Table of Contents
Preparation and characterization of Cur@SF NPs
As displayed in Fig. 1 A, curcumin was dissolved in anhydrous ethyl alcohol, and the conformation of silk fibroin changed in alcohol solution, forming cohesive nanospheres and encapsulating dissolved curcumin. SEM showed the ball-shaped shape of Cur @ SF NPs with an average atom size of approximately 150 nm ( Fig. 1 B ). The nanoparticles exhibited dear dispersibility in aqueous solvent, and their hydrodynamic size distribution was between 100 and 360 nm according to dynamic light scattering ( DLS ) ( Fig. 1 C ). In addition, curcumin alone in alcohol solution and assembled as Cur @ SF NPs both presented visible alight assimilation at 435 new mexico ( Fig. 1 D ). FTIR spectrum showed the characteristic preoccupation of the components of Cur @ SF NPs ( Fig. 1 e ). The slow-release characteristics of Cur @ SF NPs verified that curcumin released from nanoparticles was time-dependent comply protease XIV treatment ( Fig. 1 F ) .Fig. 1 homework and word picture of Cur @ SF NPs. A Preparation process of silk fibroin-loaded curcumin ( Cur @ SF NPs ). B Scanning electron microscopy ( SEM ) image of Cur @ SF NPs at two magnifications ( scale bars : 200 nm and 100 nanometer ). C Hydrodynamic size distribution of Cur @ SF NPs. D Ultraviolet and visible spectrophotometry ( UV–Vis ) spectra showing the characteristic preoccupation of curcumin in Cur @ SF NPs. E Fourier transform infrared spectroscopy ( FTIR ) of SF, curcumin and Cur @ SF NPs. The spectral peaks at 1010 cm−1 and 1046 cm−1 were assigned to the -C–O–C- shaking of curcumin. The anti-symmetric stretch of the C–O–C from curcumin is at approximately 1150 cm−1. The characteristic absorption vertex of SF was observed at 3300 cm−1, which represented the stretching shaking of the peptide shackle ( -CONH- ) of SF. particular peaks were besides observed at 1600 cm−1 ( amide I, C-O ) and 1550 cm−1 ( amide II, N–H ), which represented β-sheets of SF. The carbonyl top out of curcumin and the amide peak of SF may partially overlap, which are all attributed to the impregnable absorption at approximately 1500 cm−1. F determination of the release of curcumin from Cur @ SF NPs in type XIV collagenase solution by detecting curcumin content within 120 planck’s constant Full size image due to the dicarbonyl structure, curcumin can be complexed by Fe2+ and Fe3+, while the methoxy and phenolic resin hydroxyl structures on the benzene ring exhibited the ability to preferentially chelate Fe3+ ( Fig. 2 A ) [ 34, 35, 36 ]. We evaluated the complexation ability of curcumin with iron by measuring the decrease in optical density at 435 nanometer. The data confirmed the preference of curcumin for Fe3+, other than Fe2+ ( Fig. 2 B ), in a concentration-dependent manner ( Fig. 2 C, D ). We then tested the capacity of iron chelation by curcumin in cells. Ultrasmall iron oxide nanoparticles ( IONPs, Fe3O4 NPs ) were used to mimic aggregate cellular ferric acerb, which is deliver in cardiomyocytes of FRDA patients. Fe3O4 NPs were prepared according to the previous method acting [ 37, 38 ]. The particle sizes were determined in the rate of 10–20 nm in diameter by transmission electronical microscopy ( TEM ) ( Additional file 1 : fig. S2A ), and the moral force light scattering ( DLS ) confirmed that the hydrodynamic size of Fe3O4 NPs was 10–80 new mexico ( Additional file 1 : figure. S2B ), demonstrating the successful readiness of Fe3O4 NPs. As shown in Fig. 2 e and Additional file 1 : fig. S3, Cur @ SF NPs were able to efficiently chelate iron from IONPs to prevent their accumulation in RAW26.4 cells. The quantification result was shown in Fig. S3. We besides plotted the iron chelation curves of curcumin, SF and Cur @ SF NPs, and the resultant role showed that SF was not able to chelate iron, but curcumin and Cur @ SF NPs immediately chelated iron ( Additional file 1 : fig. S4 ).
