Collagen Peptide From Walleye Pollock Skin Promotes Proliferation Of Human Osteoblast Via CaMKK/AMPK/Runx2 Pathway

Objective:The pathological model of osteoblasts（Saos-2）injury induced by cadmi um chloride（CdCl₂）was replicated and the protective effect of collagen peptide fr om walleye pollock skin（CP）via CaMKK/AMPK/Runx2 signaling pathway was de monstrated.The method of high performance liquid chromatography（HPLC）detect ion of intracellular Pro-Hyp and Gly-Pro-Hyp.Methods:CCK8 method was used to determine the optimal concentration of CdCl₂ and measured the survival of CP in Saos-2 cell.Cytotoxicity of CP on Saos-2 cells for 24 h by LDH.The time chan ging of CdCl₂ on Saos-2 cells detected by RT-PCR.Western Blot and RT-PCR tec hniques were used to detect the expression level of mRNA and protein in different treatment groups such as p-AMPK/AMPK,Ca MKK,AMPK and Runx2 and using inhibitors,liposome transfection technology to prove its cascade relationship.The P ro-Hyp and Gly-Pro-Hyp contents in the intracellular were measured by HPLC and the purpose of determining the osteoblasts metabolic ability of CP.Statistics analys is of the data was performed with SPSS22.0.Results:The LDH assay showed tha t 200,300,400,500,600 or 700μg/m L CP incubated for 24 h,cytotoxicity was obse rved at 600μg/mL（p<0.05）.CCK-8 results showed that 2μM CdCl₂ successfully replicated the Saos-2 cell injury model for 24 h with concentration of 0.5,1,2,5μM,the cell viability decreased significantly（p<0.05）,when 125,250 and 500μg/m L CP were pre-protected for 24 h,the cell viability increased in a dose-dependent manner（p<0.05）.CdCl₂ incubated Saos-2 cell with 6,12,24,48 h CaMKK mRNA expression increased in time-dependent manner,but AMPK did not change,Runx2showed a downward trend（p<0.05）.The level of CaMKK mRNA and protein in the model group were higher than those innormal group（p<0.05）,AMPK mRNA level was unchanged,and p-AMPK/AMPK protein level,Runx2 mRNA and prote in level were lower than those in the normalgroup（p<0.05）.Suggesting that CaM KK negative correlation with cell proliferation,p-AMPK,Runx2 was positively corr elated with cell proliferation.The mRNA and protein level of CaMKK in CP grou p were lower than those in CdCl₂ group（p<0.05）.The level of p-AMPK/AMPK p rotein,Runx2 mRNA and protein were higherthan those in normal group（p<0.05）,suggesting that CP could be up-regulated by down-regulating Ca MKK.AMPK an d Runx2 promote Saos-2 cell proliferation.After si-CaMKK or STO-609 treatment,CaMKK expression was silenced or decrease in the model group,and the express ion of AMPK protein was significantly higherthan that in the normal group（p<0.05）,suggesting that Ca MKK negatively regulateAMPK.The expression of AMPK in the CP group was higher than that in the model group（p<0.05）,suggesting tha t CP can upregulate AMPK by inhibiting CaMKK.After treatment with si-AMPK or Compound C,AMPK expression in the model group wassilenced or decreased,and Runx2 expression was significantly lower than that in the normal group（p<0.05）,suggesting that AMPK is regulating Runx2.The expression of Runx2 in the CP group decreased compared with the CdCl₂ group（p<0.05）,suggesting that CP can upregulate Runx2 by activating AMPK.HPLC results showed that the linear r elationship of Pro-Hyp was y=4E+07x+15854,R²=0.9998,Gly-Pro-Hyp was y=4E+06x+15934,R²=0.9996.The average recoveries of low,medium and high concentrati ons Pro-Hyp were 97.30%、98.46%、99.94%and the respectively RSD were 0.81%、1.76%、2.03%.The average recoveries of Gly-Pro-Hyp were 98.83%、97.80%、99.20%and the respectively RSD were1.95%、2.02%、1.92%suggesting that the goo d recovery of small peptides.Compared with the normal group,Pro-Hyp content i n the model group was significantly lower（p<0.05）,Gly-Pro-Hyp,Pro-Hyp content in the 125,250,500μg/mL CP group,and500μg/m L tripeptide group.Compared w ith the model group,it was increased（p<0.05）,suggesting that osteoblasts can meta bolize CP to small short peptides.Conclusion:The Saos-2 cell damage model was successfully replicated with 2μM CdCl₂ exposure for 24 h.CP inhibit the damag e of CdCl₂-induced in Saos-2 cells and themechanism may be related to the down-regulation of CaMKK/AMPK/Runx2 signaling pathway.Osteoblast have the ability to metabolize CP to the small molecule active peptides Pro-Hyp and Gly-Pro-Hyp.