The Intervention Of Calcium And Iron To Melanogenesis In Melanocytes From Black-bone Silky Fowl

Black-Bone Silky Fowl is one of the local food resources of Jiangxi province and traditional Chinese medicine,and its health-promoting values have been well known for more than one thousand years.The most special feature of Black-bone Silky Fowl is rich in melanin.The melanin is an important functional component of Black-bone Silky Fowl.Mineral elements have important effects on melanogenesis.The contents of Ca,Fe elements were significantly higher than other mineral elements in Black-Bone Silky Fowl,so Ca and Fe may be the most important elements in melanogenesis.How to enhance and improve the synthetic amount,composition and functional activity of melanin in Black-Bone Silky Fowl,has important scientific significance to promote Jiangxi black bone chicken resource development and industrial development.The main conclusions are as follows:1.MTT experiment was used to study the growth factors such as glucose?Glc?and lipids of Yolk?LY?on the proliferation of BSF melanocytes.The date showed that concentrations of glucose was an significantly important requirement on proliferation and melanogenesis of BSF melanocytes,25 mM Glc had a significant furtherance on proliferation but disadvantage on melanogenesis.Under the conditions of high Glc?25mM?,5%FBS and 1%LY both had a significant promoting effects on proliferation,no significant difference in between,combined with each other had a synergistic effect.LY is more conducive to proliferation of BSF melanocytes than TPA according to these two growth curves.FBS is the main furtherance factor on proliferation on the conditions of 16.8 mM Glc.2.The influence of different valence state and concentrations of Ca and Fe on BSF melanocytes proliferation was studied by MTT experiment.The results showed that low concentration?0.01-0.02mmol/L?Fe³⁺and Fe²⁺had a significant promoting effect on the growth of melanocytes,and increased the concentration of Fe³⁺and Fe²⁺,there was no significant difference.On the background of 0.04 mmol/L Fe³⁺,0.04-0.16 mmol/L Fe²⁺had inhibition of growth.There is a significant difference between ferrous iron and ferric iron to melanocytes proliferation,and the cytotoxicity of ferrous iron was significantly higher than that of ferric iron.The Ca²⁺concentration?0.1-6.4 mmol/L?significantly promoted the growth of melanocytes,1.6 mmol/L was the strongest among which,the promoting effect was weakening along with increasing concentration.The tolerance of Ca²⁺was greater than Fe³⁺and Fe²⁺,which may relate to the melanin concentration in BSF melanocytes.3.Wound healing assay was used to observe the influence of Ca²⁺?Fe²⁺?Fe³⁺and Fe²⁺+Fe³⁺on BSF melanocytes migration.The results showed that FBS,LY and0.4mmol/L CaCl₂ had a significant promoting effect on the migration of melanocytes.FBS combined with LY had a significant promoting effect.Verapamil,a calcium antagonist,could block L-type calcium channel.The significant promoting effect of calcium on the migration of melanocytes,was vanished owing to the decrease of extracellular calcium influx.L-type calcium channel was an important channel for extracellular calcium influx in melanocytes.The FeCl₃ concentration?0.04-0.16mmol/L?significantly promoted the mobility rate of melanocytes,and showed iron dose dependent.The FeSO₄ concentration?0.01mmol/L?had a significant promoting effect on the mobility rate of melanocytes,and increased the concentration of Fe²⁺,there was no significant difference.0.04 mmol/L FeCl₃ combined with FeSO₄?0.08⁰.01 mmol/L?,showed significant promoting effect on the migration of melanocytes,that is to say,Fe³⁺:Fe²⁺=2:1,4:1,1:1,1:2.The promoting effect was weakening along with increasing Fe²⁺concentration.When the Fe³⁺:Fe²⁺is 4:1,the mobility rate was maximum,which was the same with 0.01mmol/L FeSO₄ alone,but was obvious lower than 0.04mM FeCl₃.So it can be seen that the combination of Fe³⁺and Fe²⁺did not have a good synergistic effect.The carnosine played an inhibitory effect by chelating Fe²⁺and Fe³⁺or reducing Fe³⁺to Fe²⁺.Thereby,that validated Fe²⁺and Fe³⁺played a significant promoting effect on the migration of melanocytes further.Protein expressions of MMP-2 and MMP-9 were assessed by and gelatin zymography in BSF melanocytes.Under the same culture conditions,the positive control group have a gray background white strip?MMPs activity bands?,but was not.That indicated that under this culture condition,the BSF melanocytes did not had the expression and activation of MMP-2 and MMP-9.4.The refined pigment was dissolved by solvent A to make the standardized melanin.They had no prominent difference in infrared spectrum,but the absorption intensity and width of characteristic absorption band were different.The alkane structure absorption peak is stronger in the standardized melanin.And the standardized melanin had amide structure and free hydroxyl,the refined pigment