Study Of The Regulation Of Kaempferol On Skin Homeostasis In Dermal Wound And Hyperplastic Scar

[Background]Normal skin homeostasis consists of skin metabolism, apoptosis, proliferation as well as functions of various cells live within, which are being orchestrated by a number of signals. This homeostasis, once damaged, will cause a series of synergetic pro-healing force. Otherwise, chronic diseases or abnormal healing occurs, for example, the inflammation results from whole-layer cutaneous injuries and the hyperplastic scar caused by abnormal healing. Cutaneous damage as deep as dermal ones, which tend to bring intense inflammation, could reach healing within short phase. And chronic inflammation would even induce other diseases for people with poor immune-system. Persistent inflammation and other infection stimulate the over proliferation of dermal fibroblasts, and the subsequent chaotic collagen accumulation, which in the end, forms scar. Such a problem entitle the key step in promotion of whole-layer cutaneous wound---inflammation regulation and the key step in suppressing hyperplastic scar---regulation of collagen metabolism as well as fibroblast proliferation. Current therapies focusing on skin wound have all adopted such approach. Based on the research of the biological properties of small molecule compounds, small molecule and stem cell have arisen as the new approach to this problem. Researches have proved the immune-regulating and pro-regenerating ability of MSC when they are recruited to wound areas. While the former ability bases on the cytokines and FasL on cell surface, the second ability is still under argument between differentiation theory and paracrine secretion theory. However, both abilities require the sufficient recruitment and engraftment of MSC.Flavone small molecule compounds have become the hot spot in regenerative medicine with many interesting properties. Our team has found, in previous study that kaempferol could exert positively on human keratinocytes, helping maintain skin homeostasis. Therefore, we in this study focus on the biological effect of kaempferol on dermal cells, with emphasis on the its pro-effect under the non-homeostasis situation. [Target]To detect the contribution of kaempferol to skin homeostasis and biological effects of kaempferol on dermal pluripotent stem cell and fibroblast and the mechanism behind as well; to explore the mechanism of how kaempferol affect skin homeostasis through MSC and of how kaempferol suppress hsFb, so as to provide theory evidence for hyperplastic scar treatment. [Methods]1.To isolate and identify mouse BMMSC, dermal fibroblast and hsFb. To detect the effect of kaempferol on such cells and each proper concentration with MTT, CFU and cytometry; to explore the relation among these cells under the kaempferol in vitro with scratch assay.2.To construct wound and non-wound mouse models and to explore the recruiting effect of kaempferol on BMMSC through intravenous transplantation of GFP+BMMSC and immune-fluorescence; to follow the BMMSC immune-regulation ability under kaempferol with PCR and in vitro co-culture model; to confirm the change of skin homeostasis under the kaempferol-induced MSC by transplanting kaempferol-induced BMMSC locally.3.To detect the kaempferol suppressive effects on hsFb and its mechanism by in vitro culture and molecular biological technic; to confirm the suppressive effects on both hyperplastic scar and inflammation of kaempferol with histological and PCR experiment. [Results]1.Mice dermal fibroblast, mBMMSCs and hsFb were harvested by isolation and cultivation in vitro. First, we chose and gathered the proliferation and migration ability as the basic phenomena of Kf regulating cell behavior, which built the basis of follow-up study. The optimal concentration of Kf on MSC and fibroblasts was decided by MTT(0.25×10-5M/L). Also previous study of Kf on MSC’s differentiation and proliferation confirmed the feasibility of this concentration: Kf promoted MSC’s proliferation and inhibited fibroblasts’ s proliferation. Flow cytometry showed that the ratios of G1/G0 of both cells were changed with different degrees, revealing that kaempferol’s regulation of proliferation was realized by regulation of cell cycle instead of induction of cell apoptosis.Conditioned medium was introduced to scratch test to explore the interplay between MSC and fibroblasts. Results gave no significant difference