The Mechanism By Which Novel Enoxparin Sodium PMMA Bone Cement Reduces Thrombosis Through Lnc RNA22719.16/miR-326-5P/CD40 Axis

Objective:1.Explore to establish the animal model of whether local thrombosis can be formed around high-viscosity bone cement,and verify that the novel material of high-viscosity cement can reduce the macroscopic appearance of local thrombosis.2.To explore the mechanism of the novel enoxaparin sodium-highviscosity bone cement in reducing thrombosis compared with ordinary highviscosity bone cement.3.Explore the differences in the expression of long non-coding RNA between different biomaterial thrombosis samples,and screen out new differential expression of long non-coding RNA.4.Preliminary verification of the relationship between the long chain non-coding RNA22719.16/ mi R-326-5p /CD40 axis affecting thrombosis.Methods:1.We established carotid arteriovenous shunt in New Zealand rabbits,implanted bone cement lines with different properties into the shunt,and observed the amount of thrombosis formed by bone cement strips between different groups within the same time.2.Total RNA was extracted using TRIzol Reagent(Invitrogen,USA),and the m RNA was reverse transcribed using Prime Script RT Reagent Kit(Takara,Japan)according to the manufacturer’s instructions.Quantitative real-time polymerase chain reaction(PCR)was performed using the SYBR Premix Ex Taq kit(Servicebio Wuhan)on Applied Biosystems’ i Q5 real-time PCR amplification instrument(Applied Biosystems USA).Each sample was amplified at 94℃ for 4min,94℃ for 30 s,60℃ for 30 s,and 72℃ for 30 s in40 cycles.Each sample was amplified at 3 repetitions and its m RNA was analyzed.3.Blood vessel samples were washed with cold PBS and then cleaved with RIPA(Beyotime,China)containing protease inhibitors.Protein lysates were separated on 10% SDS-PAGE and then transferred to polyvinylidene fluoride(PVDF)membranes(Millipore,USA).We transfused the membranewith primary antibodies including CD31(abcam,UK),CD40(abcam,UK),CD62p(abcam,UK),CD106(abcam,UK),Endothelin(abcam,UK),Thrombomodulin(abcam,UK)and then incubated the membrane overnight in a shaker at 4℃,and incubated the membrane in a medium containing the corresponding secondary antibody for 1.5 hours.Put the film in Western Lightning TM Chemiluminescence Reagent for 30 s,immediately put the film in the exposure box,and expose the photographic film in a darkroom for 1 min,then develop and fix the film.4.The frozen vascular tissues were balanced for 1 hour,fixed with 4%paraformaldehyde,conventionally dipped in wax and embedded,sliced with thickness of 8μm,paraffin sections were dewaxed to water,placed in Triton X-100(mass fraction 0.5%)for 10 minutes,and sealed with 4% goat serum albumin BAS.Primary anti-CD40-Antibody(abcam,UK)1:2000,rinsed with PBS for 3 times overnight at 4℃;secondary goat anti-rabbit Ig G antibody(abcam,UK)1:400,rinsed with PBS at room temperature for 2 hours,dried,observed and photographed under fluorescence microscope.5.Blood samples of New Zealand rabbits from different groups were collected for 4 hours after modeling,and the samples were stored with heparin sodium as anticoagulant.The supernatant was centrifuged at 1000*g for 15 minutes to obtain plasma,which was stored in a-20℃ refrigerator for later use.Set standard well and sample well,add different concentration 50μL to standard well and 50μL to sample well,do not add blank well.Add horseradish peroxidase(HRP)labeled detection antibody 100μL to each standard well and sample well,seal the membrane,incubate for 60 min at37℃,discard the liquid,dry with washing solution 350μL to each well,rest for 1min,discard the washing solution,dry with suction paper,repeat 5 times.Add 50μL of substrate A and B to each well,incubate at 37℃ for 15 min,add50μL of stop solution to each well,measure the OD value of each well at450 nm within 15 minutes.6.Common high-viscosity bone cement and novel enoxaparin