with primary amide structure.5.We found another unique solvent B to dissolve melanin.In the solvent A,the maximum solubility of the standardized melanin was 60 ug/mL?25??,with large solvent absorbance background at 405 nm.In the solvent B,the maximum solubility of the standardized melanin was 250 ug/mL?25??,with little solvent absorbance background at 405 nm.A visible light spectrophotometric method for the determination of BSF melanocytes by solvent B was developed.The melanin standard solution kept a good linearity relation in the content range of 5²50ug/mL?r=0.9995?.In the precision experiment,RSD was 1.3%-2%.The cell sample was stable in three hours,RSD was 4.86%.The repeatability of test was good,RSD was 4.4%.The average recovery of melanocytes sample ranged from 103.6%to 95.2%,the RSD was3.5%.6.The effect of tyrosine,tyrosine disodium salt,glucose,calcium,ferrous iron and ferric iron to the activity of tyrosinase was studied by methods of enzymology.The results demonstrated that tyrosine and its sodium salt have notably improved the activity of tyrosinase,and the role of tyrosine disodium salt is prominent than tyrosine.The appropriate glucose concentration will produce a synergistic effect.The high Ca²⁺concentration?0.4-1.6mM?had a significant effect on the activity of tyrosinase,so did the low Fe²⁺concentration?0.01mM?.But the high concentrations of Fe²⁺?0.08-0.16mM?had inhibition of the activity of tyrosinase.The low concentration Fe³⁺?0.01-0.04mM?had a significant promotion on the activity of tyrosinase,and enhanced with the increase of the dose.Ca²⁺,Fe³⁺and Fe²⁺all have a significant promoting effect on the activity of tyrosinase in BSF melanocytes,but the combination could weaken the promotion respectively,while the combination of Fe³⁺and Fe²⁺has no effect on the activity of tyrosinase.7.The fluorescence intensity in BSF melanocytes stimulated by Calcium and Iron was then measured in the fluorescence plate reader.It found that the Ca²⁺concentration?0.2-0.8mM?would significantly increase content of ROS in melanocytes,among them,0.4mM being remarkable.Fe²⁺and a low concentration of Fe³⁺decreased the ROS content,and high concentrations of Ca²⁺and Fe³⁺had no effect on ROS content.The concentrations of calcium and iron had no inhibitory effect on melanocytes.Although the content of ROS in BSF melanocytes changed,the intracellular ROS equilibrium was in balance under the intervention of calcium and iron.8.Using the new method for the determination of BSF melanin by solvent B to examine the effect of LY,TPA,calcium,iron on melanin synthesis in BSF melanocytes.From the results,we concluded that LY had a significant role in promoting melanogenesis,and TPA could inhibit its promotion.Calcium and iron has an obvious role in promoting the biosynthesis of melanin,with calcium being greater than iron.There was no synergistic effect between calcium and iron,but Fe³⁺and Fe²⁺had.Under the intervention of calcium and iron,the DHHP antioxidant capacity of melanin enhanced significantly,ferric iron was the strongest among them.9.The effect of calcium and iron on expression of TRP1 protein levels were assessed by Western blot analysis in BSF melanocytes.Ca²⁺caused an significant increases in TRP1 protein levels,conversely Fe²⁺and Fe³⁺with the significant inhibition.Nevertheless,the combination of Fe²⁺and Fe³⁺showed an elevation of TRP1 protein levels.If Ca²⁺combined with Fe²⁺and Fe³⁺,the auxo-action of Ca²⁺caused an significant decreases in TRP1 protein levels,and ferrous iron was stronger than ferric iron on inhibitory effect.10.With calcium antagonist verapamil blocking-up calcium channels in BSF melanocytes,intracellular calcium decreased significantly.The melanogenesis lessened,with the decline in the expression of TRP1 protein levels,but still significantly been higher than the control group.We deduced the L-type calcium channels was the main channel for the intake of calcium,but there were other ways for the intake of calcium involved in melanin synthesis.11.The effect of Glc,H₂O₂,LY,taurine and zinc citrate on HUVEC-12proliferation was studied through MTT experiment.The results showed that high glucose has little effect on cell survival within 72 hours,except for 48h.No Glc could significantly inhibit cell survival within 12-72h and showed a time dependence,so did1mM H₂O₂.There was no inhibition of cell viability,LY was 12h,taurine was 24h and zinc citrate was 12h.12.To investigate the relationship between the oxidative injury induced by no glucose and H₂O₂ and LY,taurine and zinc citrate in HUVEC-12 cells.The results showed that the low concentration LY,taurine and zinc citrate had protective effect on the damaged cells.The protective effect of the combination with each on the endothelial cells injured by hydrogen peroxide was weaker than that of zinc citrate,taurine and LY alone.Zinc citrate,taurine and LY had no protective effect on the injury of endothelial cells induced by no Glc.