Fig. 2 Iron chelating ability of curcumin in vitro. A, B The chemical structure of curcumin and the bind site where curcumin binds to iron ions ( Fe2+ and Fe3+ ). C, D UV–Vis spectrum, peaked at 435 nm, to demonstrate cast-iron chelation by curcumin in a concentration-dependent manner. Ultrasmall iron oxide nanoparticles ( IONPs, 10 μM iron ) were used as an iron source, and the absorption at 435 nanometer was quantified in D. E prussian blue stain was used to reveal Fe accretion after IONP or IONP + Cur @ SF NPs treatment in RAW 264.7 cells Full size image
Cur@SF NPs reduce the levels of oxidative stress in fibroblasts derived from FRDA patients
The antioxidative effect of curcumin is widely implicated in the improvement of neurodegenerative diseases. To see the effects in FRDA models, we first evaluated the condom of Cur @ SF NPs in lymphocytes ( GM15850, Coriell ) derived from a FRDA patient. The assiduity ( of curcumin ) was optimized to be 1–2 μM without any injury to viability after treatment for 1 to 3 days ( Fig. 3 A ). interestingly, the cast-iron contentedness of the labile iron pool was decreased in mitochondria but not in the cytosol ( Fig. 3 B ), indicating that the curcumin released from Cur @ SF NPs modulated iron homeostasis, at least partially, by chelating mitochondrial iron .Fig. 3 Iron chelation and antioxidant effect of Cur @ SF NPs in vitro. A The relative value of CCK-8 in lymphocytes ( GM15850 ) derived from FRDA patients treated with Cur @ SF NPs ( NPs ) at different concentrations for 1 or 3 days. B The relative levels of mitochondrial LIP ( labile iron pool ) measured with RPA along with vitamin b6 isonicotinoyl hydrazine ( PIH ) following drug treatment in lymphocytes GM15849 and GM15850. The relative levels of cytosolic LIP measured by calcein-AM along with 2,2′-bipyridyl ( BIP ) ( for details, see “ Materials and methods ” section ). C The subject of malondialdehyde ( MDA ). D, E The enzymatic activities of SOD and catalase. F, G The activities of mitochondrial complexes I and II. H cellular ATP message. ( *, phosphorus < 0.05 ; **, p < 0.01, GM15850 + NPs vs. GM15850 ) Full size picture furthermore, we found here that Cur @ SF NPs ( 1 μM curcumin ) importantly reduced the levels of oxidative stress in GM15850 cells, as indicated by the belittled content of malondialdehyde ( MDA, the lipid peroxidation index ) and the increase activities of antioxidant enzymes ( SOD and catalase ) ( Fig. 3 C–E ). To test whether dysfunctional mitochondria were rescued in GM15850 cells, we measured mitochondrial complex activities and ATP add. As shown in Fig. 3 F–H, the activities of mitochondrial complexes I and II and ATP production were both importantly increased. These data proved the potency of Cur @ SF NPs in improving mitochondrial officiate, identical likely through antioxidation and cast-iron chelation by released curcumin .
Cur@SF NPs restored the behavioral scores of mice
Curcumin has been widely studied in other neurodegenerative diseases [ 39, 40, 41 ]. Given that curcumin exhibits satisfying iron chelating capacity and antioxidative capability, we tried to apply Cur @ SF NPs in the discussion of FRDA mouse with FXN lack, in which mouse were developed by Dr. Mark Pook and his colleagues, and Y47 was used as a manipulate [ 42 ]. Cur @ SF NPs were injected intraperitoneally into 4-month-old YG8R mouse once every 5 days for one calendar month. then, the weight was recorded, and no significant dispute was observed between the treated and untreated groups ( Fig. 4 A ). however, the behavior assays showed that the coordination and potency were both improved, revealed by longer rotational latency to fall in the rotarod tests ( Fig. 4 B ), by shorter time to cross in the beam-walk tests ( Fig. 4 C ) and longer time to hold in hang wire tests ( Fig. 4 D ) in treated YG8R shiner than in untreated YG8R mouse. simultaneously, pace tests were performed, and the results showed a alike consequence : the intrastep distance, footstep duration, and stance distance were all significantly increased in Cur @ SF NP treated mice compared with untreated mice to levels identical close to those in Y47 command shiner ( Fig. 4 E–H ). These