Study Of Related Mechanisms About The Effect Of Endocrine Protein In Stomach On Bone Metabolism

BackgroundThe morbility of Osteoporosis(OP)is increasing in the aging society.There are a lot of related effective factors result to OP,though its mechanism still unclear,which include hereditary factor,menopause,intake or absorption deficient about calcium or phosphorus,et al.There are certainly interesting focus on the OP induced by gastrectomy or gastropathy recently.It is well known that there is certain connection between the gastrectomy and osteoporosis.By now,more and more evidences show that the extracts of oxyntic mucosa(EOM)could increase the bone mess,Both gastrin-17 and the extract of oxyntic mucosa(EOM)induce transient hypocalcaemia and stimulate an uptake of Ca into bone in rats.Gastrin was without effect in rats that had undergone gastrectomy,whereas the EOM were equally effective in unoperated and gastrectomized rats.And this hypocalcemic effect was totally abolished by prior addition with leucine aminopeptidase.That suggests that the effective component of EOM is a small peptide.We propose the name gastrocalcin for this peptide hormone,which is produced in the oxyntic mucosa and released by gastrin.It accelerates calcium uptake into bone,thereby explaining the hypocalcaemia evoked by gastrin.To the three functionally interacting factors controlling Ca2+ concentration in blood(parathyroid hormone,calcitonin,and vitamin D),we wish to add a fourth---namely,gastrocalcin.Medicine treatments for OP include anti-resorptive agents and bone-forming agents.Anti-resorptive agents,such as estrogens,calcitonin,bisphosphonates,can inhibit the bioactivity of osteoclasts and increase the bone mess relatively.Bone remodeling is an ongoing process in human bone biology that is necessary to repair micro-damage and renew skeletal integrity strength.Therefore,inhibition of bone resorption mechanism would induce the problem of bone aging.The idea medicine treatment of OP should contain both anti-resorptive agents and bone-forming agents, it is the major tend of OP management in the world.The main bone-forming agent is PTH which was approval to apply in clinic at 2002.But its cost is expensive and it would be administrated by intravenous injection only;these disadvantages limited its extensive utility.Above all,there is another bone-forming agent in the oxyntic mucosa of rats, which is named gastrocalcin.Therefore the study about gastrocalcin has great prospective.However gastrocalcin was a hypothetic peptide result from its promoting bone-forming function,its nature still unclear by now.Since that,this study about the gastrocalcin is much more important for explore the mechanism of OP introduced by gastrectomy.Objectives1,Explore the effects of EOM on the bioactivity of osteoblast.2,Screening the candidate protein of gastrocalcin and determining its nature.Methods1,Osteoblast isolation,culture,and identification:Osteoblasts were isolated from calvariae of 1-day-old Sprague-Dawley rats with the way of sequential enzymatic digestion,and than were identified by morphology,ALP staining, Alizarin red staining and fluorescence staining.2,Preparation of EOM in rats:EOM was prepared as described previously. Briefly,oxyntic mucosa was collected from six male Sprague-Dawley rats.The stomach was opened,the antrum was removed,and the oxyntic gland area (fundus)rinsed in saline and placed on a cold silica gel plate surface.The mucosa free of mucus was scraped to separate from the muscle wall,then minced by Ultra-Turrax homogenizer and homogenized in buffer,using a Potter-Elvehjem glass homogenizer.The homogenate was centrifuged and the pellet was discarded.The supernatant was passed through cheesecloth. Filtration was followed by centrifugation.After the supernatant was discarded, the pellet was resuspended in the saline(with 1%proteinase inhibitor)by ultrasonication six times(six seconds each)on the ice.At last the solution was quantified according to the instruction of BCA protein assay kit and diluted to serial concentrations:10^-1g/l,10^-2g/l,10^-3g/l with saline respectively,then kept at-80℃.3,Ca concentration in cytoplasm of osteoblasts:The cells at the third generation were digested and diluted to 1×10⁷/L,then inoculated in four special 50mm culture dishes for laser scanning confocal microscopy,for 24 hours,followed by 5μM Fluo-3Am applied into each dish to label the intracytoplasm Ca²⁺. The dishes were placed on the sample holding plate of the confocal microscope and the change of[Ca²⁺]i was investigated,under the normal(saline as control) or the influences of EOM in different concentrations.4,Proliferation of osteoblast:Cells were inoculated into 96-wells plate at the density of 2×10³ cells per well and divided into four groups and treated with low,mid,high concentrations of EOM and saline as control,respectively.24 hours after cell seeding,10μL EOM at different concentration or saline was added into each well.0,1,2 and 3 days after the treatment,cell proliferation was assessed according to the instruction of CCK-8 kit(BI Yuntian Co,China), respectively.In brief,at the end of each time point,10μL WST-8 was added to each well,and the plates were incubated for an additional 4 hours at 37℃to convert WST-8 into formazan.The absorbance of each plate was measured at 450 nm(absorbance)and 600 nm(background)with spectrophotometer.5,Reverse transcriptase polymerase chain reaction(RT-PCR)test:The cells were inoculated in 6 cm culture dishes and designed into four groups:low,mid,high concentration EOM groups and saline control group.24 