Research On Impacts Of Metformin On HaCaT Cells Proliferation Signal Pathways And Ear Psoriasis Model Of Guinea Pigs

Section Ⅰ Research on impacts of metformin on HaCaT cells proliferation signal pathwaysPurpose:As an insulin sensitizer, metformin is widely used for treating Type Ⅱ diabetes. The recent research has found that the metformin can inhibit the growth of various kinds of tumor cells, including breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and other malignant tumors and the most obvious effect of metformin on the tumor is to inhibit the tumor cell proliferation. There is obvious abnormality in the keratinocytes in vitro proliferation and differentiation in psoriasis. This research aims to discover whether the metformin could achieve the purpose of treatment of psoriasis by playing biological effects for inhibiting cell proliferation. HaCaT cell is characterized by excessive proliferation expressed by keratinocytes in skin lesion part of psoriasis and this cell is widely used as model in the research of the anti-psoriasis drug in vitro. This research studies the impacts of metformin on cell proliferation and the cell proliferation regulatory-related protein expression using human immortalized epidermal cell HaCaT cell lines and discusses the molecular mechanism of metformin in the treatment of psoriasis so as to provide theoretical basis for treatment of psoriasis by metformin.Methods:1. The morphological impacts of metformin on HaCaT cells:exert in vitro intervention on HaCaT cells by applying metformin with a concentration of0,25,50and75mmol/L respectively; after24h, place it under inverted phase contrast microscope and observe the morphological changes of the cells;2. The detection onthe impacts of metformin on HaCaT cell proliferation in vitro by CCK-8:use50mmol/L metformin to intervene HaCaT cells during logarithmic phase; collect cells respectively after24h,48h and72h and use the CCK-8colorimetric method to detect the cell proliferation activity;3. The detection on the impacts of metformin on AMPK and ERK1/2protein expressions in HaCaT cells by Western blot method:use50mmol/L metformin to intervene HaCaT cells in vitro; after24h, extract total cellular protein, and quantify the protein; use the Western blot method to detect phosphorylation level of AMPK, ERK1/2protein in the cells. The results are analyzed by using SPSS13.0statistical software.Results:1. The morphological observation after the intervention on HaCaT cells by metformin:after24h’s intervention on HaCaT cells by metformin, there are no significant changes in the shape and size of cells between0mmol/L group and25mmol/L group, except that the adhesion reduces, and some cells shed off and are floating; the cell volume in50mmol/L group is slightly smaller, intracellular granules and vacuoles are increased, the cell adhesion becomes worse, many cells shed from the sides of the bottle and float in the culture solution; all cells in75mmol/L group shed off and are floating.2. The impacts of metformin on HaCaT cell proliferation in vitro:the inhibition rate of50mmol/L metformin on HaCaT cells is36.18%after24h,12.70%after48h and10.12%after72h, which suggests that with the prolonged action of metformin, survival rate of HaCaT cells is gradually decreased.3. The impacts of metformin on AMPK and ERK1/2protein expression in HaCaT cells:Western blot results after intervention by50mmol/L metformin---before metformin intervention, the average absorbance ratio of p-AMPK and β-actin detected in HaCaT cells is2.856±0.323, while after metformin intervention, it is5.198±0.625, which shows a significant difference by contrast (p <0.05). Before metformin intervention, the average absorbance ratio of p-ERK1/2and β-actin detected in HaCaT cells is7.550±1.087, while after metformin intervention, it is10.430±1.217, which shows a significant difference by contrast (p <0.05), suggesting that metformin intervention may lead to increase in p-AMPK and p-ERK1/2expression.Conclusion:Metformin can significantly inhibit the proliferation activity of HaCaT cells in vitro, its mechanism of action is closely related to the activation AMPK signal channel and MAPK-related signal channel. Section IIResearch on the impact of metformin on psoriasis model of guinea pigsPurpose:As guinea pig ear psoriasis model can well simulate typical pathological changes in skin lesion part of psoriasis, this research studies the impact of metformin on pathological changes of guinea pig ear psoriasis models by constructing guinea pig ear psoriasis models, discusses the feasibility of metformin in treatment of psoriasis, preliminarily discusses its mechanism of action to provide preliminary data and information for the study on treatment of psoriasis by metformin.Methods:Randomly select10from the50three to four weeks old Hartley guinea pigs as the normal control group, and use the remaining40to construct the psoriasis pathological change model on guinea pig ear; prepare the normal saline control group,50mmol/L metformin group and100mmol/L metformin group. Apply5%propranolol ointment evenly on ears of guinea pigs for three times a day (continuously for4weeks), with a thickness of1.0mm to form an psoriasis sample animal model of guinea pig ears. After4weeks, apply externally the normal saline,50mmol/L metformin and1000mmol/L metformin respectively on the molding part of guinea pig ears three times a day consecutively for four weeks.4weeks later, take ear specimen of the guinea pig to make tissue pathological slice. Use Baker method to assess the histological integral of the slices in each group and detect the epidermal thickness of the guinea pig ears before and after metformin intervention. The results are analyzed statistically by using SPSS13.0statistical software.Results:1. Basic observations with naked eyes:guinea pigs in normal control group are in good spirit, quick in action, aggressive, and hair is thick and shiny, close to the skin, and the guinea pigs can eat, and relieve themselves normally. A week later, all molding animals are in poor spirit, slow in action, and like lying, eat less, lose weight, and their hair is without luster and easily sheds. After two weeks, the above symptoms of modeling animals are aggravated and the ear scratching phenomena occur; the hair at the back of ear where drug is applied sheds and is without hair roots, local skin of some animals’ ears becomes red and the telangiectasia is obvious. Four weeks later after treatment, the above symptoms of guinea pigs in metformin group have significantly alleviated.2. Measurement of the epidermis thickness of the back part of the guinea pig ears under microscope and the tissue scoring with Baker method:it is observed that, for molding group, the extensive hyperkeratosis and focal parakeratosis can be seen, and the Munro small abscess can be seen at some parakeratosis parts; the granular layer thins or disappears; the spinous layer is under significant hypertrophy, the rete ridges extend downward and the dermal papilla stretches onward; the blood capillary is dilated and congested; on dermis superficial layer, a mononuclear and polymorphonuclear cell infiltration can be seen, suggesting a successful molding. Under microscope, it is observed that the hyperkeratosis and parakeratosis phenomena are reduced, the spinous layer thins, the dermis interface is not obvious, the telangiectasia and inflammatory cell infiltration phenomenon is improved, and no Munro small abscess is observed in50mmol/L metformin group and100mmol/L metformin group. Baker scores are1.25±0.43and1.43±0.25respectively; and the epidermal thickness values on the back part of guinea pig ears are64.98±7.01and61.08±6.09. By comparing the50mmol/L metformin,100mmol/L metformin and the normal saline groups, Baker scores and epidermal thickness values of the back part of the ear differ significantly. While no significant differences show in Baker scores and epidermal thickness values of the back part of the ear in the50mmol/L metformin group and100mmol/L metformin group. Experiments show that the Baker score in metformin treatment group and the epidermis thickness on guinea pig ears are under different declination, and there is a statistically significant difference compared with the observation group after molding, suggesting that metformin may improve the pathological changes in psoriasis model of guinea pig ears.Conclusion:Metformin can significantly inhibit cell proliferation of psoriasis model in the guinea pig ears,but high concentration of metformin will not enhance therapeutic effects.