results demonstrated that Cur @ SF NPs were effective in restoring the behavioral dysfunction of FRDA mouse .Fig. 4 Cur @ SF NPs increase the behavioral scores of FRDA mice. YG8R shiner were intraperitoneally injected with Cur @ SF NPs every five days for one calendar month when the shiner were 4 months old, and then the behavioral changes were assessed. Y47 : KOKI mouse containing human FXN as wild type ; YG8R : KOKI mouse containing human FXN with ( GAA ) north slip in as FRDA model ; YG8R + SF : YG8R with silk fibroin administration as a drug delivery fomite ; YG8R + NPs : YG8R mouse treated with Cur @ SF NPs. A The body slant of mouse. The shiner were weighed every 15 days during drug administration ( newton = 10/group ). The initial long time was 4 months old. B Coordination beam-walk tests. C Coordination rotarod tests. D Strength hang cable tests. E – H Gait tests. The front metrical foot was dyed red, and the rear foot was dyed blasphemous. ( *p < 0.05 ; **p < 0.01 ; ***p < 0.001 ; ****, phosphorus < 0.0001, YG8R + NPs vs. YG8R ) Full size image
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Cur@SF NPs restore mitochondrial morphology and function in FRDA mice
Nano-assembly has significant advantages in scavenging free radicals in living organisms [ 43, 44 ]. Curcumin has been shown to activate the nuclear factor E2-related factor 2 ( Nrf2 ) nerve pathway, triggering cellular protection against oxidative injury [ 45 ]. As two members of the Nrf2 regulon, the protein levels of catalase and SOD2 were upregulated by curcumin [ 46, 47 ], which was precisely observed in our analyze ( Fig. 5 A ), while the activities of antioxidative enzymes significantly increased ( Fig. 5 B, C ). As a result, the MDA content decreased ( Fig. 5 D ) .Fig. 5 Cur @ SF NPs reduce oxidative stress and restore the mitochondrial affair of the cerebellum in YG8R mouse. All samples were from tissue cerebellum. A detection of the catalase and SOD2 protein levels. B – D The enzymatic activities of SOD, catalase and MDA content in the cerebellum after Cur @ SF NP discussion ( n = 5 ). E TEM results of cerebellar tissue to observe mitochondrial morphology. F H & E staining to evaluate the recovery of morphology and array of Purkinje neurons as indicated by the red arrows. G Immunofluorescent stain of NeuN to determine the number of neurons. H Detection of mitochondrial aconitase natural process ( purple ) and mitochondrial protein levels. mAco2 : mitochondrial aconitase ( Aco2 ) action ( empurpled ) ; Ndufs1, SDHB, and Uqcrfs1 : mitochondrial complex I/II/III subunits. I The activities of mitochondrial complexes I and II. J ATP capacity. ( *p < 0.05 ; NS no significance ; YG8R + NPs vs. YG8R, normality = 5/group ) Full size trope To further evaluate whether the excellent place of Cur @ SF NPs against oxidative tension would benefit mitochondria, we analyzed the morphology and function of mitochondria [ 13 ]. The mitochondrion in the cerebellum were imaged by TEM. Compared to the control mouse Y47, mitochondrion from FRDA mice YG8R exhibited a distinctive low concentration of cristae, but presented an improved concentration of cristae mail treatment with Cur @ SF NPs ( Fig. 5 e ). histological assays through H & E staining further demonstrated that Purkinje neurons in the edge around the dentate gyrus of the cerebellum flinch and were dark-stained but were full moon and round and light-stained after Cur @ SF NP discussion ( Fig. 5 F ). interim, the corrective nerve cell count in the cerebellum could be observed by NeuN immunolabeling in the cover YG8R mouse ( Fig. 5 G ). To track the relevant changes at the molecular level, we extracted proteins from the cerebellum and detected the formulation levels of mitochondrial complex-related proteins ( Ndufs1, SDHB, Uqcrfs1 ) and the action of mitochondrial aconitase ( mAco2 ). The upregulation of the proteins and increased mAco2 activity far confirmed the convalescence of mitochondrial function in cerebellar tissue of the treated YG8R mouse ( Fig. 5 H ). consequently, mitochondrial function was improved, as revealed by the raised activities of mitochondrial complexes ( I and II ) and ATP message ( Fig. 5 I, J ) .
