mwwXaiv lus
Tsev / Kev paub / Ntsiab lus

Kev paub

Kuwanon T Thiab Sanggenon Ib Leeg Los Ntawm Morus Alba Exert Anti-Inflammatory Effects Los Ntawm Regulating NF-κB Thiab HO-1/Nrf2 Signaling Pathways hauv BV2 Thiab RAW264.7 Cells

Mar 30, 2022

Yog xav paub ntxiv. tiv taujtina.xiang@wecistanche.com

Abstract: Peb yav dhau los tau tshawb xyuas cov tshuaj methanolic ntawm Morus alba bark thiab cim 11 lub tebchaw los ntawm cov extract: kuwanon G(1), Kuwana E (2), Kuwana T(3), sanggenon A(4), sanggenon M(5), sanggenol A(6), mulberofuran B(7), mulberofuran G(8), moracin M(9), moracin O(10), thiab norartocarpanone (11). Hauv no, peb tau tshawb xyuas qhovanti-inflammatoryCov teebmeem ntawm cov tshuaj no ntawm microglial hlwb (BV2) thiab macrophages (RAW264.7). Ntawm lawv, 3 thiab 4 tau ua rau muaj kev cuam tshuam cov lipopolysaccharide (LPS)-vim tsim cov nitric oxide hauv cov hlwb, qhia txog kev tiv thaiv kab mob ntawm ob lub tebchaw. Cov tshuaj no inhibited zus tau tej cov prostaglandin E2, interleukin -6, thiab qog necrosis factor-c, thiab qhia inducible nitric oxide synthase thiab cyclooxygenase -2 tom qab LPS stimulation. Pretreatment nrog 3 thiab 4 inhibited qhov ua kom muaj kev cuam tshuam ntawm cov teeb meem nuclear kappa B signaling txoj hauv ob hom cell. Cov tebchaw kuj tau ua rau kev qhia ntawm heme oxygenase (HO)-1 los ntawm kev ua kom muaj zog ntawm cov khoom siv hluav taws xob erythroid 2-txog yam 2. Ncua qhov kev ua ntawm HO-1 thim rov qab cov tshuaj tiv thaiv kab mob tshwm sim. los ntawm pretreatment nrog 3 thiab 4, qhia tias cov tshuaj tiv thaiv kab mob tau tswj hwm los ntawm HO-1. Ua ke, 3 thiab 4 yog cov neeg sib tw muaj peev xwm los tsim cov tshuaj kho mob thiab tiv thaiv kab mob raukab mob inflammatory.

Ntsiab lus: Morus alba; kev T; nyob rau A; BV2; RAW264.7 hlwb; anti-inflammatory teebmeem

flavonoids anti-inflammatory

Nyem qhov no kom paub ntau yam khoom

1. Taw qhia

Morus alba, cov nroj tsuag tshuaj uas yog tsev neeg Moraceae, tau siv los kho cov mob ntsws ntsws hauv cov tshuaj tsoos [1]. Nws cov cag ntoo muaj ntau yam khoom siv xws li umbelliferone, scopoletin, mucins, tannins,flavonoids(morusin, mulberrin, mulberrichromene, cyclomulberrin, moracin P, moracin O, mulberrofuran Q, Kuwana E, thiab kuwanon H)[2], 2-arylbenzofurans (moracenin D, moracin P, moracin O, thiab mulberrofuran O), thiab prenylated flavonoids (licoflavone C, cyclomulberrin, neocyclomorusin, sanggenon I, morusin, thiab Kuwana U) [3,4]. M. alba extracts thiab active compounds tuaj yeem txo cov kab mob ntsws, raws li qhia los ntawm kev tshawb fawb tsis ntev los no [5-7]. Ntxiv nrog rau cov tshuaj pharmacological ntawm M.alba ntawm lub ntsws, isoprenylated flavonoids (sanggenol Q, Kuwana T, sanggenon N, mulberrofuran G, thiab mulberrofuran C) siv cov teebmeem hepatoprotective hauv t-BHP-induced HepG2 hlwb [8]. Prenyl-flavonoids (kuwanon A, Kuwana C, Kuwana T, thiab morusin) thiab triterpenoids (betulin acid, avail, thiab -sitosterol) cuam tshuam qhov sib txawv ntawm 3T3-L1 adipocytes [9]. Morin hydrate, cov ntsiab lus tseem ceeb ntawm cov flavonoid ntawm M. alba, alleviates ntev unpredictable stress-induced memory impairment, qhia tias cov compound no yuav boost lub antioxidant tiv thaiv kab mob thiab inhibit neuroinflammatory pathways [10]. Cov kev tshawb fawb no qhia tias cov khoom muaj nyob hauv M.alba yog cov muaj peev xwm sib tw los kho ntau yam kab mob.

