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Quercetin Attenuates Brain Oxidative Alterations Induced Los ntawm Hlau Oxide Nanoparticles hauv nas

Mar 11, 2022

Hu rau:tina.xiang@wecistanche.comkom paub meej ntxiv


Abstract: Hlau oxide nanoparticle (IONP) kev kho mob muaj ntau yam txiaj ntsig kev noj qab haus huv tab sis kev siv tshuaj ntau lossis kev kho mob ntev yuav ua rau oxidative cellular raug mob, tshwj xeeb tshaj yog nyob rau hauv lub hlwb. Yog li ntawd, peb tau ua qhov kev tshawb fawb tam sim no los tshawb xyuas lub luag haujlwm tiv thaiv ntawmquercetinsupplementation tiv thaiv oxidative alterations induced nyob rau hauv lub hlwb ntawm nas vim IONPs. Plaub caug tus neeg laus txiv neej albino nas raug faib ua tsib pawg; Kev tswj hwm tau txais kev noj zaub mov zoo ib txwm muaj, pawg IONP tau txhaj tshuaj intraperitoneally nrog IONPs ntawm 50 mg / kg lub cev qhov hnyav (BW) thiab quercetin-kho pab pawg muaj IONPs ntxiv rau Q25, IONPs ntxiv rau Q50, thiab IONPs ntxiv rau Q100 uas tau hais lus supplanted nrog quercetin. los ntawm koob tshuaj ntawm 25, 50 thiab 100 mg quercetin / kg BW ib hnub, raws li, tau muab nrog tib koob tshuaj IONPs rau 30 hnub. IONPs ua rau muaj kev nce ntxiv hauv malondialdehyde (MDA) thiab txo qis glutathione (GSH) thiab oxidized glutathione (GSSG). Yog li ntawd, IONPs ua rau muaj kev raug mob loj ntawm lub paj hlwb vim yog cov hlau tso rau hauv oxidative alterations nrog rau qhov tseem ceeb ntawm lub hlwb creatine phosphokinase (CPK) thiab acetylcholinesterase (AChE). Tsis tas li ntawd, IONPs ua rau muaj kev txo qis hauv lub hlwb epinephrine, serotonin, thiab melatonin nrog kev txo qis ntawm peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1a) thiab mitochondrial transcription factor A (mtFA)mRNA. cov lus qhia. IONPs induced apoptosis hauv lub hlwb soj ntsuam los ntawm kev nce hauv caspase 3 thiab txo qis hauv B-cell lymphoma 2 (Bcl2) qhia qib. Quercetin supplementation tseem ceeb swb lub hlwboxidative puas tsuajnyob rau hauv ib tug npaum li cas ntawm koob tshuaj. Yog li, quercetin supplementation thaum lub sij hawm IONPs tau pom zoo kom tau txais txiaj ntsig ntawm IONPs nrog tsawg dua kev phom sij.

Ntsiab lus: hlau oxide nanoparticles; oxidative kev nyuaj siab; quercetin; anti-oxidant

1. Taw qhia

Hlau oxide nanoparticles (IONPs) yog siv rau kev siv tshuaj tsom, xa cov noob, kev sau npe ntawm tes, tus neeg sawv cev sib txawv hauv kev sib nqus resonance duab, thiab kev kho mob hyperthermia [1]. Nws kuj tseem siv los ua cov khoom noj ntxiv hauv cov dej haus uas muaj zog hlau thiab cov khoom noj khoom haus rau tib neeg noj [2] ntxiv rau kev siv ntau hauv kev kho dej khib nyiab, roj sensing, thiab semiconductors sorbents lubrication, pigments, thiab coatings [3]. Txawm li cas los xij, kev siv ntau yam ntawm IONPs induces hlau overload thiab pib tom qab ntawm oxidative kev nyuaj siab nyob rau hauv ntau yam kabmob [4]. Noj, nqus pa, thiab dermal nkag mus yog txoj hauv kev tseem ceeb rau kev nkag ntawm IONPs[5]. Hauv cov hlab ntsha, IONPs raug khi rau plasma proteins thiab faib rau hauv lub cev sib txawv

cov kab mob xws li lub siab, tus po, lub raum, lub ntsws, thiab lub plawv, thiab nkag mus rau hauv cov ntshav-hlwb barrier (BBB), inducing oxidative kev raug mob [6,7]. Irons tso tawm los ntawm IONPs ua rau reactive oxygen hom (ROS) tiam ua rau loj oxidative hloov nyob rau hauv lipids, proteins, thiab DNA [8,9].

