The Effect Of Oncostatinm On Proliferation And Secretion Of The Extracellular Matrix Of Human Dermal Fibroblasts Cultured With High Glucose

BackgroundWith the continuous improvement of people’s living standard and the change of lifestyle,diabetes has become the third major chronic disease that seriously threatens people’s health after tumor and cardiovascular diseases in China.Skin injury and difficult wound healing are the serious complications in the process of diabetes.Fibroblast(FB)is one of the important cellular components of skin tissue,the main repair cell secreting extracellular matrix(ECM)in the dermis,and the key and foundation of wound healing.Research shows that Human dermal fibroblasts isolated from chronic diabetic foot ulcers showed lower proliferation than normal dermal fibroblasts.Due to the decreased expression level of fibroblasts and the decreased functional activity of growth factors in diabetic wounds,the biological behavior of dermal fibroblasts in diabetes is changed,which is directly related to the local high glucose environment,and is one of the important reasons for the formation of diabetic skin damage.Therefore,for patients with diabetes,the change of fibroblast(FB)plays an important role in delayed or even non-healing of wound healing,and the normal proliferation,migration and secretion of extracellular matrix of fibroblasts are of great significance for maintaining the normal structure and physiological functions of skin.OSM was initially isolated by Robbins from the U937 lymphocyte culture supergene in 1986.It is a growth inhibiting factor on A375 melanoma cells and is mainly produced by activated T cells and monocytes.OSM is an inflammatory cytokine,which belongs to the interleukin-6 class.Il-6 is widely used in gene-immunotherapy of tumors because of its important role in immune surveillance.OSM can inhibit the function,proliferation and metastasis of various solid tumors,including pancreatic cancer,breast cancer,ovarian cancer,glioma,lung cancer,etc.As well as the differentiation of hematopoietic progenitor cells cultured in vitro.OSM can also inhibit the proliferation of liver cancer cells by inducing senescence.OSM can not only inhibit the growth of some tumor cells,but also act on a variety of other cells,participate in many physiological and pathological processes,and play an important role in a variety of diseases,including regulating inflammatory response,stimulating hematopoiesis,promoting cell dedifferentiation and other functions.OSM can promote cardiac regeneration,induce the generation of SDF1 and VEGF in cardiac muscle cells,and regulate the degradation of extracellular matrix.It has been reported that OSM can also promote the growth of fibroblasts and the proliferation of tumor cells in Kaposi.Extracellular regulatory kinase ERK is a member of the MAPK family.The signaling pathway of ERK involves the core of the signaling network that regulates cell growth and development as well as cell division.It has been confirmed by experiments that OSM can stimulate the pulmonary fibroblast receptor and then activate the mitogen-activated protein(MAPK)signal transduction pathway,and regulate the activity of lung fibroblasts through the MAPK pathway to participate in the repair of lung injury.Whether OSM can promote the proliferation,migration,extracellular matrix secretion and specific mechanisms of human dermal fibroblasts in a high glycemic environment by activating the ERK signaling pathway remains to be investigated.Therefore,the main mechanism of OSM promoting proliferation,migration and extracellular matrix secretion of human dermal fibroblasts in high glucose environment was discussed in this paper.PART Ⅰ ESTABLISHMENT OF HYPERGLYCEMIA MODEL OF HUMAN DERMAL FIBROBLASTS.ObjectiveTo investigate the optimal concentration and time of inhibition of high glucose on human dermal fibroblasts and establish a model of high glucose fibroblasts.MethodsAccording to the glucose concentration in DMEM medium,human