Kev mob, kev tiv thaiv tus kheej tsis yooj yim rau kev raug mob stimuli, ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob los ntawm kev ua kom muaj ntau lub cev tiv thaiv kab mob, suav nrog macrophages, monocytes, leukocytes, mast cells, thiab lwm hom cell [11]. Macrophages yog cov kab mob uas muaj ntau tshaj plaws thiab nthuav dav hauv lub cev tiv thaiv kab mob, thiab microglia yog cov neeg nyob hauv macrophages hauv nruab nrab paj hlwb (CNS) [12]. Macrophages thiab microglial hlwb yog cov tseem ceeb hauv cov lus teb inflammatory. Lawv tuaj yeem qhib tau rau hauv cov lus teb rau stimuli xws li lipopolysaccharide (LPS), cytokines, thiab chemokines, thiab induce inflammatory mob [13]. Nyob rau hauv cov kab mob inflammatory, ntau dhau activated macrophages thiab microglial hlwb ua rau txawv txav ntawm pro-inflammatory mediators (nitric oxide (NO), prostaglandin E2 (PGE,), inducible nitric oxide synthase (iNOS), thiab cyclooxygenase (COX) -2 ) thiab pro-inflammatory cytokines (interleukin (IL)-6 thiab qog necrosis factor (TNF)-a), los ntawm kev ua kom muaj zog ntawm cov khoom siv hluav taws xob nuclear kappa B (NF-B) qhia txoj hauv kev [14,15]. Kev nce qib ntawm cov neeg kho mob pro-inflammatory ntxiv ua rau muaj kev loj hlob ntawm cov kab mob inflammatory, uas ntxiv dag zog rau cov kab mob inflammatory, yog li ua rau lub voj voog vicious [16]. Yog li ntawd, kev tswj cov inflammatory mediators yog qhov tseem ceeb rau kev kho mob thiab tiv thaiv cov kab mob inflammatory.

Heme oxygenase (HO)-1 yog tus nqi-txheej enzyme catalyzing degradation ntawm heme rau hauv biliverdin, ferrous ion (Fe2 ntxiv), thiab carbon monoxide (CO) [17]. HO-1 induction yog tswj los ntawm kev ua kom muaj zog ntawm nuclear erythroid 2-txog yam 2(Nrf2). HO-1 tuaj yeem raug ntxias los teb rau oxidative kev nyuaj siab thiab mob los tiv thaiv cov ntaub so ntswg thiab tswj homeostasis hauv lub cev[18]. Yog li ntawd, lub hom phiaj HO-1 induction yog ib qho ntawm cov tswv yim muaj peev xwm los kho cov kab mob inflammatory.

Hauv peb qhov kev mob siab rau kev tshawb nrhiav cov neeg sib tw los ntawm cov khoom siv ntuj tsim los kho cov kab mob inflammatory, peb nthuav qhia covanti-inflammatory teebmeemntawm 11l compounds cais los ntawm M.alba hauv LPS-induced BV2 thiab RAW264.7 hlwb.

4flavonoids anti-inflammatory

2. Cov txiaj ntsig

2.1.Effects of 11 Compounds Isolated from M.alba on the Viability of BV2 and RAW264.7 Cells