Quercetin(3,3',4',5,7-pentahydroxyflavone) yog aflavonoidtam sim no nyob rau hauv txiv hmab txiv ntoo (apples, berries, cherries, thiab liab grapes) thiab zaub (dos thiab broccoli) ntxiv rau ntau cov noob, buckwheat, ceev, paj, barks, ntsuab tshuaj yej thiab txiv roj roj [10]. Quercetin muaj cov txiaj ntsig muaj txiaj ntsig zoo suav nrog kev tiv thaiv kab mob, tiv thaiv oxidant, tiv thaiv mutagenic, tiv thaiv ischemic, tiv thaiv kab mob thiab tiv thaiv kev laus [11-17]. Quercetin yog lipophilic thiab tuaj yeem nkag mus rau hauv cov ntshav-hlwb barrier (BBB) ​​[18]. Tsis tas li ntawd, quercetin muaj peev xwm tiv thaiv oxidant lub luag haujlwm los ntawm kev txhim kho ntawm txo glutathione (GSH) thiab kev txhim kho ntawm tooj liab-zinc superoxide dismutase (SOD1) [17]. Quercetin tuaj yeem chelate hlau, inhibiting Fenton cov tshuaj tiv thaiv thiab inhibiting ROS tiam [19]. Yog li ntawd, quercetin yog ib qho kev kho mob rau ntau yam kab mob neurodegenerative thiab neuronal raug mob [20]. Covanti-oxidantLub peev xwm ntawm quercetin tau txhawb kom peb ua txoj kev tshawb fawb tam sim no los tshawb xyuas qhov muaj peev xwm tiv thaiv ntawm quercetin tiv thaiv oxidative alterations induced los ntawm IONPs ntawm lub hlwb cov ntaub so ntswg.

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Nyem kom paub ntau ntxiv txog cov khoom

2. Cov txiaj ntsig

2.1.Size thiab Charge ntawm IONPs

Ib daim duab SEM ntawm IONPs, raws li pom nyob rau hauv daim duab 1, qhia ib tug kheej kheej kheej nrog ib tug nruab nrab loj ntawm 16.34-22.88 nm thaum muaj ib tug zoo zeta muaj peev xwm tus nqi ntxiv rau 22.8 mV (Daim duab 2).

SEM image of iron oxide nanoparticles (IONPs). IONPs were spherical in shape with an average size of 16.34–22.88 nm. The scale bar represents 500 nm.

IONPs had a positive zeta potential value of +22.8 mV

2.2. Lub hlwb Oxidative Stress thiab Anti-Oxidant Status

Cov qib ntawm lipid peroxidation khoom malondialdehyde (MDA) tau nce ntau hauv pawg IONPs (p<0.05) while="" they="" significantly="" decreased="" in="" ionps="" +="" q100=""><0.05) compared="" with="" the="" control="" (figure="" 3a).mda="" levels="" in="" ionps+="" q25=""><0.01), ionps+=""><0.01) and=""><0.01) were="" significantly="" decreased="" compared="" with="">

. The oxidative stress and anti-oxidant status of brain tissues. (A) Malondialdehyde (MDA) (nmol/g tissues). (B) Glutathione (GSH) (µmol/g tissue). (C) Oxidized glutathione (GSSG) (µmol/g tissue). (D) GSH/GSSG ratio. Data were analyzed with a one-way ANOVA followed by Tukey's multiple comparison test. * p < 0.05 and *** p < 0.001 vs. the control. ++ p < 0.01 and +++ p < 0.001 vs. IONPs. x p < 0.05 and xx p < 0.01 vs. IONPs + Q25. Error bars represent mean ± SD. n = 5. White color column refers to control. Black color column refers to IONPs. Colored column with different extents refers to different concentrations of quercetin supplementations to IONPs-treated groups.

GSH qib hauv hlwb homogenate tau txo qis hauv IONPs (p<0.001), ionps+=""><0.001),ionps+o50><0.001) and="" ionps+="" o100=""><0.05) compared="" with="" the="" control="" group.in="" ionps+q100,="" the="" gshlevels="" were="" significantly="" increased="" than=""><0.001) and="" ionps+="" q25(p=""><0.01), as="" represented="" in="" figure="">

Cov qib oxidized glutathione (GSSG) tau nce ntau hauv IONPs (p<0.001), ionps+q25=""><0.001),><0.001) and=""><0.05) compared="" with="" the="" control="" group="" (figure="">

Raws li cov ntaub ntawv ntawm GSH thiab GSSG, GSH / GSSG tus nqi piv tau txo qis hauv IONPs (p<0.001), ionps+="" q25,ionps+="" q50="" and="" ionps+="" o100="" compared="" with="" the="" control="" group="" (figure="" 3d).="" in="" ionps+="" q100,="" gsh/gssg="" ratio="" values="" were="" significantly="" increased="" in="" comparison="" with=""><0.01)and ionps+q25=""><>

2.3.Brain Creatine Phosphokinase (CPK) thiab Acetylcholinesterase (AChE) Kev Ua Haujlwm

CPK cov dej num tau nce ntau hauv IONPs (p<0.001), ionps="" +="" q25=""><0.001), ionps+="" q50=""><0.001) and="" ionps+="" q100=""><0.05) compared="" with="" the="" control="" group.="" in="" comparison="" with="" the="" ionp="" groups,="" cpk="" activities="" were="" significantly="" decreased="" in=""><0.01),><0.01) and="" ionps+q100=""><0.001)(figure>

Hauv daim duab 4B, AChE cov dej num tau nce ntau hauv IONPs (p<0.001), ionps=""><0.001), ionps+="" q50=""><0.001) and="" ionps+=""><0.05) compared="" with="" the="" control="" group.="" they="" significantly="" decreased="" in="" ionps+q25=""><0.01), ionps+q50=""><0.001)and ionps+q100(p=""><0.001) than="" ionps.in="" addition,="" ache="" activities="" were="" significantly="" decreased=""><0.001) in="" ionps+="" q100="" compared="" with="" ionps+="">

