diffuse distribution. Caspase-9 and -3 had maximum activation 6 h and 24 h after stress respectively, APS inhibite their activation completely(p < 0.01), closed to normal level(p > 0.05).These data suggested that H₂O₂induced cell apoptosis, apoptotic cascade events inside cells occurred with time, APS inhibited all these events, had multi-targets effects.2.2 APS reduced ROS production and protected lysosomes and mitochondriaIn the same cellular model, H2DCFHDA were used for ROS production measuement, JC- 1 for motochondrail membrane potential assay, and acrodine orange for lysosomal stability evaluate by confocal microscope scanning.Our data revealed: 1 hour after H₂O₂ withdrawal, massive ROS production occured, whileas APS treatment exhibited less reduction(p < 0.05)1 hour after H₂O₂ withdrawal, marked reduction at 3 hours(p < 0.05). H₂O₂ induced mitochondrial membrane potential collapse 1 hour after H₂O₂ withdrawal(p < 0.01), APS increase the membrane potential significantly with time dependant, the potential of 6 h and 24 h had no statistic meaning compared to Control cells. Moreover, at the early stage of oxidative stress in fibroblasts, that is 15 min after H₂O₂ withdrawal, APS protected the lysosomal stability.These indicated that APS reduced ROS production, restored mitochondrial membrane potential while had no effect on physiologic potential, thus to inhibite apoptosis in duced by oxidative stress at mitochondria and lysosome level. 2.3 Effect of APS on Cathepsin D and Bax expression in rat fibroblast induced by oxidative injury.In the same cellular model, Cathepsin D (Cat D) expression measured by Immunohistochemistry, Bax protein expression assayed by immunofluorescence methods.Our data were: Cat D had rapid higher expression 1 hour after H2O2 withdrawal, less reductionat 24th hour, while had statistic meaning by comparison with those of Control group(p < 0.01), after tratement with APS, higher expression of Cat D reduced significantly(p < 0.01), this difference continued to 24th. Bax expression reached peak 24 hours after H2O2 withdrawal with marked difference compared to those of Control(p < 0.01); significantly reduced by APS treatment(p < 0.01).The data indicated that APS inhibit Cat D and Bax expression, thus to stabilize mitochondrial membrane potential, inhibit apoptosis.These results of in vitro studies suggested that when cell undergoing apoptosis induce db oxidative stress, lysosomal membrane disruption, mitochondria assembling, capases activation, cell nuclei condensation leading to cell death, these events occurred with time. APS could inihit all these events and moreover inhibit ROS production, thus to block the cascade of apoptosis induced by oxidative stress; APS also inhibit apoptosis through regulation of Cat D and Bax expression which are the communication between lysosome and mitochondria. This thesis demonstrated the multi-targets and mechanism of APS against oxidative damages.Taken together, this thesis demostrated the effect of APS on dermal oxidative wound healing induced by EPI, APS reduced the oxidative damages during healing process; it also inhibited apoptosis of cell in the wound. The mechanism of APS against apoptosis induced by oxidative stress maybe through protecting lysosome and mitochondria, reducing ROS production, inhibiting apoptosis related enzyme and protein release.