Hauv kev tshawb fawb yav dhau los, cov cag ntoo ntawm M.alba tau muab rho tawm hauv aqueous methanol, thiab cov khoom tau txais tau muab faib ua ntu zus rau hauv EtOAc, n-BuOH, thiab H2O2. Rov ua SiO2, ODS, thiab Sephadex LH-20 kab chromatography ntawm EtOAc feem them nyiaj 11 lub tebchaw, xws li kuwanon G(1), Kuwana E(2), Kuwana T (3), sanggenon A(4), sanggenon M(5), sanggenol A(6), mulberofuran B(7), mulberofuran G(8), moracin M(9), moracin O(10), thiab norartocarpanone (11) [6,19,20]. Cov qauv tshuaj ntawm 11 lub tebchaw cais los ntawm M.alba yog qhia hauv daim duab 1. Txhawm rau txiav txim siab cytotoxic teebmeem ntawm cov tshuaj no, ib qho 3-(4,5-dimethylthiazol-2-yl){{ 26}}. (Daim duab 2). Cov tshuaj 2, 3, thiab 4 muaj tshuaj lom ntawm 80 μM, compound 5 muaj tshuaj lom ntawm 40 μM. Raws li qhov tshwm sim ntawm kev soj ntsuam toxicity, ib qho tsis muaj tshuaj lom concentration ntau tau raug xaiv rau cov kev tshawb fawb tom ntej ntawm cov tshuaj tiv thaiv kab mob (cov tshuaj 2, 3,4 ntawm 40 uM, compound 5 ntawm 200 μM, thiab lwm cov tshuaj ntawm 80 μM) .

Chemical structures of compounds 1–11 isolated from M. alba

Cytotoxic effects of compounds 1–11 isolated from M. alba on BV2 (A) and RAW264.7 (B) cells. The cells were incubated for 48 h with various concentrations of the compounds, and their viability was determined using MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 ,*** p < 0.001 compared with the control group. Figure 2. Cytotoxic effects of compounds 1–11 isolated from M. alba on BV2 (A) and RAW

2.2.Effects ofl1 Compounds cais tawm los ntawm M. alba ntawm Kev Tshaj Tawm Ntawm Cov Kabmob Kabmob thiab iNOS Protein hauv BV2 thiab RAW264.7 Cells

TSIS yog ib qho dawb radical nyob rau hauv lub plawv, hlab ntsha, thiablub cev tiv thaiv kab mob. Nws tswj hwm intracellular homeostasis, thauj cov neurotransmitters, thiab tswj kev ua haujlwm tiv thaiv kab mob thiab cytotoxicity. Txawm li cas los xij, thaum muaj ntau ntawm NO yog tsim, nws muaj kev cuam tshuam rau lub cev, suav nrog vasodilation, cytotoxicity, thiab cov ntaub so ntswg puas [21,22]. Peb mam li tshuaj xyuas cov teebmeem ntawm cov tshuaj no ntawm nitrite ntau lawm hauv LPS-induced BV2 thiab RAW264.7 hlwb. Cov hlwb raug kho nrog ntau qhov sib txawv ntawm cov tshuaj sib xyaw rau 2 teev ua ntej stimulation nrog LPS (1 ug / mL) rau 24 teev. Ntawm cov tebchaw, tsuas yog Compound 3 (kuwanonT) thiab 4 (sanggenon A) tseem ceeb inhibited nitrite ntau lawm hauv BV2 thiab RAW264.7 cell kab (Daim duab 3). Tsis tas li ntawd, peb tau ua ib qho kev sim ntxiv los sib piv cov nyhuv inhibitory ntawm nitrite ntau lawm ntawm pawg LPS-kho tom qab cov compound pretreatment thiab compound-kho pab pawg tom qab LPS pretreatment. Raws li qhov tshwm sim, tsis muaj qhov sib txawv ntawm cov nyhuv inhibitory ntawm nitrite ntau lawm ntawm ob pawg kev sim (Ntxiv daim duab S1). Yog li, hauv txoj kev tshawb no, cov kev sim hauv qab no tau ua los ntawm kev siv tshuaj ua ntej nrog cov tshuaj sib xyaw ua ke hauv 2 ~ 3 teev ua ntej kho LPS.

. Inhibitory effects of compounds 1–11 on nitrite production in BV2 (A) and RAW264.7 (B) cells. The cells were pretreated for 2 h with concentrations of compounds and stimulated for 24 h with lipopolysaccharide (LPS; 1 µg/mL). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05 and *** p < 0.001 compared with the LPS-treated group