2.4.Brain Epinephrine, Serotonin, thiab Melatonin Hormones

Epinephrine theem hauv hlwb homogenates tau txo qis hauv IONPs (p<0.001), ionps+="" q25=""><0.001) and=""><0.05) compared="" with="" the="" control="" group(figure="" 4c).in="" the="" ionps+="" q25=""><0.05),ionps+><0.001)and ionps+q100=""><0.001)groups, the="" epinephrine="" levels="" were="" significantly="" increased="" compared="" with="" ionps.="" furthermore,="" the="" levels="" were="" significantly="" increased="" in="" ionps+=""><0.05)compared with="" ionps="" +="">

Raws li tuaj yeem pom hauv daim duab 4D, qib serotonin tau txo qis hauv IONPs (p<><0.001),><0.01) and=""><0.05)compared with="" the="" control="" group.="" in="" ionps+="" q100,="" the="" levels="" significantly="" increased=""><0.05) than="" ionps="" +="">

Melatonin qib hauv hlwb homogenates tau txo qis hauv IONPs (p<0.001), ionps+=""><0.001) and="" ionps+="" q50=""><0.01) compared="" with="" the="" control="" group="" (figure="" 4e).in="" comparison="" with="" ionps,="" the="" levels="" in="" the=""><0.01) and="" ionps="" +=""><0.001)groups were="" significantly="" increased.="" it="" significantly="" increased=""><0.001)in ionps="" +="" q100="" compared="" with="" ionps="" +="">

Biochemical assessments of brain tissue. (A) Creatine phosphokinase (CPK) (U/g tissue). (B) Acetylcholinesterase (AChE) (U/g tissue). (C) Epinephrine (pg/g tissue). (D) Serotonin (ng/g tissue). (E) Melatonin (pg/g tissue). Data were analyzed with a one-way ANOVA followed by Tukey's multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control. + p < 0.05, ++ p < 0.01 and +++ p < 0.001 vs. IONPs. x p < 0.05 and xx p < 0.01 vs. IONPs + Q25. Error bars represent mean ± SD. n = 5. White color column refers to control. Black color column refers to IONPs. Colored column with different extents refers to different concentrations of quercetin supplementations to IONPs-treated groups.

2.5. Brain PGC-1a thiab mtTFA mRNA Kev nthuav qhia

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1a) mRNA qhia txog kev hloov pauv tau txo qis hauv IONPs(p<0.001) and="" ionps+q25=""><0.05) and="" significantly="" increased="" in="" ionps+="" q100=""><0.001)compared with="" the="" control="" group="" (figure="" 5a).in="" comparison="" with="" ionps,="" the="" pgc-1a="" expression="" levels="" were="" significantly="" increased="" in="" ionps+="" q50=""><0.05) and="" ionps+q100=""><0.001).in addition,="" pgc-1a="" expression="" levels="" in="" ionps+="" o100="" were="" significantly="" increased="" (p=""><0.001)compared with="" the="" ionps="" +="" q25="" and="" ionps="" +="" q50="">

mRNA relative fold change expression of brain tissue. (A) PGC-1α. (B) mtTFA. Data were analyzed with a one-way ANOVA followed by Tukey's multiple comparison test. * p < 0.05 and *** p < 0.001 vs. the control. ++ p < 0.01 and +++ p < 0.001 vs. IONPs. x p < 0.05 and xxx p < 0.001 vs. IONPs + Q25. # p < 0.05 and ### p < 0.001 vs. IONPs + Q25. Error bars represent mean ± SD. n = 5. White color column refers to control. Black color column refers to IONPs. Colored column with different extents refers to different concentrations of quercetin supplementations to IONPs-treated groups.

Hauv IONPs (p<0.001), ionps+="" q25="" (p=""><0.001)and ionps+=""><0.05), the="" mrna="" expression="" levels="" of="" mitochondrial="" transcription="" factor="" a="" (mttfa)="" were="" significantly="" decreased="" than="" in="" the="" control="" group="" while="" they="" significantly="" increased="" in="" ionps+q50=""><0.01) and="" ionps+=""><0.05)compared with="" ionps+="" q25.mttfa="" expression="" levels="" were="" significantly="" increased=""><0.05) in="" ionps+o100="" in="" comparison="" with="" ionps+q50="" (figure="">

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2.6.Hematoxylin (H) thiab Eosin(E) Staining Assessment of Brain Sections

Kev ntsuam xyuas histological ntawm cov ntaub so ntswg hauv hlwb tau qhia tias pawg tswj hwm tau pom cov qauv histological ntawm cov txheej txheem cerebellum (Daim duab 6A).Hauv pawg IONP, cerebellum pom muaj kev puas tsuaj loj ntawm Purkinje cell txheej (Daim duab 6B) thaum lub sij hawm no depletion ntawm Purkinje cell. txheej vim IONPs tau txo qis hauv IONPs ntxiv rau Q25 (Daim duab 6C), IONPs ntxiv rau Q50 (Daim duab 6D), thiab IONPs ntxiv rau Q100 (Daim duab 6E) pawg nyob rau hauv ib koob tshuaj-nyob ntawm seb.

Cerebellum histopathology. (A) Control group showing a normal histological structure of cerebellum layers. (B) IONP group showing a severe depletion of the Purkinje cells. (C) IONPs + Q25 group showing a moderate depletion of the Purkinje cells. (D) IONPs + Q50 group showing a moderate to mild depletion of the Purkinje cells. (E) IONPs + Q100 group showing a mild depletion of the Purkinje cells. Hematoxylin (H) and eosinH and (E) (×400). Scale bar = 20 µm.

