• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Enhanced macrophage polarization induced by COX-2 inhibitor-loaded Pd octahedral nanozymes for treatment of atherosclerosis

    2023-03-14 06:52:18MinXuChuchuRenYueZhouZynekHegerXioyngLingVojtechAdmNnLi
    Chinese Chemical Letters 2023年1期

    Min Xu,Chuchu Ren,Yue Zhou,Zynek Heger,Xioyng Ling,Vojtech Adm,*,Nn Li,*

    a School of Pharmaceutical Science and Technology,Tianjin University,Tainjin 300072,China

    b Department of Chemistry and Biochemistry,Mendel University in Brno,Brno CZ-613 00,Czech Republic

    Keywords:Atherosclerosis COX-2 inhibitor Pd octahedral nanozyme Macrophage polarization Anti-inflammation Antioxidation

    ABSTRACT Inhibition of foam cell formation is considered a promising treatment method for atherosclerosis,the leading cause of cardiovascular diseases worldwide.However,currently available therapeutic strategies have shown unsatisfactory clinical outcomes.Thus,herein,we design aloperine (ALO)-loaded and hyaluronic acid (HA)-modified palladium (Pd) octahedral nanozymes (Pd@HA/ALO) that can synergistically scavenge reactive oxygen species (ROS) and downregulate cyclooxygenase-2 (COX-2) expression to induce macrophage polarization,thus inhibiting foam cell formation to attenuate atherosclerosis.Due to the targeted effect of HA on stabilin-2 and CD44,which are overexpressed in atherosclerotic plaques,Pd@HA/ALO can actively accumulate in atherosclerotic plaques.Subsequently,the antioxidative effects of Pd octahedral nanozymes are mediated by their intrinsic superoxide dismutase- and catalase-like activities capable of effective scavenging of ROS.In addition,anti-inflammatory effects are mediated by controlled,on-demand near-infrared-triggered ALO release leading to inhibition of COX-2 expression.Importantly,the combined therapy can promote the polarization of macrophages to the M2 subtype by upregulating Arg-1 and CD206 expression and downregulating expression of TNF-α,IL-1β and IL-6,thereby inhibiting atherosclerosis-related foam cell formation.In conclusion,the presented in vitro and in vivo data demonstrate that Pd@HA/ALO enhanced macrophage polarization to reduce plaque formation,identifying an attractive treatment strategy for cardiovascular disease.

    Atherosclerosis,which is characterized by chronic inflammation of the arterial wall,is a major contributor to cardiovascular diseases and remains the leading cause of morbidity and mortality in industrialized countries [1,2].Lipid-regulating drugs are the primary treatment for atherosclerosis,but long-term disease remission is not achieved due to the adverse side effects [3–5].Other small molecule drugs,such as probucol,aspirin and indomethacin,have been widely used for the management of atherosclerosis [6–9].Unfortunately,these therapies are limited to mildly delaying the progression of atherosclerosis due to the nonspecific distribution throughout the body.Currently,studies have shown that macrophages play a crucial role in the development of atherosclerosis [10].In particular,different phenotypes of macrophages,including the proinflammatory classic (M1) and anti-inflammatory(M2) phenotypes,were found to affect the stability of atherosclerotic plaques [11–14].M1 macrophages are predominant during the formation of atherosclerosis,inducing inflammation by secreting proinflammatory cytokines such as tumor necrosis factor-α(TNFα),interleukin-1β(IL-1β) and interleukin-6 (IL-6) [15,16].In contrast,M2 macrophages produce anti-inflammatory cytokines and are associated with resolution of atherosclerosis [17].Therefore,alteration of the macrophage phenotype may be a strategy for the atherosclerosis management.

    Fortunately,anti-inflammatory strategies may effectively alter the balance of macrophage subtypes for treatment of atherosclerosis [18,19].Recent studies have found that cyclooxygenase-2 (COX-2),a member of the COX family,plays an important role in the inflammatory response [20,21].Interestingly,aloperine (ALO),an alkaloid extracted fromSophora alopecuroidesL.,have shown antiinflammatory activity in bothin vivoandin vitrosettings [22].As reported previously,ALO reduces the expression of COX-2,thereby exhibiting an anti-inflammatory effect and promoting the phenotypic transition of M1 macrophages to the M2 subtype,which is of utmost interest for atherosclerosis therapy [23].However,it is worth to note that efficient delivery of ALO to atherosclerotic lesions is notoriously difficult.Thus,ALO could substantially benefit from nanoparticle-based targeted delivery strategies that have been shown to provide major advantages (e.g.,improved bioavailability,efficacy and safety) not only in a therapy of cardiovascularrelated diseases [24–28].

    Scheme 1.Schematic illustration displaying (a) the preparation route of Pd@HA/ALO and (b) the therapeutic mechanism of Pd@HA/ALO in atherosclerosis treatment.

    Recent studies have indicated that nanoparticles with intrinsic antioxidative activity are promising next-generation therapies for the treatment of atherosclerosis [29–31].Specifically,through their antioxidative activity,these nanoparticles are able to regulate reactive oxygen species (ROS) for M1 to M2 phenotypic transition of macrophages,thus synergizing anti-inflammatory functions to effectively ameliorate atherosclerosis [32–35].Notably,noble metalbased nanozymes,particularly palladium (Pd) nanoparticles,have received substantial interest due to their good biocompatibility and excellent antioxidative properties,such as catalase (CAT) and superoxide dismutase (SOD) activities [36–39].More importantly,studies have shown that enzyme-like performance can be effectively adjusted by controlling the structure and crystal facets of Pd nanomaterials.In general,lower surface energy (111)-faceted Pd octahedrons have greater intrinsic antioxidative enzyme-like activities than higher surface energy (100)-faceted Pd nanocubes [40].

    In this study,we designed ALO-loaded and hyaluronic acid(HA)-modified Pd octahedral nanozymes (Pd@HA/ALO) with synergistic antioxidative and anti-inflammatory activities to alleviate atherosclerosis through M1 to M2 polarization of macrophages(Scheme 1).The as-prepared Pd@HA/ALO nanoparticles were used to treat atherosclerosis through following multifactorial mechanism: (1) Pd@HA/ALO actively targets atherosclerotic plaques through HA-modified surface exhibiting affinity to stabilin-2 and CD44,which both are highly expressed in atherosclerotic plaques.(2) Pd octahedral nanozymes exert efficient SOD- and CAT-like activities,by which they scavenge multiple ROS and promote antioxidative treatment.(3) Under near-infrared (NIR) irradiation,ALO is released from Pd@HA/ALO,thus inhibiting the expression of COX-2 to exert an anti-inflammatory effect.(4) Synergistic antioxidative and anti- inflammatory effects promote the polarization of macrophages from the M1 to M2 subtype,thereby preventing the formation of foam cells to attenuate atherosclerosis.An array of comprehensivein vitroandin vivoexperiments confirmed that the prepared nanozymes are able to prevent the progression of atherosclerosis and to stabilize atherosclerotic plaques.