Kev tsim cov NO yog nce los ntawm cov pro-inflammatory proteins inducible nitric oxide synthase (iNOS). Txawm li cas los xij, overexpression ntawm iNOS mob hnyav rau pathophysiology ntawm tus kab mob [23]. Compounds 3 thiab 4 inhibited kev qhia ntawm iNOS nyob rau hauv ib tug concentration-dependent yam (Daim duab 4). Peb tau tshuaj xyuas cov teebmeem ntawm cov tebchaw 3 thiab 4 ntawm LPS-induced qhia ntawm inflammatory yam hauv BV2 thiab RAW264.7 hlwb. Cov hlwb raug kho nrog ntau qhov sib txawv ntawm cov tshuaj sib xyaw 3 thiab 4 rau 2 teev ua ntej stimulation nrog LPS (1 ug / mL) rau 24 teev. Ob lub tebchaw ua rau muaj kev cuam tshuam LPS-induced qhia ntawm PGE2, TNF, thiab IL-6 hauv BV2 thiab RAW264.7 hlwb (Daim duab 5). Cov txiaj ntsig tau tshwm sim los qhia tias kev kho ua ntej nrog compound3 thiab 4 suppressed LPS-induced o hauv BV2 thiab RAW264.7 hlwb.

Protein expression levels of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated BV2 (A) and RAW264.7 (C) cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with LPS (1 μg/mL). Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensities are normalized to that of β-actin (B, D). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-treated group. Figure 5. Inhibitory effects of compounds 3 and 4 on the level of PGE2 (A and D), IL-6 (B and E), and TNF-α (C and F) in BV2 and RAW264.7 cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with lipopolysaccharide (LPS; 1 μg/mL). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the LPS-treated group. Figure 4. Protein expression levels of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated BV2 (A) and RAW264.7 (C) cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with LPS (1 µg/mL). Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensities are normalized to that of β-actin (B,D). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-treated group

Inhibitory effects of compounds 3 and 4 on the level of PGE2 (A and D), IL-6 (B and E), and TNF-α (C and F) in BV2 and RAW264.7 cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with lipopolysaccharide (LPS; 1 μg/mL). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the LPS-treated group. Figure 4. Protein expression levels of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated BV2 (A) and RAW264.7 (C) cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with LPS (1 µg/mL). Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensities are normalized to that of β-actin (B,D). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-treated group. Molecules 2021, 26, x FOR PEER REVIEW 5 of 16 Figure 3. Inhibitory effects of compounds 1–11 on nitrite production in BV2 (A) and RAW264.7 (B) cells. The cells were pretreated for 2 h with concentrations of compounds and stimulated for 24 h with lipopolysaccharide (LPS; 1 μg/mL). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05 and *** p < 0.001 compared with the LPS-treated group. Figure 4. Protein expression levels of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated BV2 (A) and RAW264.7 (C) cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with LPS (1 μg/mL). Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensities are normalized to that of β-actin (B, D). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-treated group. Figure 5. Inhibitory effects of compounds 3 and 4 on the level of PGE2 (A and D), IL-6 (B and E), and TNF-α (C and F) in BV2 and RAW264.7 cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with lipopolysaccharide (LPS; 1 μg/mL). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the LPS-treated group. Figure 5. Inhibitory effects of compounds 3 and 4 on the level of PGE2 (A,D), IL-6 (B,E), and TNF-α (C,F) in BV2 and RAW264.7 cells. The cells were pretreated for 2 h with various concentrations of compounds 3 or 4 and stimulated for 24 h with lipopolysaccharide (LPS; 1 µg/mL). Error bars represent mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the LPS-treated group

effects of cistanche improve immunity (33)

2.3.Effects of Compounds 3 and 4 on NF-xB Translocation in BV2 and RAW264.7 Cells

NF-kB yog qhov kev hloov pauv uas tswj hwm iNOS qhia [24]. Ib txwm NF-kB tseem nyob hauv nws daim ntawv tsis ua haujlwm los ntawm kev tsim cov txheej txheem nrog cov txheej txheem tswj hwm xws li IKB . Txawm li cas los xij, thaum nws tau qhib los ntawm LPS, IkBa yog degraded los ntawm phosphorylation, thiab NF-kB (xws li p65) yog translocated mus rau lub nucleus [25], uas txhawb cov inflammatory mediator gene thiab induces qhia inflammatory yam [26]. Txhawm rau tshuaj xyuas ntxiv cov nyhuv inhibitory ntawm cov tebchaw 3 thiab 4 ntawm kev tsim cov tshuaj tiv thaiv kab mob hauv LPS-activated BV2 thiab RAW264.7 hlwb, peb tau tshawb xyuas cov lus qhia nuclear translocation ntawm p65 hauv hlwb kho nrog cov tebchaw 3 thiab 4, LPS, lossis ob leeg siv cov tshuaj tiv thaiv-p65 FITC-labeled antibody. DAPI tau siv rau kev staining nuclear. Hauv pawg tswj hwm, p65 qhia tau pom hauv cytosol. Txawm li cas los xij, nyob rau hauv LPS-induced hlwb, p65 tsub zuj zuj tau kuaj pom nyob rau hauv lub nucleus, raws li qhia nyob rau hauv cov merged dluab ntawm DAPI thiab p65 staining. Tsis tas li ntawd, piv nrog LPS-induced hlwb, cov tebchaw 3 thiab 4 tau txo qis LPS-mediated nce hauv NF-KB (p65) DNA-binding kev ua haujlwm (Daim duab 6A, B) thiab kev hloov pauv nuclear (Daim duab 6C-F). Cov kev tshawb pom no qhia tias cov tebchaw 3 thiab 4 yog cov tswj tsis zoo ntawm LPS-stimulated NF-kB nuclear translocation.

