Effects And Mechansim Of Histatin1on The Proliferation And Migration Of Human Epidermal Cell And Human Fibroblasts In Wound Healing

BackgroundWounds in the oral cavity heal much faster and less scar formation than skin lesions in anideal way. Accelerated healing in the oral cavity has been attributed to oral tissue structureand the presence of saliva. Nowadays, some researches shew histatins as majorwound-closing factors in human saliva, can induce buccal epithelial cell lines migration.Antibacterial peptide belongs to natured immunity system, and it was found that some caninduce wound closure but defense against microorganism, which make them attractivecandidates for development as therapeutics for promoting wound healing. Since histatinsoccur only in saliva and have not been found in other body fluids, whether histatins canactivate non-oral cells and stimulate skin wound closue is unknown, and research was less.PurposeThis study will observe the effect of histatin1(Hst1)on the proliferation and migrationof human epidermal cells and human fibroblasts, and test mechanism of action, to probe theeffect of histatin1on non-oral mucosa cell, which is only present in salivary, and to provide anew therapeutic application of skin wound.MethodHuman epidermal cells and human fibroblasts was subcultured, which in exponentialphase of growth was chosen to be treated as followed.1. The ability of cell proliferation after treated by different concentration (3～100μg/mL)histatin1and(or) rhEGF at24h,48h and72h, was observed by using cytometry and MTTassay. The effects of histatin1on cell cycle was determined by flow cytometry assay. 2. The ability of cell migration after treated by different concentration (3～100μg/mL)histatin1and(or) rhEGF were observed by using wound wound healing in vitro.3. The ability of cell proliferation and migration was observed after treated by four kindsof cell antibody and histatin1by using MTT assay and wound healing in vitro in order to testpossible cell acceptor.4. Data were analyzed using ANOVA with LSD-t test and Dunnett,s T3test to determinesignificance between samples.Result1.3～100μg/mL Histatin1can induce HaCaT cell proliferation in does-and time-dependentmanner.(1)Cells had been cultured for24h, only the cell count in10ng/mL rh EGF group and30μg/mL Hst1+10ng/mL rhEGF group was significantly more than in control group(t=3.813,5.410,P＜0.05or P＜0.01).Besides at48h, in30μg/mL Hst1group and3μg/mLHst1group, the cell count in others was increased than in control group at48h and72h(witht value range from7.754to24.979, P＜0.01)..At72h, the cell count in100μg/mL Hst1group(19.21±0.59)×104was increased than in30μg/mL Hst1group(16.19±0.53)×104and3μg/mL Hst1group(15.38±0.13)×104(t=11.391,19.017, P＜0.01), and the cell count in30μg/mL Hst1+10ng/mL rhEGF group(19.75±0.35)×104was significantly more than in30μg/mL Hst1group,10ng/mL rhEGF group(19.19±0.09)×104and3μg/mL Hst1group(witht value range from4.579to34.884, P＜0.05or P＜0.01). The cell count in100μg/mL Hst1group,30μg/mL Hst1group and3μg/mL Hst1group was increased significantly, comparingat48h with at24h, and comparing at72h with at48h in the same group(with t48/24valuerange from16.629to28.333,with t72/48value range from18.218to68.891,P＜0.01).(2)Cell was cultured during24h～72h, OD in all treated groups was evidently morethan in control group(with t value range from3.160to15.576,P＜0.01or P＜0.05). At thesame time, OD decreased through concentration declined, and OD in100μg/mL Hst1groupwas all increased than in3μg/mL Hst1group in24h～72h(with t value range from4.264to7.234,P＜0.01).OD in all groups at48h and72h was evaluated than at24h in the samegroup(with t value range from6.219to22.308,P＜0.01).