    The preparation process of Pd@HA/ALO is shown in Scheme 1a.First,Pd nanocubes were prepared based on a solution method using PVP,AA and KBr as a stabilizing agent,reductive agent and capping agent,respectively.The obtained Pd nanocubes exhibited uniform cubic shapes with average sizes of 32 nm,as confirmed by TEM (Fig.1a).Next,the as-prepared Pd nanocubes were used as seeds to synthesize Pd octahedrons,demonstrated in the TEM micrograph (Fig.1b).Specifically,Pd atoms were deposited on the (100) facet through thermodynamic control,leading to the transformation of Pd nanocubes into octahedra dominated by the (111) facet [41].Finally,HA and ALO were subsequently adsorbed onto the surface to fabricate Pd@HA/ALO.This resulted in an increased particle size of ~50 nm (Fig.1c).It is worth to note that dynamic light scattering revealed larger diameter (68.67 nm) compared to TEM micrographs.This was likely caused by the HA coating increasing the hydrodynamic diameter of Pd@HA/ALO (Fig.S1 in Supporting information).Moreover,the zeta potential decreased from -18.9 ± 0.6 mV (Pd octahedron) to-27.1 ± 1.15 mV (Pd@HA/ALO),indicating the successful coating of HA (Fig.S2 in Supporting information).In addition,the highresolution TEM (HRTEM) micrographs (Figs.1d and e) revealed lattice fringes of 0.20 and 0.22 nm,attributed to the (100) plane of the Pd nanocubes and the (111) plane of the Pd octahedrons,respectively.In addition,elemental mapping confirmed that Pd,O and N were homogeneously dispersed throughout the Pd@HA/ALO(Fig.1f).Moreover,the powder XRD spectrum indicated preferential crystal growth along the (111) direction for the Pd octahedrons,which was consistent with the HRTEM results (Fig.1g).In the XPS (Fig.1h),the split energy between Pd 3d3/2(341.1 eV)and Pd 3d5/2(335.8 eV) was 5.3 eV,which indicated the existence of Pd0in the final products.The UV-vis spectra further showed that Pd@HA/ALO presented a broad absorption band from the UV to NIR window (Fig.1i),suggesting the potential of Pd@HA/ALO to serve as a photothermal material.Additionally,the stability of Pd@HA/ALO was investigated in water,phosphate-buffered saline(PBS,pH 7.4),Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS).After 5 days,the size and dispersion properties of Pd@HA/ALO were well maintained,demonstrating that Pd@HA/ALO was stable enough for relatively long storage (Fig.S3 and Table S2 in Supporting information).

    Fig.1.Preparation and characterization of the Pd@HA/ALO.(a) TEM image of Pd nanocubes.(b) TEM image of Pd octahedrons.(c) TEM image of Pd@HA/ALO.(d) HRTEM image of Pd nanocubes.(e) HRTEM image of Pd octahedrons.(f) Element mapping of Pd@HA/ALO.(g) XPS spectra of Pd@HA/ALO.(h) XRD patterns of Pd@HA/ALO.(i) UV-vis spectra of Pd cubes,Pd octahedrons and Pd@HA/ALO.

    We further assessed the release kinetics of ALO from Pd@HA/ALO-based drug delivery systemsin vitro.First,we detected the photothermal effect of Pd@HA/ALO upon 808 nm laser irradiation.As shown in Fig.2a,the Pd@HA/ALO solution exhibited a concentration-dependent temperature increase with a maximum temperature of 43.8°C under 808 nm laser irradiation,which was monitored by an IR camera (Fig.2b).Moreover,the photothermal conversion efficiency of Pd@HA/ALO was determined to be 27.83%(Fig.2c),indicating its excellent photothermal conversion properties.Then,we used NIR light to trigger the release of ALO from Pd@HA/ALO.Without the 808 nm laser,the amount of released ALO in pH 7.4 buffer was only 9.1% and 10.0% at 12 and 24 h,respectively (Fig.S4 in Supporting information).In contrast,after exposure to an 808 nm laser,the release percentage increased to 28.5% at 12 h and further reached 30.3% at 24 h,revealing that the NIR laser could efficiently control the release of ALO.This phenomenon is plausibly due to the rapid increase in local temperature generated from Pd@HA/ALO that increases the thermal vibration to weaken the interactions between ALO and Pd@HA to accelerate the release of ALO [42,43].

    To investigate the ROS scavenging abilities of Pd@HA/ALO,we detected two representatives of ROS,H2O2and superoxide anion (O2·-).As expected,Pd@HA/ALO possessed high H2O2scavenging activity in a concentration- and time-dependent manner(Fig.2d and Fig.S5 in Supporting information).Specifically,more than 70% of H2O2was eliminated within 5 min after exposure to 100 μg/mL Pd@HA/ALO.In addition,the CAT-like activity of Pd@HA/ALO (100 μg/mL) to scavenge H2O2was approximately equal to that of 9.47 ± 0.61 U/mL CAT (Fig.2e).Furthermore,typical EPR spectra were chosen to confirm the effect of Pd@HA/ALO on scavenging H2O2.The characteristic signal intensities of H2O2were reduced with increasing Pd@HA/ALO concentrations and incubation times,revealing the excellent H2O2scavenging capacity of Pd@HA/ALO (Figs.2f and g).

    Subsequently,we focused on the O2·-scavenging ability of Pd@HA/ALO.In this study,the O2·-concentration was reduced with increasing Pd@HA/ALO concentration and incubation time(Fig.2h and Fig.S6 in Supporting information).Specifically,approximately 60% of the O2·-was decomposed after treatment with 200 μg/mL Pd@HA/ALO.In addition,the SOD-like activity of Pd@HA/ALO was shown to be concentration-dependent (Fig.2i).The O2·-scavenging efficiency of Pd@HA/ALO (200 μg/mL) was determined to be approximately equal to that of 15.70 ± 1.55 U/mL SOD.Moreover,the EPR spectra further demonstrated that Pd@HA/ALO exhibited a high O2·-scavenging capacity (Figs.2j and k).Overall,these results confirmed that Pd@HA/ALO provided dual enzyme-like activity (SOD and CAT) to scavenge ROS,which is highly beneficial for antioxidative-based therapeutic interventions.