Effects of compounds 3 and 4 on and NF-κB DNA-binding activity (A,B) and NF-κB (p65) localization (C–F) in BV2 and RAW264.7 cells. The cells were pretreated with compounds 3 or 4 for 2 h and stimulated with liposaccharide (LPS; 1 µg/mL) for 1 h. Experiments were performed using a commercially available enzyme-linked immunosorbent assay kit, as described in the Materials and Methods section. ** p and *** p < 0.001 compared with the LPS-treated group

2.4.Effects of Compounds 3 and 4 on the Nrf2/HO-1 Pathway in BV2 and RAW264.7

Cells Heme oxygenase-1(HO-1) yog lub hom phiaj ntawm nuclear factor E2-txog yam 2(Nrf2), Nrf2/HO-1 txoj hauv kev yog lub zog muaj zog tiv thaiv kab mob rau kev txhawb nqa dawb hlau hauv carbon monoxide (CO), biliverdin, thiab heme [27]. Cov pa roj carbon monoxide, lub gaseous metabolite ntawm heme catabolism, qhia txog kev tswj cov teebmeem ntawm vasodilation thiab proinflammatory teb [28]. Tsis tas li ntawd, HO-1 kuj tseem paub tias ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv cov hlwb los ntawm kev mob thiab oxidative kev nyuaj siab thiab tswj cov nitrite ntau lawm hauv activated macrophages [29]. Peb tau ua Western blotting los tshawb xyuas seb cov tebchaw 3 thiab 4 nce qib kev qhia ntawm HO-1, nyob rau hauv qhov paub zoo HO-1 inducer cobalt protoporphyrin (CoPP) tau siv los ua kev tswj xyuas zoo nce HO{{ 16}} protein qhia. [30]. Cov txiaj ntsig tau qhia tias cov tebchaw 3 thiab 4 kuj tau tswj hwm qhov kev qhia ntawm HO-1 (Daim duab 7). Peb tau tshawb nrhiav ntxiv seb cov tebchaw 3 thiab 4 tswj kev ua haujlwm ntawm Nrf2. Kev hloov pauv ntawm Nrf2 mus rau lub nucleus tau nce ntxiv nyob rau lub sijhawm, qhia tias cov tebchaw 3 thiab 4 tau txhawb nqa Nrf2/HO-1 txoj hauv kev hauv BV2 thiab RAW264.7 hlwb (Daim duab 8).

ffects of compounds 3 and 4 on heme oxygenase (HO)-1 expression in BV2 (A) and RAW264.7 (C) cells. The cells were treated with compounds 3 or 4 or CoPP (10 μM) for 12 h. Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensity was normalized to each total form expression (B and D). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. Figure 7. Effects of compounds 3 and 4 on heme oxygenase (HO)-1 expression in BV2 (A) and RAW264.7 (C) cells. The cells were treated with compounds 3 or 4 or CoPP (10 µM) for 12 h. Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensity was normalized to each total form expression (B,D). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group.