Cov nas nyob rau hauv pawg tswj tau pom ib txwm histological qauv ntawm meninges thiab cerebrum cortex (Scheme 1A) thaum lub meninges ntawm nas kho nrog IONPs pom congestion ntawm submeningeal hlab ntsha (Scheme 1B).Nyob rau hauv cov pab pawg neeg IONP, lub cerebrum ntawm lub hlwb. Cov nas pom tau tias Satellitosis, neuronophagia (Scheme 1C), gliosis (Scheme 1D), thiab spongiosis (Scheme 1E) ntxiv rau qhov congestion ntawm choroid plexus (Scheme 1F).

Raws li tuaj yeem pom hauv Scheme iF, lub hlwb ntawm nas nyob rau hauv IONPs ntxiv rau Q25 pab pawg pom spongiosis (cov xub luv) thiab congestion ntawm cov hlab ntsha submeningeal. Ntxiv mus, cerebrum ntawm nas nyob rau hauv IONPs ntxiv rau Q50 pab pawg pom me me spongiosis thiab hlab ntsha congestion (Scheme 1H). Lub hlwb ntawm nas nyob rau hauv IONPs ntxiv rau Q100group tau pom ib qho kev ua haujlwm ntawm cov qog nqaij hlav thiab cerebrum cortex (Scheme 1I).

Histopathology assessment. (A) The brain of a rat showing the normal histological structure of the meninges and cerebrum cortex, H and E (×200). (B) The meninges of a rat treated with IONPs showing the congestion of the submeningeal blood vessels (star), H and E (×200). (C) The cerebrum of a rat treated with IONPs showing satellitosis (arrows) and neuronophagia (arrowhead), H and E (×400). (D) The cerebrum of a rat treated with IONPs showing gliosis (A), H and E (×400). (E) The cerebrum of a rat treated with IONPs showing spongiosis (short arrows), H and E (×400). (F) The choroid plexus of a rat treated with IONPs showing severe congestion (stars), H and E (×400). (G) The brain of a rat in the IONPs + Q25 group showing spongiosis (short arrows) and the congestion of the submeningeal blood vessels (stars), H and E (×200). (H) The cerebrum of a rat in the IONPs + Q50 group showing mild spongiosis (short arrows) and the congestion of blood vessels (star), H and E (×400). (I) The brain of a rat in the IONPs + Q100 group showing a relatively normal histological structure of the meninges and cerebrum cortex, H and E (×200). Scale bar = 20 µm.

2.7. Prussian Blue Staining Assessment of Brain Sections

Lub paj hlwb ntawm pawg tswj tau pom qhov tsis zoo Prussian xiav staining (Daim duab 7A). Hauv pawg IONPs, Prussian xiav staining me ntsis tau lees paub hauv cov hlwb (Daim duab 7B). Ntawm qhov tsis sib xws, qhov kev siv ntawm Prussian xiav staining me ntsis ntawm ntu ntawm pawg IONPs tau txo qis los ntawm quercetin hauv IONPs ntxiv rau Q25 (Figure7C), IONPs ntxiv rau Q50 (Daim duab 7D) thiab IONPs ntxiv rau Q100 (Daim duab hauv 7E) koob tshuaj-dependent yam.

Brain tissue stained with Prussian blue (Black arrows). (A) Control group showing a negative Prussian blue stain. (B) IONPs group showing blue spots representing the iron contents by Prussian blue stain. (C) IONPs + Q25 group showing moderate blue spots representing the iron contents by Prussian blue stain. (D) IONPs + Q50 group showing a few blue spots representing the iron contents by Prussian blue stain. (E) IONPs + Q100 group showing very few dispersed blue spots representing the iron contents by Prussian blue stain. ×400. Scale bar = 20 µm

2.8. Caspase 3 thiab Bcl2 Protein Levels nyob rau hauv lub hlwb

Cov pab pawg tswj tau pom qhov tsis zoo ntawm cov qib caspase 3 cov protein ntau (Daim duab 8A) thaum lawv tau qhia ntau heev hauv IONPs (Daim duab 8B).Quercetin khopab pawg (Daim duab 8C-E) tau qhia qhov qhia qis ntawm caspase 3 piv nrog IONPs. Ntawm qhov tsis sib xws, Bcl2 tau nthuav tawm hauv IONPs ntxiv rau Q25 (Daim duab 9C), IONPs ntxiv rau Q50 (Daim duab 9D) thiab IONPs ntxiv rau Q100 (Daim duab 9E) piv nrog IONPs (Daim duab 9B) thiab pawg tswj hwm (Daim duab 9A).

Immunohistochemistry of caspase 3 in brain tissue. (A) Control group showing a caspase 3 negative (black arrow) immunostained neuron. (B) IONPs group showing strong (red arrows), moderate (blue arrow) and weak (green arrow) caspase 3 positive brown immunostained neurons. (C) IONPs + Q25 group showing strong (red arrow), moderate (blue arrow) and weak (green arrow) caspase 3 positive brown immunostained neurons. (D) IONPs + Q50 group showing strong (red arrow), moderate (blue arrows) and weak (green arrow) caspase 3 positive brown immunostained neurons. (E) IONPs + Q100 group showing negative (black arrows) and weak (green arrow) caspase 3 positive brown immunostained neurons. ×400. Scale bar = 20 µm.

