Effects Of Heparin Loaded Magnetic Nanoparticles On Glomerular Filtration Rate In Rats With Acute Hepatic Failure

Objective:Hepatorenal syndrome(HRS)is an acute functional renal failure due to severe liver damage and portal hypertension.It is one of the common serious complications of advanced liver disease.The prognosis of patients with severe hepatitis and cirrhosis combined with HRS is extremely poor.The case fatality rate is about 70%.Although clinically active treatment measures such as volume expansion and vasoconstriction have been taken in patients with HRS,the renal function of most patients still cannot be improved.The prognosis of HRS in patients with cirrhosis indicates a poor.If there is no liver transplantation,the renal failure is prognosis irreversible.HRS is the result of activation of the vasoconstrictor system in the renal circulatory system.The functional status of RASMCs(Rat arterial smooth muscle cells)directly affects the relaxation and contraction of the renal arterioles.The increase of cytosolic free Ca2⁺concentration([Ca2+]i)is closely related to the contraction of GMCs and RASMCs.When severe liver failure occurs,the active substances that constrict blood vessels in the patient's body increase significantly.These substances have the ability to cause the increase of inositol triphosphate(IP₃)in many tissue cells,which in combination with IP₃Rs releases intracellular calcium,which in turn causes renal artery smooth muscle cells and mesangial cells to contract.The above reactions can be inhibited by blocking agents,and heparin is an easily available,low-cost,commonly used clinical drug,which can competitively block IP₃ induced calcium ion release.But heparin also has many adverse reactions,such as bleeding,mostly caused by excessive.Its membrane permeability is poor,so a larger amount is required to play a therapeutic role.This is limiting its clinical application.If heparin can only be applied to the targeted site through a drug carrier to reduce adverse reactions.It may be used to treat HRS.Fe₃O₄ nanoparticles are currently the most widely used magnetic nanomaterials due to its low toxicity,chemical stability is good,surface is easy to modify,and relatively low costs.Studies have found that heparin can be linked to ferric oxide nanoparticles with amino and carboxyl groups on the surface of the modified surface.Kyoung Ah Min et al.Found that by adsorbing glycine on the surface of magnetic nanoparticles,and then combining heparin with the surface of nanoparticles by polymerizing the electrostatic reaction between glycine and heparin.The ability to pass through cell membranes and polyester membranes has increased 100 times in a magnetic field.If heparin can be targeted to the kidney through the drug-loading system.Its membrane permeability can increase.Enter the cell to blocking the release of Ca²⁺induced by IP₃Rs and inhibiting vasoconstriction.The heparin Dosage can can be reduced.Increased drug effect while reducing adverse effects.The therapeutic effect of hepatorenal syndrome was achieved by blocking the TNF?-induced IP₃Rs expression pathway in liver failure rats.Materials and methods:1.Materials1)Rat GMCs(HBZY-1)(CRL-2573)was obtained from CCTCC(China Center for Type Culture Collection).Cell was cultured at 37?in a 5%CO2 humidified incubator with high glucose DMEM medium(containing 10%fetal bovine serum(FBS),100U/I penicillin,100mg/l streptomycin).2)Male SPF SD rats weighting around 140±5g were house in stainless steel mesh cages under controlled conditions of temperature(23±3?)and light 12h a day.The animals were allowed access to food and tap water throughout the acclimatization for 2 days and then experiment.2.Methods1)Carboxylated Fe3O4 magnetic nanoparticles and heparin completed drug loading according to the EDC/NSH catalytic amidation between the carboxylic acid end groups on the surface of the magnetic nanoparticles and the amine groups on heparin.1%agarose gel electrophoresis was used to compare the drug loading nanoparticles before and after the reaction.2)The nanoparticle before and after drug congjuanction is observed and mesured by transmission electron microscope(TEM).3)Magnetic nanoparticle was proven by Fourier Transform Infrared spectroscopy(FTIR)(Shanghai microspectrum chemical technology service Co.LTD.)4)The drug loading rate of heparin nanoparticles was measured indirectly by Anti-II factor activity method.5)MTS method was used to evaluate the effect of magnetic nanoparticles(MNP)and heparin-loaded nanoparticles(Hep-MNP)on the proliferation rate of HBZY-1 cells.6)The cell was co-cultured with MNP and Hep-MNP,and the intracellular iron content was detected to determine the cell entry efficiency.7)Western blot was used to study the effect of drug-loaded nanoparticles on the expression of IP₃R1 protein in HBZY-1.8)Realtime PCR was used to detect the IP₃R1 mRNA in HBZY-1 after Hep-MNP.9)Measure the intracellular calcium concentration([Ca²⁺]i)of the HBZY-1cell.Test the effect of Hep-MNP o in HBZY-1 on[Ca²⁺]i release.10)Rats were randomly divided into 5 groups:The microosmotic pumps with FITC-inulin were implanted into the bilateral abdominal cavity of SD rats.Permanent magnets were implanted subcutaneous near kidneys.5 days later,MNP,Hep-MNP and heparin were injected into tail vain 30min before the injection of Gal N/LPS.11)To construct an animal model of acute liver failure and kidney injury,D-Gal N800mg/kg+LPS32 g/kg),with a volume of 5ml/kg.The dead rats could not collect blood,only the death time of rat was calculated.12)Routine HE staining was performed on liver and kidney tissues.the liver tissue was observed under light microscope.Routine HE staining was performed to observe the no changes in renal tissue structure under light microscope.13)Serum