between(Kf+Fb) conditioned medium and Kf while migration ability of MSC was decreased using(DMSO+Fb) conditioned medium. MTT showed that fibroblast had no effect on MSC’s migration when its proliferation was inhibited. However, fibroblast wouldn’t promote MSC’s migration when fibroblast was disinhibited.Compared with MSC, fibroblasts’ s migration was mainly observed in the areas of skin wound and inflammation, and it was affected by MSC and other cells in a more complicated way. Therefore, more in vivo conditions were simulated in the scratch test. The results exhibited that conditioned medium of young mice BMMSCs(YB-CM)didn’t inhibit fibroblast’s proliferation. However, the inhibition by Kf could be neutralized by YB-CM. Conditioned medium of senile mice BMMSCs didn’t promote fibroblast’s migration and this effect couldn’t be changed by Kf. The results of Kf on hsFb’s proliferation showed that Kf inhibited hsFb’s proliferation in vitro, offering us some clues of treating human skin scar by Kf.2.In vivo tests demonstrated the BMMSC-recruiting milieu induced by kaempferol. Thus we examined several reported BMMSC chemotactic factors through RT-PCR. No candidate being found, we decided to transfer our exploration to the MSC’s impact on skin. On the whole-layer cutaneous wound model on mouse we constructed, we proved that kaempferol could bring more MSC on the wound bed and further eased inflammatory state.We demonstrated that the up-regulated FasL caused by kaempferol contributed most to the strengthened immune-regulating ability of MSC. We, from the angle of cell therapy, conceived the transplantation of kaempferol-induced BMMSC on mouse models, proving the therapeutic potential of kaempferol-induced BMMSC. However, the long cycle of mouse model experiments and our schedule deprived us of the transplantation of MSC that was FasL-down-regulated and the confirmation of kaempferol effect on ulcer models, which needed further endeavor in the future.3.Our study on the in vitro expression of fibroblast showed down-regulated COL-Ⅰand up-regulated MMP, implying a faster collagen metabolism for kaempferol-induced fibroblasts. We demonstrated the mechanism behind the suppression effect of kaempferol on fibroblasts was the down-regulation of phosphorylated Smad2/3 in TGF-β pathway, and the increase of collagen metabolism both of which indicated a potential of hyperplastic scar treating strategy. Therefore, we adopted a rabbit ear model to confirm the suppression of kaempferol on hyperplastic scar fibroblasts. Our results demonstrated that besides the refined appearance, the thickness of scar has also drastically declined, with a significantly lower SEI to control group. PCR yielded consistent results that inflammation in kaempferol scar was much lighter than that in control scar. But whether did this declination stem from kaempferol-recruited MSC needed further study. [Conclusion]We in this study proved that kaempferol, with a certain concentration could exert important effect on in vitro cultured MSC and fibroblast. It suppressed the proliferation and immigration of fibroblast but not BMMSC. Kaempferol could also recruit endogenic and exogenous BMMSC to contribute to the homeostasis of skin in intact mouse model.Kaempferol also contributed to cutaneous homeostasis on wounded mouse model, in which MSC played a vital role. We found that kaempferol could increase the FasL expression on MSC in vitro, thus causing more T cell apoptosis in co-culture model. Kaempferol also recruited more BMMSC to wound bed on wounded mouse model, probably due to certain chemotactic factors released by kaempferol affected skin cells. Kaempferol-derived lower inflammation in situ entitled us another in vivo experiment which consisted transplantation of kaempferol-induced BMMSC into a cutaneous wound mouse model. Results showed that kaempferol-MSC brought much lighter inflammation, confirming our conclusion that kaempferol contributed to homeostasis through MSC immune-regulating ability.Kaempferol’s suppression on mouse fibroblast entitled us to explore the mechanism behind hsFb proliferation suppression which according to our results was the down-regulation of phosphorylated-Smad2/3 in TGF-β pathway. This suppressive effect was later confirmed on rabbit ear model that surface smeared kaempferol resulted in thinner scar and lighter inflammation in scar tissue, with the inflammation lighting mechanism requested further study.