Immunohistochemistry of Bcl2 in brain tissue. (A) Control group showing negative (black arrow) and strong (red arrow) Bcl2 positive brown immunostained neurons. (B) IONPs group showing negative (black arrows) and a few strong (red arrows) and weak (green arrow) Bcl2 positive brown immunostained neurons. (C) IONPs + Q25 group showing negative (black arrows) Bcl2 brown immunostained neurons. (D) IONPs + Q50 group showing moderate (blue arrows) and weak (green arrows) Bcl2 positive brown immunostained neurons. (E) IONPs + Q100 group showing strong (red arrows) Bcl2 positive brown immunostained neurons. ×400. Scale bar = 20 µm.

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3. Kev sib tham

Kev raug rau IONPs coj mus rau hlau thiab nws cov deposition nyob rau hauv cov ntaub so ntswg mos, tshwj xeeb tshaj yog nyob rau hauv lub hlwb [21]. Hauv txoj kev tshawb fawb tam sim no, peb tau lees paub cov hlau tso rau hauv lub hlwb ua pov thawj los ntawm Prussian xiav staining thiab morphology alterations soj ntsuam los ntawm H thiab E staining kev ntsuam xyuas. Dhakshinamoorthy et al. [22] pom tau hais tias cov ntsiab lus hlau tau nce ntau hauv lub hlwb ntawm IONP-kho pab pawg piv nrog kev tswj hwm. Qhov no tau soj ntsuam los ntawm Prussian xiav staining ntawm lub hlwb thaj tsam thiab ua pov thawj los ntawm cov pob xiav nyob rau hauv lub frontal cortex, hippocampus, thiab cerebellum. El-Sayed et al. [23] kuj tau hais txog qhov nce ntxiv ntawm cov hlau hauv cov ntaub so ntswg hauv hlwb vim IONP kev tswj hwm hauv nas.

IONPs tuaj yeem muaj peev xwm nkag mus rau BBB thiab ua rau lub hlwb raug mob dua li lwm cov kabmob hauv nruab nrog cev [24,25]. Lwm qhov kev piav qhia yuav yog qhov nce qib hlau

nyob rau hauv lub hlwb vim yog kev khi ntawm hlau mus rau transferrin ua rau lub upregulation ntawm hlau receptors nyob rau hauv lub hlwb thiab, yog li ntawd, thauj hlau hla BBB [26].

Dawb Feions teb nrog H2O2 los tsim ROS hauv Fenton cov txheej txheem tshuaj tiv thaiv [27]. Qhov siab ROS nce permeability ntawm lub mitochondrial membrane sab nrauv, lipid peroxidation, protein puas, thiab DNA saw tawg [28]. Covoxidative kev nyuaj siabntawm ROS ua rau nce hauv lub hlwb theem ntawm cov khoom lipid peroxidation, MDA, raws li tau hais hauv txoj kev tshawb fawb tam sim no hauv IONP-kho nas. Ib yam li ntawd, Dhakshinamoorthy et al, [22] qhia txog kev nce qib hauv MDA hauv lub hlwb ntawm IONP-kho nas. Reddy thiab al.[26] kuj tau hais tias MDA qib tau nce ntau hauv cov ntaub so ntswg hauv hlwb hauv cov koob tshuaj ntau tab sis qhov tsis tseem ceeb nce hauv cov tshuaj tsawg hauv IONP-kho nas. Gaharwar thiab Paulraj [5] tau qhia tias MDA qib tau nce siab hauv IONP-kho nas. Ib yam li ntawd, IONPs induced oxidative raug mob ntawm cardiomyocytes soj ntsuam los ntawm ib tug siab MDA ntau lawm thiab txo GSH concentrations [29].

Cov kws sau ntawv hauv ntau qhov kev tshawb fawb tau tshawb xyuas cov txiaj ntsig kev tiv thaiv ntawm cov khoom ntuj lossis lawv cov tshuaj tiv thaiv kev mob tshwm sim nrog IONPs suav nrog Echinacea purpurea [30] thiab Antlriscus sulvestris [31] extracts. Hauv txoj kev tshawb fawb tam sim no, peb tau tshawb xyuas lub luag haujlwm tiv thaiv ntawm quercetin tiv thaiv cov tshuaj lom ntawm IONPs hauv lub hlwb ntawm nas. Quercetin supplementation rau IONP-kho nas txo qis MDA qib hauv cov ntaub so ntswg hlwb thiab peb cov txiaj ntsig tau sib haum nrog cov tau txais los ntawm Dong li al. [32] uas tau hais tias quercetin txo qis lipid peroxidation los ntawm kev txo qis MDA qib hauv cov hlwb hauv cov nas. Qhov no yuav piav qhia los ntawm lub peev xwm ntawm quercetin kom txo tau ROS, yog li ntawd, inhibiting lipid peroxidation thiab tiv thaiv kev tsim ntawm MDA [33]. Qhov no tau raug ntaus nqi rau pawg catechol (B nplhaib) thiab OH pawg ntawm txoj hauj lwm 3 ntawm lub nplhaib A thiab C, uas muaj qhov zoo tshaj plaws dawb radical scavenging [17].