dermal fibroblasts were divided into 5 groups: A: Control group:containing 25 mmol/L glucose;B:High glucose 1 group: containing 30mmol/L glucose;C:High glucose 2 groups: containing 35 mmol/L glucose;D:High glucose 3 groups: containing 40 mmol/L glucose;E:High glucose 4groups: containing 45 mmol/L glucose.1)The effect of high glucose on the activity of human dermal fibroblasts was detected by MTT method.2)The effect of high glucose on human dermal fibroblast’s cell cycle was detected by flow cytometry.3)Western Blot(WB)was used to detect the expression of ERK,p-ERK and CyclinD1 proteins.Results1)MTT results showed that the proliferation activity of dermal fibroblasts decreased with the increase of glucose concentration in a concentration-dependent manner.The results showed that there was a statistically significant difference between normal control group and40mmol/L high glucose culture for 48h(P<0.01).2)Flow cytometry results showed that compared with the normal control group,the 40 mmol/L high-glucose treatment group showed significant G1 phase block(P<0.05)and decreased cell number in S phase(P<0.01).3)WB results showed that after 48 h of cell culture,the expression of ERK,p-ERK and CyclinD1 in the 40 mmol/L high-glucose culture group was significantly decreased compared with the normal control group(P<0.01;P< 0.01;P < 0.01);The protein expression of ERK,p-ERK and CyclinD1 was significantly decreased in the 40 mmol/L high-glucose group after 48 h cell culture compared with the 40 mmol/L high-glucose group after 36 h cell culture.ConclusionHigh glucose can inhibit the activity of human dermal fibroblasts.When the concentration is 40 mmol/L,the proliferation of human dermal fibroblasts will be significantly inhibited,and the expression of ERK,p-ERK and CyclinD1 proteins in fibroblasts will be significantly inhibited.PART Ⅱ THE EFFECT OF OSM ON PROMOTION THE PROLIFERATION OF HUMAN DERMAL FIBROBLASTS IN HIGH GLUCOSE ENVIRONMENTObjective To investigate the effect of OSM on the proliferation of human dermal fibroblasts in high glucose environment.Methods1)the hyperglycemic model of human dermal fibroblasts cultured with 40 mmol/L glucose treated with different concentrations of OSM,and the human dermal fibroblasts were divided into:A:High glucose group(40mmol/L glucose),B:OSM 1 group(40mmol/L glucose +25ng/ml OSM),C:OSM 2 group(40mmol/L glucose +50ng/ml OSM),D:OSM 3group(40mmol/L glucose +100ng/ml OSM),E:OSM 4 group(40mmol/L glucose +200ng/ml OSM).After the culture of human dermal fibroblasts was completed,the activity of human dermal fibroblasts was detected by MTT method.The cells were further divided into groups: A: control group(25mmol/L glucose);B: high glucose group(40mmol/L glucose);C: high glucose +OSM group(40mmol/L glucose + 100ng/ml OSM);D: high glucose + OSM + PD98059(40mmol/L glucose + 100ng/ml OSM +PD98059).To explore the mechanism of OSM promoting the proliferation of human dermal fibroblasts.2)Flow cytometry was used to detect the effect of OSM on human dermal fibroblast’s cell cycle in high glucose environment.3)WB was used to detected the expression of cell cycle regulatory proteins ERK1/2,p-ERK1/2 and CyclinD1.4)Real-time Quantitative PCR(RT-qPCR)was used to detect the m RNA expression of ERK1/2 and CyclinD1.Results1)MTT results showed that OSM could significantly promote the proliferation of human dermal fibroblasts in the high glucose model.The cell activity increased significantly in the 100ng/ml OSM treatment group(P<0.01),and the cell activity decreased in the 200ng/ml OSM treatment group compared with the 100ng/ml OSM treatment group(P<0.01).The cell activity of 100ng/ml OSM group increased significantly after 48 h treatment compared with 24 h and 36 h treatment(P<0.01;P < 0.05).2)Flow cytometry showed that: compared with the normal control group,the number of cells in the S phase decreased(P<0.05)and the number of cells in the G1 phase increased(P<0.05)in the high glucose group.Compared with the high-glucose group,the