    Fig.2.Photothermal effect and multienzyme-like antioxidative activities of Pd@HA/ALO.(a) Temperature curves of different concentrations of Pd@HA/ALO in 5 min under 808 nm laser irradiation (1.0 W/cm2).(b) Photothermal images of water and Pd@HA/ALO under 808 nm laser irradiation (1.0 W/cm2).(c) Linear correlation of the cooling times versus negative natural logarithm of driving force temperature.(d) H2O2 scavenging ability after treated with different concentrations of Pd@HA/ALO for 5 min.(e) The CAT-like activity of Pd@HA/ALO.Data are mean ± SD (n=3).(f) Concentration-dependent EPR spectra of Pd@HA/ALO for H2O2 scavenging.(g) Time-dependent EPR spectra of Pd@HA/ALO for H2O2 scavenging.(h) O2·- scavenging ability after treated with different concentrations of Pd@HA/ALO for 30 min.(i) SOD-like activity of Pd@HA/ALO.Data are mean ± SD (n=3).(j) Concentration-dependent EPR spectra of Pd@HA/ALO for O2·- scavenging.(k) Time-dependent EPR spectra of Pd@HA/ALO for O2·- scavenging.

    Encouraged by the outstanding ROS scavenging ability of Pd@HA/ALO,we next studied its biocompatibilityin vitro.First,we assessed the hemocompatibility of Pd@HA/ALO after incubation with erythrocytes (Fig.3a).No significant hemolysis (approximately 10%) of Pd@HA/ALO was observed even when the concentration was 200 μg/mL,which is in line with a negative surface charge of Pd@HA/ALO.Subsequently,analyses of cytotoxicity in RAW264.7 cells showed that Pd@HA/ALO induced only negligible cytotoxicity even at the maximum concentration (200 μg/mL),suggesting excellent cytocompatibility (Fig.3b).Notably,Pd@HA/ALO induced slight cytotoxicity after exposure to the NIR laser due to induced hyperthermia (Fig.3c).

    Subsequently,we investigated the cellular uptake behavior of Pd@HA/ALO using confocal microscopy and TEM.For this purpose,RAW264.7 macrophages were incubated with fluorescein isothiocyanate (FITC)-labeled Pd@HA/ALO for 1,3,6,9 and 12 h.Spatial FITC fluorescence (green color) exhibited a gradually increasing trend and reached a maximum at 9 h,confirming the successful cellular internalization of Pd@HA/ALO (Fig.3d and Fig.S7 in Supporting information).According to the colocalization analysis,although the green fluorescence signals were largely co-localized with the red color of LysoTracker after 9 h of incubation (Pearson’s correlation coefficient 0.55 ± 0.02),separated green fluorescence was observed at 12 h (Pearson’s correlation coefficient 0.39± 0.05),indicating that the Pd@HA/ALO could effectively achieve endolysomal escape after internalization (Fig.S8 in Supporting information).Furthermore,after 12 h,penetration of the endolysosomal membrane by Pd@HA/ALO was observed leading to the release of Pd@HA/ALO into the cytoplasm (Fig.3e).Therefore,these results confirmed that Pd@HA/ALO could be endocytosed effectively by RAW264.7 macrophages,which is a crucial prerequisite for a successful treatment of atherosclerosis.

    Compared with other cells,during atherosclerosis,macrophages are more susceptible to oxidative stress.Therefore,the macrophage-mediated protection against ROS-induced oxidative damage and subsequent initiation of a cascade of pathological processes during the early stage of atherosclerosis significantly attenuate its progression.Thus,we further examined antioxidative activity of Pd@HA/ALO in RAW264.7 macrophages.As shown in the calcein-AM and propidium iodide (PI) co-staining assays,the addition of Pd@HA/ALO led to an efficient inhibition of induction of apoptosis triggered by H2O2treatment (Fig.3f).Subsequently,flow cytometric analysis was performed to further validate the cytoprotective ability of Pd@HA/ALO (Fig.3g).Compared with that of the model group,a higher viable proportion (84.1%) and a lower apoptotic proportion (5.2%) of RAW264.7 macrophages were achieved after Pd@HA/ALO+NIR treatment,which is consistent with the results of the calcein-AM/PI co-staining.

    Fig.3. In vitro cytotoxicity,cellular internalization and antioxidation of Pd@HA/ALO.(a) Hemolysis of red blood cells after incubation with different concentrations of Pd@HA/ALO for 2 h.Inset: hemolysis photograph after centrifugation.Data are mean ± SD (n=3).(b) Relative cell viability of RAW264.7 cells after treated with different concentrations of Pd@HA/ALO without NIR laser irradiation for 24 and 48 h.(c) Relative cell viability of RAW264.7 cells after treated with different concentrations of Pd@HA/ALO with NIR laser irradiation for 24 and 48 h.Data are mean ± SD (n=5).**P<0.01.(d) CLSM images of time-dependent cellular uptake of FITC-labeled Pd@HA/ALO.Scale bar: 10 μm.(e) Representative bio-TEM images of RAW264.7 cells after incubated with Pd@HA/ALO for 6 and 12 h.(f) CLSM images of calcein-AM and PI co-staining RAW264.7 cells after different treatments.Scale bar: 100 μm.(g) Flow cytometry analysis of RAW264.7 cells after different treatments.

    Further,we also studied the directin vitroROS scavenging activity of Pd@HA/ALO in RAW264.7 cells.As shown in Fig.4a,the intracellular total ROS (green fluorescent signal),H2O2(green fluorescent signal) and O2·-(red fluorescent signal) levels increased dramatically after LPS treatment.Comparatively,the intracellular fluorescent signals of total ROS,H2O2and O2·-decreased significantly when the cells were incubated with Pd@HA/ALO.Notably,after irradiation with an NIR laser,RAW264.7 cells in the Pd@HA/ALO group presented lower free radical fluorescence than those without irradiation (Figs.S9-S11 in Supporting information).Quantitative analysis of intracellular total ROS levelsviaflow cytometry further confirmed this observation (Fig.4b).Taken together,these results demonstrate that Pd@HA/ALO nanoparticles significantly reduce intracellular ROS levels and protect RAW264.7 macrophages against ROS-induced oxidative damage.

    Subsequently,we also investigated whether Pd@HA/ALO possessed an anti-inflammatory effect in macrophages.As shown in Fig.4c,LPS-stimulated RAW264.7 cells showed significantly increased mRNA expression of COX-2,which plays a crucial role in the inflammatory behavior of activated macrophages.In contrast,the expression level of COX-2 was decreased after Pd@HA/ALO+NIR treatment,suggesting that the released ALO prominently inhibited COX-2 expression.Consequently,our results verified that Pd@HA/ALO could attenuate oxidation and inflammation in macrophages by inhibiting intracellular ROS and COX-2 production,respectively.