Effects of compounds 3 and 4 on Nrf2 activation in BV2 (A,C) and RAW264.7 (B,D) cells. The cells were treated with compounds 3 or 4 for 0.5, 1, and 1.5 h. Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensity was normalized to each β-actin or PCNA. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, and ### p < 0.001, compared with the control group

Effects of compounds 3 and 4 on Nrf2 activation in BV2 (A,C) and RAW264.7 (B,D) cells. The cells were treated with compounds 3 or 4 for 0.5, 1, and 1.5 h. Representative blots from three independent experiments are shown. Immunoblots were quantified using the ImageJ software. Band intensity was normalized to each β-actin or PCNA. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, and ### p < 0.001, compared with the control group

Txhawm rau tshuaj xyuas ntxiv seb puas muaj cov tshuaj tiv thaiv neuroinflammatory thiab tshuaj tiv thaiv kab mob ntawm cov tebchaw 3 thiab 4 yog cuam tshuam nrog HO-1 qhia hauv BV2 thiab RAW264.7 hlwb, peb tau ua ib txheej kev sim nrog tin protoporphyrin-IX (SNPP) uas yog ib qho kev xaiv inhibitor ntawm HO-1. Siv SNPP uas tuaj yeem txo qis qhov kev qhia ntawm HO-1, peb sim txiav txim siab seb HO{11}} kho kom haum rau cov nyhuv inhibitory ntawm cov tebchaw saum toj no ntawm cov lus teb inflammatory induced los ntawm LPS 【31】. Tom qab kho cov hlwb nrog 20 μM ntawm cov tebchaw 3 thiab 4 rau 2 h nrog lossis tsis muaj 50 um SNPP lawv tau kho nrog LPS rau 24 teev. Txawm hais tias cov tebchaw 3 thiab 4 txo cov nitrite ntau lawm hauv LPSinduced BV2 thiab RAW264.7 hlwb, lawv cov teebmeem tiv thaiv tau thim rov qab los ntawm kev kho SNPP (Daim duab 9). SNPP ib leeg tsis cuam tshuam rau nitric oxide (NO) ntau lawm tom qab LPS stimulation, qhia tias cov tshuaj tiv thaiv kab mob ntawm cov tebchaw 3 thiab 4 raug tswj los ntawm HO-1 qhia.

image

Inhibitory effects of compounds 3 and 4 on nitrite production through the regulation of HO-1 activity in BV2 (A,C,E) and RAW264.7 (B,D,F) cells. The cells were treated with 50 µM of tin protoporphyrin-IX (SnPP) or compounds 3 or 4 and stimulated for 24 h with lipopolysaccharide (LPS; 1 µg/mL). Data are presented as mean ± standard deviation of three independent experiments. ** p < 0.01 and *** p < 0.001

3. Kev sib tham

Txoj kev tshawb fawb tam sim no tau pom tias Kuwana T thiab sanggenon A tawm dag zog tiv thaiv LPS-induced o hauv BV2 thiab RAW264.7 hlwb. Cov tshuaj no inhibited LPS-induced zus tau tej cov NO, PGE2, thiab pro-inflammatory cytokines, xws li IL-6 thiab TNF-, thiab kev qhia ntawm iNOS thiab COX-2 nyob rau hauv ob qho tib si BV2 thiab RAW264.7cells. Cov txiaj ntsig no tau qhia tias Kuwana T thiab sanggenon A tau siv lawv cov tshuaj tiv thaiv kab mob los ntawm kev ua haujlwm tsis zoo ntawm NF-kB txoj kev taw qhia. Tsis tas li ntawd, cov tshuaj no tau ntxias cov lus qhia ntawm HO-1 los ntawm kev ua haujlwm ntawm Nrf2. Ntxiv rau, cov tshuaj no tau ntxias cov lus qhia ntawm HO-1 los ntawm Nrf2 ua kom, thiab cov nyhuv no kuj tau lees paub tias muaj feem xyuam nrog anti-inflammatory kev ua.