hours later,the new culture medium added in to culture system,together with 200μl EOM at different concentration or saline.After another 24 hour culture,total RNA was extracted from each group's osteoblasts using a TRIzol kit.The reverse transcription reaction was performed using 4μg of total RNA and oligo(dT) primer,and collagen typeâ… ,osteocalcin,P-actin(reference gene)gene were amplified using two oligonucleotide primers.The PCR products were separated electrophoretically in a 2%agarose DNA gel stained with ethidium bromide. Semi-quantitative analysis was performed using computing densitometer and Image Quant software.6,Western-blot test:Cells were cultured in 6 cm culture dishes and treated with EOM.After 48 hours,the total proteins were extracted from each group. Proteins concentration were assessed by BCA protein assay kit,according to the manufacturer's instructions.Equal proteins(60μg)were separated electrophoretically in SDS-PAGE for collagen typeâ… (138kDa)andβ-actin (43kDa)or Tricine-SDS-PAGE for osteocalcin(5.8kDa).The proteins were transferred to immobilon polyvinyldifluoride(PVDF)membranes.After blocked with 5%BSA for 1 h at room temperature,the membrane was incubated with rabbit anti-mouse antibodies against collagen typeâ… ,osteocalcin orβ-actin(for reference protein)(1:1000)for 1 h at room temperature,washed 6 times(5 minutes each)with TBS buffer containing 0.2%TW-20,followed by incubation with anti-rabbit peroxidase-conjugated secondary antibody(1:3000) for 1 h at room temperature and wash as above.The proteins recognized by antibodies were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film.Quantitative data were obtained using computing densitometer and Image Quant software.7,Proteomics experiment:Total protein was extracted from the gastric mucosa tissue of experiment and the control group respectively.And then total protein was treated for desalination and quantitation.The protein samples were separated with fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE),and the different proteins were identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS).8,Analysis by bioinformatics technology:The identified different proteins were estimated further by the technology of bioinformatics through data base and literature.9,The result of 2D-DIGE was reconfirmed by western-blot test.10,The location and the distribution of candidate protein defined by 2D-DIGE were explored by immunohistochemistry test.Results1,Osteoblast identification:According to the morphology observation and the staining of ALP,AZR and fluorescence,the isolated and cultured cells from rat cranial bone have the characters of osteoblasts.2,Intracytoplasm Ca2+of osteoblast assay:Under laser scanning confocal microscope,four quadrants total to 20 cells of each visual field were chosen randomly for intracytoplasm[Ca²⁺]i recording,before or after administration of different stimulate factors.None cell changed in control group,but 15%(three out of twenty cells)in 10^-3g/L EOM group,35%(seven out of twenty cells)in 10^-2g/Lgroup and 95%(nineteen out of twenty cells)in 10^-1g/Lgroup had demonstrated a kind of response that the[Ca²⁺]i in those cells showed increasing rapidly and temporally just after different concentration of EOM was administered。3,Osteoblast proliferation assay:The number of cells in three experimental groups increased as compared with the control group,This difference is significant after being analysised by statistics(p＜0.05).4,RT-PCR and Western-blot test:Contrast to the control group,the mRNA expression of collagen typeâ… and osteocalcin increased in three experimental groups This difference is significant after being analysised by statistics (p＜0.05).5,Proteomics experiment:There are 50 different proteins expressed in experimental groups compared as control groups,among those different proteins,21 proteins increased expression and 29 decreased expression.22 different proteins were identified by MS.Analysis by bioinformatics technology: The 22 different proteins were located detailly into subcellular structure by WOLF PSORT data base.The interactivity of 22different proteins were predicted by STRING data base.Among the 22 different proteins,our interesting focus on the PDIA3 protein,which is highly expressed after the administration of gastrin,and plays an important role in the mechanism of mineral salt.In intestinal epithelial cells and perfused duodena,the PDIA3 also known as PDIA3/1,25D3-MARRS receptor mediates the rapid(seconds to minutes)stimulation of phosphate and calcium.And it is notable that osteoblasts,osteoclasts,and odontoblasts have been reported to contain PDIA3/1,25D3-MARRS.6,According to the result of western-blot test,the expression of PDIA3/1,25D3-MARRS in rat stomach mucosa was increasing after the administration of gastrin.This result is coincident with 2D-DIGE.That is the confirmation for 2D-DIGE.7,According to the result of immunohistochemistry,the expression of PDIA3/ 1,25D3-MARRS is positive in the rat mucosa,and the location of expression is in the glandular tube.Conclusions1,We have demonstrated that EOM elevated intracellular[Ca²⁺]i,after the administration of EOM with doses relationship.2,EOM enhanced osteoblast cells' proliferation significantly.3,EOM increased the expression of collagen type I and osteocalcin in primary cultured osteoblasts,which means it can promote the bioactivity of osteoblasts.4,PDIA3 was regarded as the key factor of promoting bone-forming derives from gastric mucosa.5,PDIA3/1,25D3-MARRS can be regard as the endocrine protein screted from stomach mucosa.