sodiumhigh-viscosity bone cement thrombus samples were taken,and Tianmo#tr205-200 kit was used for RNA extraction of the samples.Total RNA extracted was examined by Agilent Bioanalyzer 2100(Agilent Technologies,Santa Clara,CA,US)for RNA integrity.And using Qubit?3.0 Fluorometer(Life Technologies,CA,USA)and Nanodrop One spectrometer(Thermo Fisher Scientific Inc.,USA)to detect the concentration and purity of total RNA.The reference genome version used was Ory Cun2.0.102.7.Classic ce RNA prediction was performed by sequencing companies for the new differentially expressed Lnc RNA and CD40 gene screened out from the thrombosis samples of the novel enoxparin sodium-high-viscosity bone cement group and the ordinary high-viscosity bone cement group.Lnc RNA and mi RNA that can regulate CD40 gene were predicted and preliminarily verified by dual luciferase reporter gene assay,and FISH assay was performed to verify Lnc RNA probe in vascular tissue.Results:1.It has been verified in animal models that high-viscosity bone cement can form local thrombosis.Compared with ordinary high-viscosity bone cement,the novel enoxparin sodium-high-viscosity bone cement can significantly reduce the amount of local thrombosis,with significant statistical differences.2.The expression of CD40 in 6 proteins related to thrombosis in vascular tissue endothelial cells in the novel enoxparin sodium-high-viscosity bone cement group was significantly lower than that in the ordinary high-viscosity bone cement group,but higher than that in the sham group,with statistical significance.3.The CD40 fluorescence intensity of vascular endothelial cells in the novel enoxaparin sodium-high-viscosity bone cement group was significantly lower than that in the ordinary high-viscosity bone cement group,and was higher than that in the sham group.There was no significant difference in plasma s CD40 L expression among the three groups.4.The thrombus samples were good,and the novel enoxaparin sodium–high-viscosity bone cement was successfully screened out for its new differential expression of long non-coding RNA compared with ordinary highviscosity bone cement.5.The co-transfected ocu-mi R-326-5p mimics inhibited luciferase expression in fireflies(P<0.05),while the ocu-mi R-326-5p inhibitors increased luciferase activity in fireflies(P<0.05).To confirm that this inhibition or promotion depends on the predicted mi R-326-5p site,we tested pmirglo-CD40 m RNA 3?-UTR-mut and pmirglo-lnc RNA MSTRG.22719.16-mut,whose recognition sites contain mutations.This mutant did not respond to either ocu-mi R-326-5p mimics(P<0.05)or inhibitors(P<0.05).In FISH experiment,the fluorescence brightness of lnc RNA MSTRG.22719.16 probe was significantly reduced in the novel enoxaparin sodium bone cement group.Conclusion:1.It was verified by the animal model of New Zealand rabbit external arteriovenous shunt that ordinary high-viscosity bone cement could to form local attached thrombosis.2.The novel enoxparin sodium-high-viscosity bone cement could also produce local attached thrombosis,but compared with ordinary high-viscosity bone cement,the amount of thrombosis by the novel material was significantly reduced.3.Compared with ordinary high-viscosity bone cement,the novel enoxaparin sodium-high-viscosity bone cement can reduce local thrombosis by reducing the expression of CD40 protein in vascular endothelial cells.4.Novel enoxaparin sodium-high-viscosity bone cement and ordinary high-viscosity bone cement have little effect on s CD40 L in blood circulation.5.Differentially expressed long non-coding RNA were found in different samples of bone cement thrombosis,and some new differentially expressed long non-coding RNA were obtained.It is inferred that these differentially expressed long non-coding RNA play a regulatory role in reducing the process of thrombosis.6.The new differentially expressed lnc RNA MSTRG.22719.16 can regulate CD40 gene through ocu-mi R-326-5p.