Cur@SF NPs eliminate cardiac iron accumulation and improve cardiomyocyte hypertrophy
The heart is another badly affected tissue for FRDA patients, and approximately 60 % of patients die from deadly congestive heart failure and supraventricular cardiac arrhythmia [ 9 ]. Given that iron retention is the independent campaign of myocardial dysfunction in FRDA patients and curcumin can chelate cast-iron, we further examined the iron levels in myocardium after Cur @ SF NP administration. The iron content was importantly lower in the hearts of tempered YG8R mouse than in the hearts of untreated YG8R shiner, as revealed by ferrozine assays and ICP ( Fig. 6 A, B ). In consistency with this, ferritin expression decreased following treatment with Cur @ SF NPs ( Fig. 6 C ). prussian blue stain of the slice weave further confirmed iron deposit in the hearts of YG8R mouse and was hardly detectable after treatment ( Fig. 6 D ). Again, curcumin increased the protein levels and enzymatic activities of catalase and SOD2 ( Fig. 6 E–G ). The results were consistent with those in the cerebellum accompanied by decrease MDA capacity after treatment ( Fig. 6 H ). To directly examine the antioxidative capability of Cur @ SF NPs, we isolated primary macrophages from YG8R shiner. DCFH-DA fluorescence showed the reduced oxidative stress after Cur @ SF NPs treatment ( F one g. 6 I ), confirming the antioxidative efficacy of Cur @ SF NPs .Fig. 6 Cur @ SF NPs eliminate cardiac iron accumulation and reduce oxidative stress levels. All samples were from tissue affection. A Ferrozine assays for iron content. B The relative levels of iron detected with ICP. C signal detection of the ferritin protein degree. D prussian staining to detect cast-iron in cardiomyocytes. E detection of the catalase and SOD2 protein levels. F – H The enzymatic activities of SOD, catalase and MDA content in weave hearts after Cur @ SF NP treatment ( nitrogen = 5 ). I primary coil macrophages were stained with the green fluorescent dye DCFH-DA after 2 μM Cur @ SF NPs treatment for 24 h. ( *p < 0.05 ; **p < 0.01 ; ***p < 0.001, YG8R + NPs vs. YG8R ) Full size picture
FXN deficiency-induced iron overload is powerfully associated with myocardial hypertrophy. After H & E tarnish, we counted the cell diameter to evaluate cardiomyocyte hypertrophy. The median diameter of cardiomyocytes was larger in YG8R mouse than in Y47 shiner but significantly reduced compared with untreated mouse ( P < 0.001 ) to be close to that in Y47 mouse ( Fig. 7 A, B ). The low density of mitochondrial cristae signally became dense in YG8R heart following Cur @ SF NP treatment, as revealed by TEM ( Fig. 7 C ). interestingly, we found that the sizes of the mitochondrion, in general, were larger in the cardiomyocytes of YG8R than in those of Y47, which was diminished in the process group ( Fig. 7 C ). total proteins were extracted from the heart and used to detect the construction levels of Ndufs1, SDHB, and Uqcrfs1 and the natural process of mAco2. merely Ndufs1 expression was significantly increased, although not SDHB and Uqcrfs1, which remained constant under the three conditions. however, the enzymatic action of mAco2 was greatly higher in YG8R mouse after discussion, even better than that in Y47 mouse ( Fig. 7 D ). consequently, ATP capacity and the activities of mitochondrial complexes ( I and II ) were restored in the hearts of the tempered YG8R mouse ( Fig. 7 E–G ). Taken together, Cur @ SF NP treatment is greatly beneficial to the convalescence of mitochondrial morphology and function in FRDA mouse .Fig. 7 Cur @ SF NPs significantly improved myocardial officiate. A, B H & E staining to evaluate the improvement of cardiomyocyte hypertrophy by counting the diameter of cardiomyocytes. C TEM results to observe the mitochondrial morphology and inner ultrastructure. D Detection of aconitase activeness ( purple ) and protein levels of mitochondrial proteins. mAco2 : mitochondrial aconitase ( Aco2 ) activeness. E, F The activities of mitochondrial complexes I and II. G ATP subject. H A schematic diagram depicting Cur @ SF NPs alleviating FRDA through synergetic iron chelation and antioxidation. ( *p < 0.05 ; ****p < 0.0001, YG8R + NPs vs. YG8R, n = 5/group ) Full size prototype