    We next evaluated thein vitromacrophage polarization (M1 to M2 phenotypic transition) after incubation with Pd@HA/ALO.As shown in Fig.4c,qRT-PCR analysis indicated that mRNA expression of proinflammatory M1 markers,including TNF-α,IL-1βand IL-6,was significantly increased after treatment with LPS.However,compared with the Pd@HA/ALO group,the Pd@HA/ALO+NIR group showed substantially reduced expression levels of proinflammatory M1 markers,indicating the increased release of ALO by NIR-induced hyperthermia.Moreover,the expression of representative M2 markers,including Arg-1 and CD206,was upregulated in the Pd@HA/ALO treatment group compared with the other groups.This results verified that Pd@HA/ALO could successfully promote the M1 to M2 phenotypic transition of macrophages through synergistic antioxidant and anti-inflammatory effects.

    Subsequently,we explored the inhibitory effect of Pd@HA/ALO treatment on foam cell formation.For this purpose,RAW264.7 macrophages treated with 50 μg/mL oxLDL for 24 h presented many intracellular lipid droplets and significant foam cell formation,as illustrated by staining with Oil Red O (ORO) (Fig.4d).Compared with the model group,the Pd@HA/ALO group showed notably suppressed foam cell formation,especially after NIR irradiation.Quantification of intracellularly deposited ORO supported this microscopic observation (P<0.01) (Fig.4e).Taken together,these results demonstrated that Pd@HA/ALO could suppress the formation of foam cells by inducing phenotypic alteration of macrophages from M1 to M2.

    Fig.4.Effect of Pd@HA/ALO on antioxidation,anti-inflammation and foam cell formation.(a) CLSM images showing intracellular ROS of RAW264.7 cells after different treatments.Intracellular total ROS,H2O2 and O2·- were stained by DCFH-DA,H2O2 probe and DHE,respectively.Scale bar: 20 μm.(b) ROS levels of RAW264.7 cells after different treatments.(c) mRNA expression of M1 (TNF-α,IL-1β,and IL-6),M2 (Arg-1 and CD206) macrophage markers and COX-2 in RAW264.7 cells after different treatments,as evaluated by qRT-PCR analysis.Data are mean ± SD (n=3).*P<0.05.(d) Optical microscopy images showing oxLDL-induced foam cell formation in RAW264.7 cells after different treatments.Ⅰ: Control; Ⅱ: Model; Ⅲ: Pd@HA/ALO; Ⅳ: Pd@HA/ALO+NIR.Scale bar: 50 μm.(e) Quantified contents of ORO in foam cells derived from RAW264.7 cells after different treatments.Data are mean ± SD (n=3).**P<0.01,***P<0.001,ns: no significance.

    To study the anti-atherosclerotic effectsin vivo,we first investigated the pharmacokinetic profiles of Pd@HA/ALO in C57BL/6 mice.All animal care and experimental protocols were approved by The Animal Ethics Committee of the Chinese Academy of Medical Sciences Institute of Radiology.After i.v.injection,fluorescence imaging results suggested that the signal of ZnPc-labeled Pd@HA/ALO increased with time (Figs.5a and b).This trend was different from the standard exponential damping of i.v.injected drugs,which was mainly reflected the non-linear profile of fluorescence signal accumulation and fluorescent substance decay.Then,we investigated thein vivotargeting ability in atherosclerotic plaque-bearingApoE-/-mice.After i.v.injection for 4 h,fluorescence imaging showed that ZnPc-labeled Pd@HA/ALO accumulated in isolated entire aortas (Fig.5c).At 12 h post-injection,imaging showed significantly stronger fluorescent signals in the isolated aortas,indicating that Pd@HA/ALO could target atherosclerotic plaques (Figs.5c and d).Moreover,the accumulation of Pd@HA/ALO in major organs,including the heart,liver,spleen,lung,and kidneys,showed a timedependent profile (Figs.S12 and S13 in Supporting information).In addition,the ability of Pd@HA/ALO to target atherosclerotic aortas was further demonstrated by immunofluorescence analysis.In this study,fluorescence imaging showed the substantial distribution of ZnPc-labeled Pd@HA/ALO in the aortic root and aortic arch (Fig.5e,Figs.S14 and S15 in Supporting information).In particular,the red fluorescence (ZnPc) of Pd@HA/ALO displayed relatively high colocalization with the green fluorescence of CD68+macrophages and CD31+endothelial cells,revealing that the HA modification enhanced the atherosclerotic plaque-targeting capabilityin vivo.Thus,Pd@HA/ALO with targeted drug delivery allowed subsequent local drug release and efficient atherosclerosis management,resulting from decreased phagocytosis and sustained local drug release.

    In addition,the ability of Pd@HA/ALO to target atherosclerotic aortas was further demonstrated by immunofluorescence analysis.In this study,fluorescence imaging showed the substantial distribution of ZnPc-labeled Pd@HA/ALO in the aortic root and aortic arch (Fig.5e,Figs.S14 and S15).In particular,the red fluorescence (ZnPc) of Pd@HA/ALO displayed relatively high colocalization with the green fluorescence of CD68+macrophages and CD31+endothelial cells,revealing that the HA modification enhanced the atherosclerotic plaque-targeting capabilityin vivo.Thus,Pd@HA/ALO with targeted drug delivery allowed subsequent local drug release and efficient atherosclerosis management,resulting from decreased phagocytosis and sustained local drug release.

    After confirming the significant accumulation of Pd@HA/ALO within plaques,we assessed the therapeutic effect inApoE-/-mice.ApoE-/-mice were fed a high-fat diet for 10 weeks.At 1 week,the animals were randomized into 4 groups with different treatments: control (C57BL/6 mice),model (saline),Pd@HA/ALO (10 mg/kg) and Pd@HA/ALO (10 mg/kg)+NIR.Mice received twiceweekly i.v.injections for a total of 10 weeks.After treatment,the whole aortas were harvested and stained with ORO.Atherosclerotic lesions identified by ORO staining were present in the model group,and the lesion area of the plaque in the Pd@HA/ALO treatment group was significantly reduced compared with that of the model group (Fig.5f).Importantly,the highest decrease in the ORO-stained areas in aortas was observed for the mice in the NIR irradiation group.Consistent with this result,the ORO-stained cryosections from the aortic root also revealed the extent of plaque reduction in the vascular lumen (Fig.5g).Further quantification of the ORO-stained plaques showed that the ratio of the plaque area to that of the whole aorta was decreased from 21.29% to 14.56% after treatment with Pd@HA/ALO (Fig.5h).In addition,Pd@HA/ALO+NIR treatment achieved a significantly higher therapeutic efficacy,as shown by a plaque ratio of 9.24%,revealing that the NIR-triggered release of ALO strongly inhibits atherosclerotic development.Compared with that of the model group (40.00%),the plaque area ratio decreased to 32.93% and 24.90% after treatment with Pd@HA/ALO and Pd@HA/ALO+NIR,respectively (Fig.5i),confirming that Pd@HA/ALO could effectively attenuate the progression of atherosclerosis.