GSH yog ib feem tseem ceeb ntawm cellular tiv thaiv antioxidant uas ncaj qha reacts nrog ROS thiab lwm yam reactive hom [34]. Hauv txoj kev tshawb no, lub hlwb GSH qib tau txo qis hauv IONP-kho nas tab sis GSSG qib tau nce ntau. Reddy et al. nce. Kev sib koom ua ke ntawm IONP-kho nas nrog quercetin rov qab los ntawm kev nce GSH thiab txo qis GSSG qib hauv cov ntaub so ntswg hlwb. Cov txiaj ntsig no tau sib haum xeeb nrog Singh li al. [35] uas tau tshaj tawm qhov siab ntawm GSH qib hauv cov ntaub so ntswg hauv hlwb qhia tias quercetin's antioxidant muaj peev xwm. Dong et al. [32] kuj tau nthuav tawm tias quercetin hloov pauv cov khoom siv hluav taws xob nuclear erythroid 2- cuam tshuam txog 2 (Nrf2) gene qhia. Nrf2 thiaj li txhawb nqa cov tshuaj tiv thaiv oxidant enzyme hauv cov ntaub so ntswg hlwb.

Hauv txoj kev tshawb fawb tam sim no, CPK tau nce ntau hauv lub hlwb ntawm IONP-kho nas. Hauv cellular zog buffering thiab lub zog kis tau tus mob, CPK / phosphocreatine plays lub luag hauj lwm tseem ceeb, tshwj xeeb tshaj yog nyob rau hauv lub hlwb uas muaj siab thiab fluctuating zog xav tau kev pab xws li neurons [36]. Yog li, kev ua kom CPK / phosphocreatine system thiab CPK qhia kev hloov pauv tuaj yeem yog qhov qhia ua ntej ntawm oxidative thiab bioenergetic stress nyob rau hauv lub cell [37] raws li kev them nyiaj rau txo qis zog vim oxidative kev nyuaj siab. Kev sib koom ua ke ntawm IONP-kho nas nrog quercetin txo CPK hauv cov ntaub so ntswg hlwb, Lemmens li al. [38] pom kev txo qis ntawm CPK kev ua ub no vim quercetin supplementation.

AChE kev ua haujlwm hauv cov ntaub so ntswg lub paj hlwb yog lub luag haujlwm rau hydrolysis ntawm acetylcholine (Ach) rau choline ntawm synapses thiab neuromuscular hlws ris [39]. Hauv kev tshawb fawb tam sim no, AChE tau nce ntau hauv IONP-kho nas thiab qhov txiaj ntsig no tau sib haum nrog Dhakshinamoorthy li al. [22] uas lees paub qhov nce ntxiv hauv AChE cov haujlwm hauv lub hlwb ntawm IONP-kho nas nyob rau hauv uas cov hlau txuam los ntawm IONPs hloov cholinergic system.

Neurotransmitters yog cov tshuaj endogenous uas tso cai rau cov neurons sib txuas lus thoob plaws hauv lub cev; lawv pab kom lub hlwb ua haujlwm sib txawv hauv cov tshuaj synaptic kis tau tus mob [40]. Hauv kev tshawb fawb tam sim no, epinephrine, serotonin, thiab melatonin concentrations hauv cov ntaub so ntswg hlwb tau txo qis hauv IONP-kho nas.

Yousef et al. [41] qhia tias qib serotonin thiab dopamine tau txo qis hauv cov ntaub so ntswg hauv hlwb hauv IONP-kho nas. Hauv qhov sib piv, quercetin nce epinephrine, serotonin, thiab melatonin concentrations hauv hlwb cov ntaub so ntswg piv nrog IONPs. Singh et al. [35l tau hais tias quercetin nce serotonin, norepinephrine, thiab dopamine.

Mitochondrial biogenesis suav tias yog lub luag haujlwm tseem ceeb hauv kev tswj hwm mitochondrial homeostasis kom tau raws li kev xav tau ntawm cov cellular physiological xav tau ntawm cov hlwb neural [42]. PGC-1a yog ib qho kev tswj hwm kev sib txuas lus uas ua rau mitochondrial biogenesis thiab txhawb kev qhia ntawm mtTFA [43]. Hauv txoj kev tshawb fawb tam sim no, qib kev qhia ntawm PGC-1a thiab mtTFA tau txo qis hauv cov ntaub so ntswg hauv hlwb hauv IONP-kho nas. Peb cov txiaj ntsig tau pom zoo nrog cov tau txais los ntawm Yousef li al. [44]. Lawv tau piav qhia tias mitochondrial biogenesis sawv cev los ntawm PGC-la thiab mtTFA txo qis lawv cov kev qhia hauv hlwb hauv IONP-tso nas qhia tias txo qis mitochondrial biogenesis thiab mtDNA replication thiab transcription uas yuav ua rau mitochondrial dysfunction. Kev sib koom ua ke ntawm IONP-kho nas nrog quercetin tau kho qhov txo qis los ntawm kev txhawb nqa ob qho tib si PGC-1a thiab mtTFA kab lus hauv cov ntaub so ntswg thiab peb cov txiaj ntsig tau sib haum nrog Sharma li al.[45] leej twg tau tshaj tawm tias quercetin tau nce qhov kev qhia ntawm PGC-1a, NRF-1, NRF-2, thiab mtTFA.Lawv tau hais tias vim qhov txo qis ROS los ntawm quercetin-induced PGC{{17 }}kev txhawb nqa, qhov no ua rau kev ua kom NRF-1 thiab NRF{19}} txhawb nqa kev qhia ntawm mtTFA.