(3) At24h and48h, comparing with in control group(0.48±0.01,0.54±0.01), PI in100μg/mL Hst1group (0.62±0.01,0.70±0.01)and30μg/mL Hst1group (0.52±0.01, 0.66±0.01)was increased (with t value range from4.752to16.104, P＜0.01). PI in alltreated groups was increased with the concentration increased(with t value range from3.690to17.459, P＜0.01),but PI in30μg/mL Hst1group at72h was similar with3μg/mL Hst1(with t value0.273,P＞0.05). PI was increased significantly in the same group comparing48h with24h(with t value range from7.607to15.582,P＜0.01),but PI was decreasedsignificantly in the same group comparing72h with48h(with t value range from-29.672to-18.657,P＜0.01).2.30μg/mL Hst1can induce HaCaT cell migration in vitro.Untreated by Mitomycin C, the healed area rate(75.87±3.94)%in30μg/mL Hst1groupat16h was significantly more than in control group(52.98±3.50)%(with t value12.241,P＜0.01).The healing area rate at16h in30μg/mL Hst1+10ng/mL rhEGF group(94.98±4.08)%,15μg/mL Hst1+5ng/mL rhEGF group (97.05±3.67)%,15μg/mLHst1+10ng/mL rhEGF group(80.55±5.94)%(with t value from-11.324to-2.502, P＜0.01or P＜0.05)。The healing area rate at16h in all groups was similar with at24h(with t valuefrom0.990to1.771,P＞0.05). After Mitomycin C treated, which restrained the cellsproliferation, the cells healing area at16h(59.89±3.41)%was more elevated in30μg/mLHst1group than that in control group(38.40±4.22)%(with t value7.790,P＜0.01), but thehealing area was declined at16h than that of Mitomycin C untreated (with t value-10.863,P＜0.01). The healing area in combination groups was no significantly difference with that in30μg/mL Hst1group(with t value from0.0614to2.030,P＞0.05). The healing area rate at16h in all groups was similar with at24h(with t value from0.925to1.750,P＞0.05).3.3～100μg/mL Histatin1can induce HF proliferation in time-dependent manner.(1)Cell was cultured in24h～72h, and the cell count in100μg/mL Hst1group,30μg/mL Hst1group and3μg/mL Hst1group was more than in the control group at the sametime(with t value from4.062to47.370,P＜0.01or P＜0.05). At24h, the cell count in100μg/mL Hst1group(3.89±0.18)×10~4was increased than in3μg/mL Hst1group(3.13±0.53)×10~4(with t value4.040,P＜0.05). The cell count in30μg/mL Hst1group (5.02±0.71)×10~4and3μg/mL Hst1group (5.00±0.35)×10~4was more than in100μg/mL Hst1group(4.13±0.18)×10~4(with t value3.661,6.680,P＜0.05or P＜0.01) at48h. At72h, the cell count in30μg/mL Hst1group (12.37±0.18)×104was increased than in100μg/mL Hst1group (9.50±0.71)×10~4and3μg/mL Hst1group(10.13±1.24)×10~4(with t value11.808,5.349,P＜0.01). In the same contentrition group comparing at48h with at24h, and comparing at72h with at48h,the cell count was increased(with t value from2.854to30.055,P＜0.01or P＜0.05). Atthe same time, the cell count in30μg/mL Hst1+10ng/mL rhEGF group was significantlymore than in Hst1group or rhEGF group(with t value from3.857to20.045,P＜0.01).(2)Cell was cultured in24h～72h, OD in treated groups was evidently more than incontrol group at the same time(with t value range from3.712to18.262,P＜0.01). At24h,OD was increased comparing in100μg/mL Hst1group(0.50±0.02) with30μg/mL Hst1group(0.47±0.02), and comparing in30μg/mL Hst1group with in3μg/mL Hst1group(0.42±0.03)(with t value3.187,5.036,P＜0.01). At48h, OD in all groups was nosignificantly difference(with t value range from1.279to2.375,P＞0.05).At72h,OD in100μg/mL Hst1group (0.70±0.04)and3μg/mL Hst1group(0.62±0.03) was evidently decreased,comparing with in30μg/mL Hst1group(0.79±0.04)(with t value-6.484,-7.512,P＜0.01). Inthe same contentrition group comparing at48h with at24h, and comparing at72h with at48h,OD was increased(with t value from4.298to25.956,P＜0.01).