    Fig.5. In vivo targeting ability and therapeutic effects of Pd@HA/ALO.(a) Ex vivo images of whole blood collected at various time points after i.v.administration of ZnPclabeled Pd@HA/ALO.(b) Quantified data of fluorescence intensities.Data are mean ± SD (n=3).(c) Ex vivo fluorescence images and (d) quantitative data of fluorescence signals accumulated in the aorta at 4 and 12 h points after i.v.administration of ZnPc-labeled Pd@HA/ALO.Data are mean ± SD (n=3).(e) Immunofluorescence analysis of the colocalization of ZnPc-labeled Pd@HA/ALO with CD68+ macrophages in the aortic root.Scale bar: 100 μm.(f) Representative ORO-stained images of aortas in each group.(g) Representative ORO-stained images of aortic roots sections in each group.Scale bar: 100 μm.(h) Quantitative data of the atherosclerotic plaque area.(i) Quantitative data of the atherosclerotic plaque area in the aortic root sections.Data are mean ± SD (n=5).(j) Serum levels of LDL in ApoE-/- mice after different treatments.(k) Serum levels of total cholesterol in ApoE-/- mice after different treatments.Data are mean ± SD (n=3).*P<0.05,**P<0.01,***P<0.001,ns: no significance.

    Subsequently,the composition of atherosclerotic plaques in aortic root sections was detected by H&E staining and immunohistochemistry.Examination of the H&E-stained sections of aortic roots showed that plaques in the model group largely consisted of acellular and lipid-rich necrotic cores (Fig.S16a in Supporting information).After treatment with Pd@HA/ALO,the necrotic area was notably decreased.Specifically,the quantitative analysis indicated that the necrotic core area was decreased from 26.86% to 16.42% after treatment with Pd@HA/ALO+NIR (Fig.S16b in Supporting information).Further separate immunohistochemical analyses for CD68 (a macrophage marker) and MMP-9 indicated that the number of macrophages and the level of MMP-9 were dramatically reduced in the atherosclerotic plaque area after treatment with Pd@HA/ALO,particularly after NIR irradiation (Figs.S17a-c in Supporting information).These findings indicated that Pd@HA/ALO can effectively suppress the progression of atherosclerosis due to the blockade of macrophage infiltration and the stabilization of vulnerable plaques.Concordant with these findings,staining with Masson’s trichrome showed a higher content of collagen around plaques of the Pd@HA/ALO-treated groups,resulting in enhanced fibrous cap thickness (Figs.S17a and d in Supporting information).Moreover,the immunohistochemical analyses forα-SMA (an SMC marker) demonstrated a high accumulation of SMCs in plaques in the Pd@HA/ALO-treated groups,indicating that vascular SMC proliferation was beneficial at various stages of atherogenesis (Figs.S17a and e in Supporting information) [44].Accordingly,these results demonstrated that Pd@HA/ALO lowers the accumulation of macrophages in plaques,prevent the formation of vulnerable plaques and increase the number of SMCs,thereby effectively delaying the development of atherosclerosis.

    Finally,the possible adverse effects of Pd@HA/ALO were examined after 10 weeks treatment.The clinical biochemical analysis of ALT,AST,ALP,BUN and CREA revealed normal values,suggesting that the functions of the liver and kidney were barely impaired after treatment with different formulations (Fig.S18 in Supporting information).Additionally,the levels of HDL,LDL,TGs and TC were examined.Compared to the model groups,the Pd@HA/ALO and Pd@HA/ALO+NIR groups displayed lower levels of LDL and TC,whereas no distinguishable changes were found in the TGs and HDL levels (Fig.S18,Figs.5j and k).In addition,examination of H&E-stained sections of major organs (heart,liver,spleen,lung and kidney) showed no obvious tissue pathologies in the treated mice of all groups,which further confirmed the good biocompatibility of Pd@HA/ALO (Fig.S19 in Supporting information).Together,these findings indicated that Pd@HA/ALO possessed a good safety profile for long-term treatment.

    In summary,we successfully engineered an endogenous targeted Pd@HA/ALO nanozyme to enhance macrophage polarization(M1 to M2) for treatment of atherosclerosis.Pd@HA/ALO innately exhibited multiple antioxidant enzyme activities to degrade intracellular ROS,leading to an antioxidative effect.Moreover,the ALO released from Pd@HA/ALO inhibited the expression of COX-2,which further relieved the inflammatory state.Synergistic antioxidant and anti-inflammatory effects effectively promoted the polarization of macrophages from the M1 to M2 subtype,thus suppressing the formation of foam cells.Furthermore,Pd@HA/ALO displayed a desirable safety profile without notable side effects,even after long-term administration in mice.Overall,Pd@HA/ALO could be a blueprint for next-generation nanomedicine in cardiovascular disease treatment and prevention.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    This work was supported by the Young Elite Scientists Sponsorship Program by Tianjin (No.0701320001) and the Major Special Projects (No.0402080005).Furthermore,the financial support from the CEITEC 2020 Project (No.LQ1601) and the Ministry of Education,Youth and Sports of the Czech Republic under the National Sustainability Programme II and by ERDF (No.CZ.02.1.01/0.0/0.0/16_025/0007314) is highly acknowledged.

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2022.06.008.