Quercetin txo qis cov nyhuv apoptotic ntawm IONPs los ntawm kev txo qis hauv caspase 3 thiab nce Bcl2 qhia qib hauv lub hlwb immunohistochemical seem. Qhov kev tshawb pom no txhawb nqa lubanti-oxidantmuaj peev xwm ntawm quercetin. Ib yam li ntawd, kev kho quercetin txo qis qhov nce ntawm caspase 3 hauv ischemic hlwb raug mob hauv nas [46,47] thiab Bcl2 hauv lipopolysaccharide-kho nas [48].

1flavonoids antioxidant

4. Cov ntaub ntawv thiab cov txheej txheem

4.1.Reagents thiab Tshuaj

Hlau (ⅡI) oxide nanoparticle hmoov tau txais los ntawm Sigma-Aldrich (Louis, Mo, USA), uas tau solubilized hauv dej deionized ua ntej siv. Quercetin yog ib daim ntawv hmoov kuj tau los ntawm Sigma-Aldrich, uas yog yaj nyob rau hauv dimethyl sulfoxide (DMSO) thiab distilled dej ntawm ib tug ratio ntawm 1:20, feem, ua ntej siv. DMSO Ntau dua lossis sib npaug rau 99.6 feem pua ​​​​tau yuav los ntawm Sigma-Aldrich. Lwm yam tshuaj thiab dej deionized tau yuav los ntawm Lub Chaw rau Kev Kawm Kawm Tiav thiab Kev Tshawb Fawb ntawm Alexandria University.

4.2. Nqe lus hais txog Ethics

Txoj kev tshawb no tau pom zoo los teb rau cov lus qhia ntawm "NIH Guide for the Care and Use of Laboratory Tsiaj" los ntawm Kws Qhia Ntawv ntawm Veterinary Medicine Ethics Committee ntawm Alexandria University, Egypt.

4.3. Characterization ntawm IONPs

Lub morphology thiab qhov luaj li cas ntawm IONPs tau tshuaj xyuas los ntawm kev tshuaj ntsuam electron microscope (SEM). Lub particle tus nqi kuj tau txiav txim los ntawm zeta muaj peev xwm siv ZetaSizer Nano ZS (Malvern Instruments, Malvern, UK).

4.4. Tsiaj txhu, vaj tse, thiab kev sim tsim

Plaub caug tus neeg laus noj qab nyob zoo txiv neej albino nas hnyav 150 ± 20 lub cev qhov hnyav (BW) tau yuav los ntawm Lub Tsev Saib Xyuas Tsiaj, Kev Tshawb Fawb Kev Kho Mob ntawm Alexandria University, Egypt. Cov tsiaj tau khaws cia rau hauv lub tawb hlau nyob rau hauv kev tswj hwm ib puag ncig nrog qhov kub siab (23 ± 2), av noo (55 ± 5) thiab lub voj voog tsaus / lub teeb (12 h), thiab pub dawb rau kev noj zaub mov basal (Table 1) thiab haus dej. Txhua tus tsiaj tau nyob hauv ob lub lis piam ua ntej qhov kev sim rau acclimatization. Cov nas tau muab faib ua tsib pawg (yim nas txhua tus); Kev tswj hwm tau txais kev noj zaub mov zoo li qub thiab dej ad libitum, IONPs pawg tau txhaj tshuaj intraperitoneally nrog IONPs 50 mg / kg BW peb zaug hauv ib lub lis piam[49], IONPs ntxiv rau Q25 mg pawg tau muab tshuaj nrog tib koob tshuaj.

ntawm IONPs thiab gavaged nrog 25 mg quercetin / kg BW txhua hnub, IONPs ntxiv rau Q50 pab pawg tau tswj hwm nrog tib koob tshuaj IONPs thiab gavaged nrog 50 mg quercetin / kg BW txhua hnub thiab IONPs ntxiv rau Q100group tau tswj hwm nrog tib koob tshuaj IONPs thiab gav. nrog 100 mg quercetin / kg BW txhua hnub [17]. Tag nrho cov kev kho mob tau khaws cia rau 30 hnub.

Ingredients of the basal diet

4.5. Qauv

Thaum kawg ntawm qhov kev sim, cov nas tau yoo mov rau 12 teev thiab tshuaj loog siv cov tshuaj intraperitoneal ntawm ketamine / xylazine (100 mg / kg / 10 mg / kg) tom qab ntawd euthanized thiab lub paj hlwb raug txiav tam sim ntawd, yaug nrog chilled ib txwm saline 0.9 feem pua ​​thiab muab faib ua peb ntu; thawj tus yog siv rau kev tshuaj ntsuam biochemical. Qhov thib ob tau khaws cia ntawm -80 degree rau mRNA extraction thiab RT-PCR kev ntsuam xyuas thaum lub xeem ntu tau ntws nrog phosphate buffer saline (PBS, pH 7.4) thiab tsau hauv 4 feem pua ​​paraformaldehyde yaj hauv PBS rau 48 h rau qauv fixation.