(3)At24h, PI in100μg/mL Hst1group (0.16±0.01)and30μg/mL Hst1group(0.16±0.01)was increased than in control group(0.12±0.01)(with t value6.790,6.052,P＜0.01).At48h PI in other groups was evaluated than in control group(with t value from5.497to20.927,P＜0.01). At72h, PI in all groups was no significantly difference(with F value2.583,P＞0.05).At24h, PI in100μg/mL Hst1group was increased than in30μg/mL Hst1group and3μg/mL Hst1group (with t value7.662,5.828,P＜0.01or P＜0.05). At48h, PI in30μg/mL Hst1group was increased than in100μg/mL Hst1group and3μg/mL Hst1group(with t value11.875,15.430,P＜0.05). PI was increased in different concentration Hst1,comparing at48h with at72h and24h(with t value from5.688to26.912,P＜0.01).4.30μg/mL Hst1can induce HF wound heal in vitro.Untreated by Mitomycin C, the healed area ratein30μg/mL Hst1group (3,7.50±2.22)%at8h was significantly more than in control group(26.00±5.60)%(with t value6.037,P＜0.01).The healing area rate at8h in15μg/mL Hst1+10ng/mL rhEGF group(45.24±3.26)%was increased in30μg/mL Hst1group(with t value6.170, P＜0.05)。After Mitomycin Ctreated, which restrained the cells proliferation, the cells healing area at8h was nosignificantly difference comparing with each other(with t value1.822,P＞0.05), but thehealing area was declined at8h than that of Mitomycin C untreated (with t value from -12.067to-5.824,P＜0.01). The healing area rate at16h in all groups was similar with at8h(with t value from0.442to4.121,P＞0.05).5. Four kinds of antibody can inhibit Hst1induction on HaCaT cell proliferation andmigration.Cell treated by antibody, OD in all antibody group was significantly decreased at24h～72h than in30μg/mL Hst1group(with t value from3.424to34.583,P＜0.05or P＜0.01).And the heal rate in all antibody group [(41.15±0.95)%,(53.15±2.07)%,(46.24±2.51)%,(47.06±1.33)%]was also declined than in30μg/mL Hst1group (71.02±2.42)%in vitro(witht value from-36.333to-17.724,P＜0.01), the heal rate in EGFR antibody group wassignificantly reduced than in other three antibody group(with t value from-16.644to-6.000,P＜0.01). After Mitomycin C treated, the heal rate in all antibody group[(34.05±0.96)%,(45.76±2.02)%,(2.31±0.23)%,(4.75±0.36)%] was also declined than in30μg/mL Hst1group(50.41±1.17)%in vitro(with t value from-127.235to-6.284,P＜0.01), and the heal rate inSyndecan-4and Syndecan-1antibody group was significantly reduced than in other twoantibody group(with t value from102.068to-63.082,P＜0.01).6. Syndecan-4can inhibit Hst1induction on HF proliferation.Cell treated by antibody, OD in Syndecan-4antibody group (0.37±0.03),(0.53±0.03),(0.64±0.03))was significantly decreased at24h～72h than in30μg/mL Hst1group((0.50±0.02),(0.65±0.03),(0.72±0.04))(with t value from2.868to11.462, P＜0.01).And the heal rate in Syndecan-4antibody group (21.83±2.19)%was also declined than in30μg/mL Hst1group (31.23±2.66)%in vitro(with t value-7.132,P＜0.01), and wassignificantly reduced than in other three antibody group(with t value from-5.209to-5.315,P＜0.01). The healing area rate at16h in all groups was similar with at8h(with t value from0.184to1.242,P＞0.05).Conclusion1. Histatin1can induce the proliferation and migration of human epidermal cells, andrhEGF can promote the proliferation and wound healing in vitro by histatin1inducing, but noeffect to migration by histatin1inducing.2. Histatin1can induce the proliferation of human fibroblasts, and rhEGF can promotethe proliferation and wound healing in vitro by histatin1inducing.3. EGFR,TGF R Ⅱ, Syndecan-1and Syndecan-4can be relevant with the effect of the histatin1on the proliferation and migration of human epidermal cells. Syndecan-4can berelevant with the effect of the histatin1on the proliferation of human fibroblasts.