    亚洲精品日韩在线中文字幕| 国产高清有码在线观看视频| 在现免费观看毛片| 老熟女久久久| 国产成人午夜福利电影在线观看| 日本黄大片高清| 精品国产一区二区久久| 最近中文字幕高清免费大全6| 99热6这里只有精品| 99热这里只有是精品50| 国产免费福利视频在线观看| 精品午夜福利在线看| 免费大片18禁| 国产69精品久久久久777片| 青春草视频在线免费观看| 国产av码专区亚洲av| 少妇丰满av| 观看美女的网站| 麻豆成人午夜福利视频| 久久这里有精品视频免费| 男女免费视频国产| 一本—道久久a久久精品蜜桃钙片| 毛片一级片免费看久久久久| 久久久久久久国产电影| 亚洲av二区三区四区| 少妇人妻一区二区三区视频| 久久99热这里只频精品6学生| 亚洲精品第二区| 久久狼人影院| 亚洲精品乱码久久久久久按摩| 亚洲国产欧美在线一区| 美女cb高潮喷水在线观看| 精品亚洲乱码少妇综合久久| 老司机影院毛片| 又大又黄又爽视频免费| av卡一久久| 欧美 日韩 精品 国产| 国产伦在线观看视频一区| 男女啪啪激烈高潮av片| av天堂久久9| 亚洲精品第二区| 赤兔流量卡办理| 肉色欧美久久久久久久蜜桃| 又粗又硬又长又爽又黄的视频| 一区二区三区乱码不卡18| 亚洲精品乱码久久久v下载方式| 人妻制服诱惑在线中文字幕| 免费播放大片免费观看视频在线观看| 欧美日韩视频高清一区二区三区二| 国产成人aa在线观看| av专区在线播放| 人妻夜夜爽99麻豆av| 亚洲一级一片aⅴ在线观看| 午夜91福利影院| 亚洲不卡免费看| 亚洲中文av在线| 日韩伦理黄色片| 亚洲欧美日韩卡通动漫| 免费在线观看成人毛片| 丁香六月天网| 春色校园在线视频观看| 一区二区三区精品91| 99视频精品全部免费 在线| 免费人妻精品一区二区三区视频| 日韩制服骚丝袜av| 多毛熟女@视频| 国产免费视频播放在线视频| 少妇人妻精品综合一区二区| 午夜影院在线不卡| 亚洲成人av在线免费| 亚洲av中文av极速乱| 色吧在线观看| 中文字幕av电影在线播放| 国产乱来视频区| 国产日韩欧美在线精品| 日本免费在线观看一区| 日韩制服骚丝袜av| 国产成人a∨麻豆精品| 成人二区视频| 少妇的逼水好多| 成人免费观看视频高清| 你懂的网址亚洲精品在线观看| 国产 一区精品| 欧美少妇被猛烈插入视频| 在现免费观看毛片| videos熟女内射| 岛国毛片在线播放| 男女免费视频国产| 亚洲国产av新网站| 精品一区在线观看国产| 婷婷色综合www| 伊人久久国产一区二区| 午夜视频国产福利| 看十八女毛片水多多多| 日产精品乱码卡一卡2卡三| 国产精品国产三级专区第一集| 久久免费观看电影| 亚洲av福利一区| 亚洲情色 制服丝袜| 成人无遮挡网站| 一本大道久久a久久精品| 99热6这里只有精品| 亚洲av成人精品一区久久| 免费看光身美女| 国产成人精品无人区| 亚洲天堂av无毛| 久久人人爽人人爽人人片va| 精品少妇久久久久久888优播| 精品卡一卡二卡四卡免费| 麻豆乱淫一区二区| 亚洲精品国产av蜜桃| 午夜视频国产福利| 久久免费观看电影| 国产亚洲午夜精品一区二区久久| 精品国产国语对白av| 我要看黄色一级片免费的| 亚洲人与动物交配视频| 最近手机中文字幕大全| 日韩 亚洲 欧美在线| 欧美3d第一页| 赤兔流量卡办理| 你懂的网址亚洲精品在线观看| 91aial.com中文字幕在线观看| 亚洲av中文av极速乱| 国产精品久久久久久久电影| 69精品国产乱码久久久| 人人澡人人妻人| 51国产日韩欧美| 成年av动漫网址| 久久亚洲国产成人精品v| 蜜桃在线观看..| 久久这里有精品视频免费| av有码第一页| 国产一区二区三区av在线| 国产亚洲午夜精品一区二区久久| 亚洲综合精品二区| 特大巨黑吊av在线直播| 高清毛片免费看| 少妇精品久久久久久久| 岛国毛片在线播放| 国产极品天堂在线| www.色视频.com| 久久久久人妻精品一区果冻| 国产在视频线精品| 下体分泌物呈黄色| 乱人伦中国视频| 国产精品人妻久久久久久| 三级经典国产精品| 男人舔奶头视频| av又黄又爽大尺度在线免费看| 人人妻人人添人人爽欧美一区卜| 男人和女人高潮做爰伦理| 欧美成人精品欧美一级黄| 国产免费又黄又爽又色| 日韩熟女老妇一区二区性免费视频| 日韩av在线免费看完整版不卡| 亚洲欧洲国产日韩| 国产综合精华液| 日韩一区二区三区影片| 国产黄片美女视频| 夫妻午夜视频| 乱人伦中国视频| 欧美精品一区二区大全| 六月丁香七月| 国产又色又爽无遮挡免| 99久久中文字幕三级久久日本| 简卡轻食公司| 成人免费观看视频高清| 日韩强制内射视频| 国产乱人偷精品视频| 天天操日日干夜夜撸| 国产日韩一区二区三区精品不卡 | 精品一区二区免费观看| 人妻少妇偷人精品九色| 亚洲av二区三区四区| 一级片'在线观看视频| 国产中年淑女户外野战色| 日韩,欧美,国产一区二区三区| 成人国产av品久久久| 晚上一个人看的免费电影| 九九在线视频观看精品| 久久综合国产亚洲精品| 一级爰片在线观看| 日本色播在线视频| 在线 av 中文字幕| 国产白丝娇喘喷水9色精品| 自拍欧美九色日韩亚洲蝌蚪91 | 国产极品粉嫩免费观看在线 | 最近的中文字幕免费完整| 日韩,欧美,国产一区二区三区| 亚州av有码| 久久久久国产精品人妻一区二区| 欧美日韩综合久久久久久| 国产男女内射视频| 久久精品国产鲁丝片午夜精品| 中文天堂在线官网| 嫩草影院入口| 国产毛片在线视频| 国产男女内射视频| 涩涩av久久男人的天堂| 免费人妻精品一区二区三区视频| 免费观看的影片在线观看| 欧美高清成人免费视频www| 国产伦精品一区二区三区视频9| 国产淫片久久久久久久久| 国产精品99久久久久久久久| 国产精品三级大全| 日日啪夜夜爽| 91aial.com中文字幕在线观看| 亚洲欧美清纯卡通| 亚洲性久久影院| 各种免费的搞黄视频| 麻豆乱淫一区二区| 日韩欧美 国产精品| 日本av手机在线免费观看| 又黄又爽又刺激的免费视频.