creatinine(Cr),alanine aminotransferase(ALT),asthenate aminotransferase(AST),potassium(K⁺),sodium(Na⁺)and chloride(Cl^-)were detected.14)The fluorescence intensity of serum was detected by luciferase marker,and GFR,GFR1(ml·min),GFR1=R/[Iss],GFR2(ml·min·kg BW),GFR3(ml·min·kg BW),to observe the effect of Hep-MNP,heparin and other drugs on glomerular filtration rate in rats with acute liver failure.15)The glomerulus was separated and the changes in glomerular volume(GIS1/GIS2)before and after the treatment of ET-1 were measured by the method of glomerular Inulin volume measurement(GIS),which indirectly reflected the vasoconstriction of the glomerulus.16)The glomerulus was extracted,and the calcium level in the glomerulus before and after the action of ET-1 was detected by intracellular calcium determination,and the calcium release in the glomerulus was reflected by the multiple increase of calcium in the glomerulus.17)Western blot and real-time PCR were used to detect the expression of IPR1protein and mRNA in the glomeruli of the rats in each group.Results:1.Carboxylated magnetic nanoparticles(MNP-COOH)and heparin were catalyzed by EDC/NHS:compared with the reaction product Hep-MNP,the separation of Hep-MNP and liquid could be separated by magnetic field.2.In TEM:The diameter of MNP was about 15nm,and the distribution was dispersed without obvious condensation.Heparin-MNP diameter was about 15-18nm,no obvious condensation.3.FTIR method verified the reaction products:compared with MNP,the FTIR spectra of Hep-MNP showed partial characteristic peaks of heparin.4.Drug loading ability of magnetic nanoparticles:1 mg MNP can load 8.02 units heparin.The anticoagulant activity 1 mg of Hep-MNP was equivalent to that of 3.08units of heparin.5.The MTS method of proliferation of HBZY-1 cells,the two dose groups of Hep-MNP,MNP,1U/ml heparin and 5 U/ml heparin were not significantly decreased(P>0.05).25,125 U/ml heparin had show inhibitory effect on cell proliferation(P<0.05).6.Through check the intracellular iron,it was found that Hep-MNP can entered the cell faster than that of MNP,and the magnetic field could accelerate MNP and Hep-MNP.7.Effects of Hep-MNP,heparin and MNP on the expression of IP₃R1 protein in HBZY-1 cells.There was no significant difference in the expression level of IP3R1protein in Hep-MNP,,heparin and MNP groups compared with the NS group(P>0.05),TNF group was significantly higher than that in the NS group(P<0.01).8.Effects of Hep-MNP,heparin and MNP on IPR1 mRNA expression in HBZY-1cells.There was no significant difference in IP3R1mRNA level between Hep-MNP,heparin and MNP group and NS group(P>0.05),TNF group was significantly higher than that in NS group(P<0.01).9.Effects of Hep-MNP,heparin and MNP on intracellular calcium release of HBZY-1:Hep-MNP and heparin could inhibit intracellular calcium release(P<0.05).Compare with NS group,MNP group show no difference(P>0.05).10.There was no significant difference in the mortality rate among other groups.11.Under the influence of Gal N/LPS.The damaged was characterized by massive hepatocellular necrosis without liver cell regeneration.There was no obvious injury in glomerulus,proximal tubule and distal tubule in kidney pathology.12.Under the influence of Gal N/LPS,there was a markedly increase in serumt ALT and AST.There was no significant difference in Cr value between G/L+heparin group and G/L+Hep-MNP group(P>0.05),and no significant difference in Cr value between G/L+MNP group and NS group(P>0.05),no significant difference in K⁺,Na⁺and Cl^-between 5 groups(P>0.05).13.GFR of the rats in each group was significantly lower in G/L+NS group,G/L+MNP group,G/L+heparin group and G/L+Hep-MNP group than in the physiological group(P<0.05).G/L+Hep-MNP group and G/L+heparin group showed improvement in GFR1,GFR2 and GFR3 compared with G/L+NS group and G/L+MNP group(P<0.05).14.Compared with the physiological group,G/L+NS group,G/L+MNP group,G/L+Hep-MNP group the multiple ratio of increased calcium concentration in the glomerulus of rat is higher(P<0.05).G/L+heparin group and G/L+Hep-MNP group was significantly lower than that of G/L+NS group and G/L+MNP group(P<0.05).The Hep-MNP group showed more significant improvement than the heparin group(P<0.05).15.GIS changes in G/L+Hep-MNP,G/L+NS group and G/L+MNP group were significantly increased compared with physiological group(P<0.05).GIS changes in G/L+heparin group and G/L+Hep-MNP group were significantly decreased compared with G/L+NS group(P<0.05).No difference between G/L+heparin group and G/L+Hep-MNP group(P>0.05).16.IP₃R1 mRNA expression in G/L+NS group,G/L+MNP group,G/L+heparin group and G/L+Hep-MNP group was significantly higher than that in physiological group(2.31±0.14,P<0.05).32.24±0.10,P<0.05).There was no significant difference between them(P>0.05).17.IP₃R1 protein expression in G/L+NS group,G/L+MNP group,G/L+heparin and G/L+Hep-MNP group was significantly higher than that in the physiological group(P<0.05).Conclusion:1.Through the catalytic reaction of EDC/NHS,heparin can be loaded by the carboxylated Fe₃O₄ nanoparticles.The nanoparticles have no obvious cytotoxicity or proliferation inhibition after drug loading.and can be absorbed by RGMCs(HBZY-1).2.1mg Hep-MNP can carry about 8U of Heparin.the anticoagulant activity of 1mg Hep-MNP is equivalent to 3 U of Heparin.The expression of IP₃R1mRNA and protein can not be Influenced.It could act on the intracellular IP₃R1 receptor to Inhibit the release of calcium ions and reduce intracellular calcium concentration.3.Compared with normal group,glomerular filtration rate in rats with acute liver failure was significantly lower and Cr was significantly higher,while Hep-MNP and Heparin could significantly improve GFR and Cr levels in rats with acute liver failure.