4.6. Biochemical Analyzes

Ib feem ntawm txhua lub paj hlwb tau homogenized hauv txias phosphate buffer saline (PBS) thiab centrifuged rau 10 min ntawm 4 degree ntawm 1435 × g. Malondialdehyde (MDA), GSH, oxidized glutathione (GSSG), thiab creatine phosphokinase (CPK) tau txiav txim los ntawm cov khoom siv coj mus muag ntawm Biodiagnostic Co.(Giza, Egypt). Acetylcholinesterase (AChE) enzyme kev ua ub no hauv lub hlwb homogenates tau txiav txim los ntawm cov khoom siv tshuaj ntsuam xyuas xim (BioVision Co., Milpitas, CA, USA). Epinephrine, serotonin, thiab melatonin qib hauv hlwb homogenates kuj tau txiav txim siab siv cov khoom siv ELISA (BioVision Co., Milpitas, CA, USA).

4.7.mRNA Extraction thiab RT-PCR

Tag Nrho RNA tau muab rho tawm los ntawm cov qauv siv cov chaw tsim khoom yooj yim-RED Total RNA Extraction Kits (iNtRON Biotechnology, Inc., Gyeonggi-do, Kaus Lim Qab Teb). Thawj-strand cDNA tau ua tiav siv HiSen Script cDNA(iNtRON Biotechnology, Inc.) pob. Cov primers tshwj xeeb tau siv los nthuav tawm cov noob xaiv nrog glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ua cov noob tu vaj tse ruaj khov (Table 2).mRNA kev qhia tau ua tiav siv Strata gene MX3005P PCR (Agilent Technologies, Santa Clara, CA, USA) thiab TOP real TM PreMIX SYBR Green qPCR master blend (cat. RT 500, Enzynomics, Daejeon, South Kauslim) ua raws li cov chaw tsim khoom cov lus qhia. Cov txheeb ze ntawm cov noob qhia cov ntsiab lus tau raug tshuaj xyuas siv 2- txoj kev AAct raws li tau piav qhia los ntawm Pfaffl [50].

Sequences of primers.

4.8. Kev kuaj Histopathological

Siv cov txheej txheem kev sib xyaw ua ke ntawm cov tshuaj paraffin, cov qauv ruaj khov tau qhuav los ntawm kev nce qib ntawm ethanol, tshem tawm mus rau hauv peb lub xylene hloov, thiab ua tiav los ntawm paraffin embedding ntawm 65 degree C. Ntu 4 um tuab tau stained nrog Hematoxylin thiab Eosin (H thiab E) [51].

Cov hlwb hauv hlwb tau hydrated los ntawm kev txo qis cawv ntau thiab muab tso rau hauv 20 feem pua ​​​​poov tshuaj ferrocyanide tov nrog 20 feem pua ​​​​HCl tov rau 30 min. Cov slides tau ntxuav, lub cev qhuav dej, tshem tawm nrog xylene, thiab pom nyob rau hauv lub tshuab tsom iav rau xiav staining o hlau txheem [52].

4.9.Immunohistochemical Examination

Tus txheej txheem horseradish peroxidase (HRP) immunohistochemistry txheej txheem tau siv rau qhov zoo nqi swb ntawm cov ntaub so ntswg paraffin. Raws li cov chaw tsim khoom cov lus qhia, luav anti-rat caspase 3 (Lab Vision, Fremont, CA, USA) thiab Bcl2 (DAKO, Glostrup, Denmark) tau siv. Tsib um-thick seem ntawm cerebrum, cerebellum, qaum qaum, thiab sciatic paj hlwb tau dewaxed, rehydrated, thiab pretreated nrog 3 feem pua ​​hydrogen peroxide (H2O2) los thaiv endogenous peroxidase kev ua. Antigen retrieval tau ua tiav los ntawm kev tso cov swb hauv lub microwave rau 10 min hauv 10 mM sodium citrate tsis kam (pH6.0). Slides tau incubated nrog thawj cov tshuaj tiv thaiv thiab tom qab ntawd yaug nrog Tris-buffer saline thiab cov tshuaj tiv thaiv theem nrab. Slides raug incubated nrog ib tug 3,3/-diaminobenzidine (DAB) substrate chromogen tov thiab ces counterstained nrog Mayer's hematoxylin. Cov duab ntawm 10 qhov chaw sib txawv tau pom ntawm qhov loj ntawm × 400.

4.10. Kev txheeb cais

Ib txoj kev ANOVA nrog Tukey's post hoc ntau qhov kev xeem tau siv rau kev txheeb xyuas cov ntaub ntawv siv GraphPad Prism v.5https://www.graphpad.com/, nkag mus rau 10 Lub Peb Hlis 2021 (GraphPad, San Diego, CA, USA) . Tag nrho cov lus tshaj tawm ntawm qhov tseem ceeb nyob ntawm p<0.05. 5.="">

Hlau oxide nanoparticles (IONPs) intraperitoneally txhaj los ntawm ib koob ntawm 50 mg / kg BWinduced oxidative alterations nyob rau hauv lub hlwb thaum quercetin nyob rau hauv koob tshuaj ntawm 25, 50, thiab 100 mg / kg BWalleviated hlwb oxidative raug mob tshwm sim los ntawm IONPs. Yog li, quercetin yog ib qho kev cog lus pub ntxiv nrog rau kev kho IONP.

Cov ntaub ntawv

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