| 国产极品天堂在线| 国产精品久久久久久精品电影小说| 精品人妻熟女毛片av久久网站| 亚洲怡红院男人天堂| 精品久久久精品久久久| 午夜影院在线不卡| 亚洲经典国产精华液单| 人妻 亚洲 视频| 久久久午夜欧美精品| 丰满乱子伦码专区| 免费av中文字幕在线| 水蜜桃什么品种好| 乱人伦中国视频| 麻豆成人午夜福利视频| 人妻一区二区av| 日本午夜av视频| 91精品伊人久久大香线蕉| 欧美日韩亚洲高清精品| 久久国产精品大桥未久av | 国产深夜福利视频在线观看| 人人妻人人澡人人爽人人夜夜| 五月伊人婷婷丁香| 国产精品女同一区二区软件| 欧美成人精品欧美一级黄| 亚洲中文av在线| 极品人妻少妇av视频| 亚洲欧美清纯卡通| 成人毛片60女人毛片免费| 一区二区av电影网| 国产高清国产精品国产三级| 另类精品久久| 精品酒店卫生间| 女人精品久久久久毛片| 午夜福利影视在线免费观看| 精品一区二区三卡| 亚洲精品自拍成人| 久久午夜综合久久蜜桃| 在线免费观看不下载黄p国产| 成人午夜精彩视频在线观看| freevideosex欧美| 久久久精品94久久精品| 9色porny在线观看| 亚洲欧美一区二区三区国产| 精品一区二区三卡| 日韩av不卡免费在线播放| 哪个播放器可以免费观看大片| 激情五月婷婷亚洲| 一区二区三区乱码不卡18| 麻豆精品久久久久久蜜桃| 亚洲久久久国产精品| 自拍偷自拍亚洲精品老妇| 久久午夜综合久久蜜桃| 国产69精品久久久久777片| 内射极品少妇av片p| 美女主播在线视频| 国产av国产精品国产| 狂野欧美白嫩少妇大欣赏| 国产精品女同一区二区软件| 性色av一级| 久久av网站| 又大又黄又爽视频免费| 亚洲国产最新在线播放| 日日啪夜夜爽| 日本黄大片高清| 色哟哟·www| 在线免费观看不下载黄p国产| a级片在线免费高清观看视频| 69精品国产乱码久久久| 亚洲婷婷狠狠爱综合网| 99热这里只有是精品50| 久久热精品热| 丰满迷人的少妇在线观看| 亚洲av综合色区一区| 五月开心婷婷网| 国产精品国产三级国产av玫瑰| 男人和女人高潮做爰伦理| 蜜桃在线观看..| 午夜av观看不卡| 国产精品一区www在线观看| 大陆偷拍与自拍| 黄色怎么调成土黄色| 国产在线男女| 丰满人妻一区二区三区视频av| 亚洲国产成人一精品久久久| 日本色播在线视频| 婷婷色av中文字幕| 国产一区二区三区av在线| 少妇人妻精品综合一区二区| 男女啪啪激烈高潮av片| 亚洲国产精品成人久久小说| 成人毛片60女人毛片免费| 亚洲综合精品二区| 在线天堂最新版资源| 午夜福利视频精品| 国产精品.久久久| 天堂8中文在线网| 午夜视频国产福利| 久久久精品94久久精品| 大又大粗又爽又黄少妇毛片口| 少妇人妻久久综合中文| 久久精品国产亚洲av天美| 哪个播放器可以免费观看大片| 韩国av在线不卡| 亚洲av欧美aⅴ国产| 能在线免费看毛片的网站| 国产精品一区二区三区四区免费观看| 91在线精品国自产拍蜜月| 国产伦理片在线播放av一区| 一级二级三级毛片免费看| 免费人成在线观看视频色| 精品久久久久久电影网| 中文字幕免费在线视频6| 日本与韩国留学比较| 狂野欧美白嫩少妇大欣赏| 成人毛片60女人毛片免费| 春色校园在线视频观看| 精品熟女少妇av免费看| 我要看黄色一级片免费的| 久久国产精品大桥未久av | 欧美老熟妇乱子伦牲交| 18禁在线播放成人免费| 亚洲性久久影院| 成人影院久久| 蜜桃在线观看..| 欧美国产精品一级二级三级 | 日韩成人av中文字幕在线观看| 蜜桃在线观看..| 国产精品欧美亚洲77777| 亚洲三级黄色毛片| 一级a做视频免费观看| 日韩视频在线欧美| 日本猛色少妇xxxxx猛交久久| 国产精品麻豆人妻色哟哟久久| 草草在线视频免费看| 熟妇人妻不卡中文字幕| 极品人妻少妇av视频| 亚洲精品乱码久久久v下载方式| 五月伊人婷婷丁香| 成年av动漫网址| av福利片在线| 蜜桃久久精品国产亚洲av| 26uuu在线亚洲综合色| 欧美 日韩 精品 国产| 欧美日韩视频精品一区| 丝袜在线中文字幕| 哪个播放器可以免费观看大片| 免费看av在线观看网站| 欧美日韩精品成人综合77777| 国产精品伦人一区二区| 一区在线观看完整版| 在线观看www视频免费| av在线观看视频网站免费| www.av在线官网国产| 久久久午夜欧美精品| 亚洲精品第二区| freevideosex欧美| 成人二区视频| 国产极品粉嫩免费观看在线 | 国产亚洲av片在线观看秒播厂| 国产成人91sexporn| 亚洲精品乱码久久久v下载方式| 国内揄拍国产精品人妻在线| 日日啪夜夜爽| 日韩中文字幕视频在线看片| 黄色欧美视频在线观看| 免费av中文字幕在线| 少妇被粗大的猛进出69影院 | 亚洲欧美日韩东京热| 大码成人一级视频| 亚洲欧美清纯卡通| 亚洲成人一二三区av| 十八禁网站网址无遮挡 | 久久精品国产鲁丝片午夜精品| 少妇被粗大猛烈的视频| 国产日韩欧美视频二区| 亚洲国产精品一区二区三区在线| 亚洲色图综合在线观看| 在线播放无遮挡| 免费久久久久久久精品成人欧美视频 | 国产精品一区二区三区四区免费观看| 一级a做视频免费观看| 中文字幕人妻熟人妻熟丝袜美| 国内揄拍国产精品人妻在线| 极品教师在线视频| 免费高清在线观看视频在线观看| 久久久久精品性色| 肉色欧美久久久久久久蜜桃| 久久韩国三级中文字幕| 色5月婷婷丁香| 成人特级av手机在线观看| 大又大粗又爽又黄少妇毛片口| 丰满人妻一区二区三区视频av| 黑人巨大精品欧美一区二区蜜桃 | 日本欧美视频一区| 国产欧美日韩综合在线一区二区 | 18+在线观看网站| 日韩中文字幕视频在线看片| 