Ceria Nanoparticles Prophylactic Used For Renal Ischemia-Reperfusion Injury Treatment By Attenuating Oxidative Stress And Inflammatory Response

Background:Renal ischemia-reperfusion injury(RIRI),the major cause of acute kidney injury(AKI),occurs in many clinical scenarios and is a fatal disease that kills many patients each year.RIRI generally includes two stages:ischemia and reperfusion,during which excessive oxidative stress and uncontrolled inflammatory response are the two core factors leading to the occurrence and development of the disease.Although the research on the pathological mechanism has made great progress,there has so far been no definitive therapy except for dialysis to treat RIRI,and untimely diagnosis and intervention are the main reasons for advanced renal fibrosis.With the development of nanotechnology,some nanomaterials with nanoenzyme activity have been widely used in scavenging reactive oxygen species(ROS)and regulating inflammatory responses,among which ceria nanoparticles(CNPs)have attracted much attention due to their unique superoxide dismutase(SOD)and hydroxyl radical antioxidant capacity(HORAC)and are widely used in inflammation-related diseases.Moreover,the distribution of nanoparticles during inflammation is size-dependent and organ-specific,and nanoparticles of different sizes have different enzymatic activities and immunomodulatory abilities.Based on the above problems,this topic proposes the following ideas:1.Prophylactic use of CNPs for the treatment of RIRI and subsequent renal fibrosis.2.Find better size nanoparticles by comparing the retention characteristics and therapeutic effects of CNPs of different sizes.To test this hypothesis,CNPs with different sizes were successfully synthesized and characterized.At the same time,based on in vivo and in vitro models,its metabolic characteristics,therapeutic effect and mechanism of action were evaluated in depth.This study aims to provide a new perspective for clinical prevention and treatment of RIRI and renal fibrosis.Materials and medthods:(1)Synthesis and characterization of CNPs:Three different sizes of CNPs(NP46,NP81,NP118)were synthesized by a novel thermal decomposition method and then analyzed by transmission electron microscopy(TEM),X-ray photoelectron spectra(XPS),X-ray powder diffraction(XRD),elemental mapping,enzymatic activity detection(SOD,HORAC)and N2 adsorption and desorption examination of the properties.(2)Detection of the therapeutic effect of CNPs prophylactic used for RIRI.The right nephrectomy and left kidney ischemia-reperfusion(IR)models of C57BL/6J mice were constructed.Mice were administered with CNPs(1 mg/kg)by intravenous injection 2 h before operation,and serum biochemical indexes(serum creatinine,blood urea nitrogen(BUN)),tissue damage(H.E staining,kidney injury index,KIM-1 immunohistochemical staining),cell apoptosis(TUNEL staining of renal tissue)and renal ultrasound(renal vascular RI index)were detected 24 hours after operation.(3)Verify the regulation of CNPs on oxidative stress and inflammatory responses in vivo.Determination of malondialdehyde(MDA)and total superoxide dismutase(T-SOD)levels in IR kidneys,and detection of the activation of the Nrf2 pathway(Nrf2,GCLC,GPX2,NQO1)by western blot(WB)to verify the effect of CNPs on oxidative stress;Immunohistochemistry was used to detect CD45+ leukocytes infiltration in IR kidney,and the main subsets(CD4+T,CD8+T,macrophages,B cells,M1 macrophages)of leukocytes were identified by flow cytometry.Moreover,RT-qPCR and WB detection were used to detect inflammation-related genes(Cxcl1,Il1b,Il6,Tnf)and NF-κb pathway proteins(p65,p-p65,IL-1β)respectively to confirm CNPs’ inflammation regulatory ability.(4)Verify the regulation of CNPs on oxidative stress and inflammatory responses in vitro.In IR mimicking condition,we used human renal tubular epithelial cells(HK2)line,human umbilical vein endothelial cells(HUVEC)line,Primary human renal tubular epithelial cells(RTEC),mouse aortic endothelial cells(MAEC)line to verify the ability of CNPs of attenuating oxidative stress,including the detection of cell viability,intracellular ROS content,mitochondrial cytochrome c and MMP;LPS stimulated RAW264.7 cells were used to verify the ability of inflammatory regulation of CNPs,such as intracellular ROS content,mitochondrial cytochrome c,M1-type polarization(CD86+)ratio,inflammation-related genes(Il1b,Il16,Tnf)and inflammation-related proteins(IL-1β,IL-6,TNF-α)expression.