cell number in the S-phase was significantly increased in the high-glucose + OSM group(P<0.05),and the cell number in the G1 phase was decreased(P<0.05).Compared with the high glucose group,there was no significant difference in the cell number of S and G1 phase in the high glucose +OSM + PD98059 group.3)Western blot results showed that the protein expression of ERK,p-ERK and CyclinD1 in the high-glucose group was significantly lower than that in the normal control group(P<0.01;P < 0.01;P < 0.01);The protein expressions of ERK,p-ERK and CyclinD1 were significantly increased in the high glucose + OSM group compared with the high glucose group(P <0.01;P < 0.01;P < 0.01);There was no significant difference in protein expression between the group with high glucose + OSM+ PD98059 and the group with high glucose.4)PCR results were consistent with Western blot results,with statistically significant differences(P<0.01).Conclusion OSM has the effect of promoting the proliferation of human dermal fibroblasts,and the results showed that the 100ng/ml OSM treatment of human dermal fibroblasts inhibited by high glucose for 48 h had the most significant effect on the proliferation of cells(P < 0.001).OSM promotes the G1-phase to S-phase transformation of hyperglycemia fibroblasts,thereby promoting mitosis,possibly through the ERK signaling pathway.OSM can promote the expression of CyclinD1 in human dermal fibroblasts at the level of genes and proteins,and this effect may through the ERK pathway.PART Ⅲ THE ROLE OF OSM IN PROMOTION EXTRACELLULAR MATRIX SECRETION OF HUMAN DERMAL FIBROBLASTS IN HIGH GLUCOSE ENVIROMENTObjective To investigate the effect of OSM on extracellular matrix secretion of human dermal fibroblasts in high glucose environment.Methods1)The cells were divided into A: Normal control group(25mmol/L glucose),B: High glucose group(40mmol/L glucose),C:OSM1 group(40mmol/L glucose +25ng/ml OSM),D:OSM2 group(40mmol/L glucose+50ng/ml OSM),E:OSM 3 group(40mmol/L glucose +100ng/ml OSM),and F:OSM4 group(40mmol/L glucose +200ng/ml OSM).The protein expression levels of Collagen Ⅰ,Ⅲ and Fibronectin in each group were detected by western-blot,and the optimal concentration of OSM promoting extracellular matrix secretion in human dermal fibroblasts in a high glucose environment was selected.2)The cells were then divided into: A: Control group(25mmol/L glucose);B: High glucose group(40mmol/L glucose);C: High glucose+OSM group(40mmol/L glucose + 100ng/ml OSM);D: High glucose +OSM + PD98059(40mmol/L glucose + 100ng/L OSM + PD98059).m RNA and protein expression levels of Collagen Ⅰ,Ⅲ and Fibronectin in each group were detected by q-PCR and western-blot.Results1)The relative expressions of Collagen Ⅰ,Collagen Ⅲ and fibronectin in the higher glucose culture groups of 100ng/ml OSM treatment group were significantly enhanced(P<0.01;P< 0.01;P< 0.01)The relative expression of Collagen Ⅰ,Collagen Ⅲ and fibronectin in the OSM group of200ng/ml was significantly lower than that in the OSM group of 100ng/ml(P<0.01;P < 0.01;P < 0.01).2)Western blot and PCR were used to detect the protein and m RNA expression of Collagen Ⅰ,Collagen Ⅲ and Fibronectin in the cells of each group.Compared with the normal control group,the protein expression of Collagen Ⅰ,Collagen Ⅲ and Fibronectin in the high-glucose group was significantly reduced(P<0.05;P< 0.01;P < 0.01);The protein expressions of Collagen Ⅰ,Collagen Ⅲ and Fibronectin in the high glucose + OSM group were significantly higher than those in the high glucose group(P<0.01;P < 0.01;P < 0.01);There was no significant difference in protein expression between the group with high glucose + OSM + PD98059 and high glucose group.The results of PCR and WB were basically the same,and the differences were statistically significant(P<0.01).Conclusion OSM promotes the secretion of Collagen Ⅰ,Ⅲ,and Fibronectin in the extracellular matrix of human dermal fibroblasts in a high glycemic environment,possibly through the ERK signaling pathway.