亚洲av不卡在线观看| 人妻夜夜爽99麻豆av| 黄色毛片三级朝国网站 | 97在线视频观看| 欧美精品高潮呻吟av久久| 精品一区二区免费观看| 免费看av在线观看网站| 日日啪夜夜撸| 最近2019中文字幕mv第一页| h日本视频在线播放| 久久久精品94久久精品| 国产精品国产三级国产专区5o| 国产成人免费无遮挡视频| 日韩精品免费视频一区二区三区 | 日本av手机在线免费观看| 中文字幕久久专区| 亚洲欧美成人综合另类久久久| 国产乱来视频区| 国产女主播在线喷水免费视频网站| av卡一久久| a级一级毛片免费在线观看| 一本—道久久a久久精品蜜桃钙片| 香蕉精品网在线| 婷婷色麻豆天堂久久| 老司机亚洲免费影院| 最近中文字幕2019免费版| 亚洲,一卡二卡三卡| 赤兔流量卡办理| 在线观看三级黄色| 高清在线视频一区二区三区| 欧美日韩精品成人综合77777| 国产精品国产三级国产专区5o| 国产精品无大码| 亚洲国产成人一精品久久久| 丰满人妻一区二区三区视频av| 精品视频人人做人人爽| 99九九线精品视频在线观看视频| 亚洲,欧美,日韩| 热re99久久国产66热| 免费观看在线日韩| 少妇熟女欧美另类| av免费观看日本| 久久久久久人妻| 国产精品国产三级国产av玫瑰| 亚洲中文av在线| 亚洲成人手机| 夜夜看夜夜爽夜夜摸| 乱系列少妇在线播放| 黄片无遮挡物在线观看| 精品亚洲乱码少妇综合久久| 九色成人免费人妻av| a级片在线免费高清观看视频| 国精品久久久久久国模美| 精品国产露脸久久av麻豆| 欧美日韩av久久| 亚洲美女黄色视频免费看| 国产欧美日韩一区二区三区在线 | 最后的刺客免费高清国语| 九九在线视频观看精品| 国产精品久久久久久精品电影小说| 亚洲精品亚洲一区二区| 在线亚洲精品国产二区图片欧美 | av不卡在线播放| 天天躁夜夜躁狠狠久久av| 另类精品久久| 色吧在线观看| 蜜桃在线观看..| 91精品国产国语对白视频| 精品国产露脸久久av麻豆| 黄色视频在线播放观看不卡| 观看av在线不卡| 日本91视频免费播放| 久久久精品94久久精品| 欧美激情极品国产一区二区三区 | 久久韩国三级中文字幕| 国产成人精品无人区| 在线观看www视频免费| 日韩制服骚丝袜av| 人妻 亚洲 视频| av国产精品久久久久影院| 国产有黄有色有爽视频| 99热6这里只有精品| 日韩精品有码人妻一区| 久久精品熟女亚洲av麻豆精品| 视频中文字幕在线观看| 肉色欧美久久久久久久蜜桃| 国产精品一二三区在线看| 黑人巨大精品欧美一区二区蜜桃 | 久久精品国产亚洲av涩爱| 亚洲国产最新在线播放| 这个男人来自地球电影免费观看 | 亚洲av二区三区四区| 麻豆成人午夜福利视频| 婷婷色综合大香蕉| 欧美97在线视频| 久久综合国产亚洲精品| 美女脱内裤让男人舔精品视频| 97精品久久久久久久久久精品| 日本av免费视频播放| 极品教师在线视频| 亚洲av福利一区| 99re6热这里在线精品视频| 插阴视频在线观看视频| 免费高清在线观看视频在线观看| 久久精品国产鲁丝片午夜精品| 欧美国产精品一级二级三级 | 中文乱码字字幕精品一区二区三区| 中文欧美无线码| 日日啪夜夜爽| av天堂久久9| 黑丝袜美女国产一区| 亚洲欧美中文字幕日韩二区| 观看av在线不卡| 日本欧美视频一区| 久久久久久久精品精品| 成年美女黄网站色视频大全免费 | 国产成人freesex在线| 大香蕉97超碰在线| 亚洲一区二区三区欧美精品| 亚洲精品一区蜜桃| av天堂久久9| 成人亚洲欧美一区二区av| 在线免费观看不下载黄p国产| 搡女人真爽免费视频火全软件| 99久久精品一区二区三区| 91久久精品国产一区二区成人| 亚洲精品aⅴ在线观看| 极品教师在线视频| 2018国产大陆天天弄谢| 精品酒店卫生间| 熟女人妻精品中文字幕| 日韩不卡一区二区三区视频在线| av视频免费观看在线观看| 亚洲成人手机| 日日啪夜夜撸| 91成人精品电影| 国产男女内射视频| 日韩熟女老妇一区二区性免费视频| 国产 精品1| 日韩制服骚丝袜av| 亚洲精品一二三| 日韩免费高清中文字幕av| 久久久亚洲精品成人影院| 亚洲精品乱码久久久v下载方式| 免费av中文字幕在线| .国产精品久久| 欧美xxxx性猛交bbbb| 国内揄拍国产精品人妻在线| 少妇的逼水好多| 亚洲国产最新在线播放| 中文欧美无线码| 黄色欧美视频在线观看| 国产成人免费无遮挡视频| 日韩av不卡免费在线播放| 97在线人人人人妻| 青青草视频在线视频观看| 成年美女黄网站色视频大全免费 | 精品酒店卫生间| 97精品久久久久久久久久精品| 菩萨蛮人人尽说江南好唐韦庄| 亚洲精品一二三| 高清欧美精品videossex| av免费观看日本| 午夜免费观看性视频| 好男人视频免费观看在线| 久久99精品国语久久久| 免费少妇av软件| 黄色毛片三级朝国网站 | 成年av动漫网址| 狂野欧美激情性xxxx在线观看| 国产高清不卡午夜福利| 欧美3d第一页| 免费久久久久久久精品成人欧美视频 | 日韩中字成人| 高清在线视频一区二区三区| av又黄又爽大尺度在线免费看| 日韩精品免费视频一区二区三区 | 国产乱人偷精品视频| 国产成人精品婷婷| 熟女av电影| 午夜福利视频精品| 寂寞人妻少妇视频99o| 免费黄色在线免费观看| 国产精品无大码| 久久精品国产鲁丝片午夜精品| av又黄又爽大尺度在线免费看| 免费看不卡的av| 中文字幕av电影在线播放| 国产熟女欧美一区二区| 狂野欧美白嫩少妇大欣赏| 日本午夜av视频| av在线老鸭窝| 欧美少妇被猛烈插入视频| 亚洲欧美日韩东京热| 亚洲精品国产av成人精品| 日韩av不卡免费在线播放| 18禁动态无遮挡网站| 色婷婷av一区二区三区视频| 一级毛片aaaaaa免费看小| 欧美日韩视频高清一区二区三区二| av天堂中文字幕网| a 毛片基地|