(5)Detection of the therapeutic effect of CNPs on renal fibrosis after RIRI:The IR model of the left kidney of C57BL/6J mice was constructed.Mice were administered with CNPs(10 mg/kg)by intravenous injection 2 h before operation,and the levels of NP46,NP81,and NP118 were detected by ICP-MS,including metabolic characteristics in the blood circulation,heart,liver,spleen,lungs and kidneys.The potential therapeutic effect of CNPs on renal fibrosis was validated in a right nephrectomy and left kidney IR model.(Masson’s trichrome staining and Sirius red staining)Results:(1)TEM detection showed that we synthesized three kinds of spherical and uniformly distributed nanoparticles with diameters of 46 nm,81 nm and 118 nm,respectively.XRD and elemental mapping analysis proved that they are composed of oxygen and cerium elements and conform to the characteristics of CNPs.XPS and enzymatic activity assays demonstrated that their surfaces are enriched in Ce3+ and Ce4+,thus endowing them with excellent SOD and HORAC enzymatic activities.Finally,N2 adsorption/desorption experiments showed that NP118 had the largest specific surface area,thus endowed it with the strongest enzymatic catalytic activity.(2)The prophylactic use of CNPs alleviated the injury of RIRI.Compared with the control group,the serum creatinine and BUN in the CNPs pretreatment group decreased.H.E,immunohistochemistry and TUNEL staining also proved that the degree of renal injury was alleviated,and the expression of the injury-related protein KIM-1 and cell apoptosis were reduced in CNPs treated group.In addition,CNPs also ameliorated RIRI-induced renal microcirculation damage by renal ultrasound.Notably,NP118 showed the best therapeutic effect.(3)In vivo experiments demonstrated that CNPs reduced IR-induced oxidative stress.Compared with the control group,CNPs pretreatment led to lower MDA and higher T-SOD levels.At the same time,the Nrf2 pathway and its related proteins have stronger expression and activation;On the other hand,CNPs also alleviated the inflammatory response,including the decrease in the infiltration of CD45+ immune cells,among which the decrease in macrophages was the most significant,and the macrophages tended to M1 type polarization was significantly suppressed.The effect of CNPs on immune cells is ultimately reflected in the down-regulation of inflammation-related genes(Cxcl1,Hl1b,Il6,Tnf)and the inhibition of inflammation-related NF-κb pathway.(4)CNPs were found to alleviate oxidative stress by reducing intracellular ROS content through reducing MMP and directly scavenging ROS in an IR mimicking condition in vitro,thereby enhancing cell survival.Meanwhile,activation of RAW264.7 cells under LPS-stimulated conditions found that CNPs scavenged intracellular ROS,thus inhibited the polarization of M1 macrophages,and ultimately leaded to inflammation-related genes(Il1b,Il6,Tnf)and proteins(IL-1β,IL-6,TNF-α)expression were down-regulated.(5)ICP-MS confirmed that NP46,NP81,and NP118 have similar metabolic characteristics in major organs and circulation,and can be rapidly excreted in healthy kidneys for a short period of time.But it is worth noting that in the damaged kidney,NP118 can remain stable for more than 21 days.Masson trichrome staining and sirius red staining also proved that CNPs can effectively inhibit the progression of RIRI-induced renal fibrosis,and NP118 has the best effectConclusion:In conclusion,prophylactic use of CNPs can effectively prevent and treat severe renal injury caused by RIRI.On the one hand,CNPs alleviate the level of oxidative stress by directly scavenging ROS and reducing MMPs in renal tubular epithelial cells and vascular endothelial cells.On the other hand,CNPs reduce the synthesis and release of cytokines and chemokines by inhibiting the infiltration of macrophages and M1-type polarization and eventually inhibit inflammation.This dual regulation of CNPs well protected the kidneys from RIRI-induced damage.At the same time,by comparing the metabolic characteristics of NP46,NP81,and NP118,we confirmed that NP118 can stay in the injured kidney for a long time to inhibit the occurrence of fibrosis after RIRI,and it is rapidly excreted in the healthy kidney without causing drug accumulation.Our study demonstrates for the first time the superior therapeutic effect of CNPs on RIRI treatment,filling a gap in the related research field.This study aims to provide a new perspective for clinical prevention and treatment of RIRI and renal fibrosis.