Nitric oxide synthase (NOS) has three isoforms: neuronal NOS (nNOS; NOS1), iNOS (NOS2), and endothelial eNOS (NOS3)[32]. iNOS is an inducible form that is upregulated in response to various stimuli, including LPS, cytokines, chemokines, and stress, while nNOS and eNOS are constitutive forms that catalyze continuous NO secretion at basal concentrations J33]. Similar to iNOS, COX-2 is also an inducible form upregulated by various inflammatory stimuli, such as cytokines, growth factors, tumor promoters, and bacterial LPS[34]. Its other isoform, COX-1, is constitutively expressed in most tissues under normal physiological conditions [35].COX-2 exerts an enzymatic effect on the conversion of prostaglandin H2 (PGH2), which converts arachidonic acid to PGE, prostaglandin 12(PGI2), prostaglandin F2a (PGF>.), and thromboxane A2(TXA,)[361. Nyob rau hauv cov mob inflammatory, theem ntawm iNOS thiab COX-2 nce, ua rau overproduction ntawm NO thiab PGE2, raws li, thiab ua rau exacerbation ntawm inflammatory kab mob [37]. Nyob rau hauv txoj kev tshawb no, peb thawj zaug soj ntsuam 11 lub tebchaw raug rho tawm los ntawm M.alba los txiav txim seb TSIS muaj ntau lawm yog inhibited hauv LPS-stimulated BV2 thiab RAW264.7 hlwb. Peb cov txiaj ntsig tau pom tias kuwanon T thiab sanggenon A tau ua rau muaj kev cuam tshuam loj tshaj plaws hauv BV2 thiab RAW264.7 hlwb (Daim duab 3). Hauv peb txoj kev tshawb fawb dhau los, moracin M tau muaj kev tiv thaiv kab mob ntawm alveolar macrophages, tab sis peb cov txiaj ntsig tau pom qhov txawv. qauv. Txawm li cas los xij, txij li txhua lub xov tooj muaj cov yam ntxwv sib txawv, cov haujlwm sib txawv yuav tshwm sim txawm tias muaj tib lub tswv yim J38]. Yog li ntawd, raws li nyob rau hauv cov txiaj ntsig ntawm daim ntawv no, nws tau lees paub tias moracin M tsis muaj cov tshuaj tiv thaiv kab mob hauv BV2 thiab RAW264.7 hlwb mus txog qhov kev kho mob concentration. Yog li ntawd, kuwanon T thiab sanggenon A raug xaiv rau kev sim ntxiv.

Cytokines muaj kev tswj hwm kev cuam tshuam ntawm kev mob thiab kev tiv thaiv kab mob [39]. Kev ua kom cov macrophages thiab microglial hlwb txhim kho qhov tso tawm ntawm pro-inflammatory cytokines J40, thiab qhov kev tso tawm ntau ntxiv ua rau cytokine tso tawm ntxiv [41]. IL-6 yog ib qho ntawm cov cytokines loj thiab yog tus neeg nruab nrab soluble lub luag haujlwm rau o, tiv thaiv kab mob, thiab hematopoiesis [42]. IL-6 signaling yog tswj los ntawm ob txoj kev sib txawv, suav nrog kev khi rau daim nyias nyias IL-6 receptor (mblL6R) thiab kev lees paub ntawm soluble IL-6 receptor (sIL6R)[43]. Ob lub tswv yim muaj feem xyuam nrog glycoprotein (GP)130 ua kom, uas ua rau ua kom cov dej ntws qis qis, suav nrog Janus kinase (IAK) / signal transducer thiab activator of transcription (STAT)kinases, phosphoinositide 3-kinase (PI3K). ), thiab mitogen-activated protein kinase (MAPK) [44]. IL-6 yog siv los kwv yees thiab ntsuas qhov mob ntawm cov neeg mob qog noj ntshav, kab mob, kab mob autoimmune, kab mob pancreatic, thiab kab mob plawv.

TNF-, lwm qhov loj cytokine, kuj ua lub luag haujlwm tseem ceeb hauv ntau lub cev tiv thaiv kab mob thiab kab mob, xws li kev tiv thaiv kab mob hauv lub cev, apoptosis, necrosis, thiab ciaj sia taus. Cov teebmeem lom ntawm TNF- raug tswj los ntawm kev khi rau ob qhov sib txawv receptors, qog necrosis factor receptor (TNFR)1 thiab TNFR2. TNFR1 yog tus neeg nruab nrab tseem ceeb ntawm TNF- kev ua haujlwm hauv cov hlwb feem ntau, vim tias TNFR2 tsis muaj kev sib raug zoo rau TNF-, ua rau TNF- dissociating yooj yim dua los ntawm TNFR2 dua li ntawm TNFR1. Kev txawv txav hauv TNF- kev taw qhia thiab kev tsim tawm ntau dhau ntawm TNF- ua rau muaj ntau yam kab mob, suav nrog rheumatoid mob caj dab, psoriasis, Crohn's kab mob, atherosclerosis, sepsis, ntshav qab zib, thiab rog [45,46. Yog li ntawd, nws yog ib qho tseem ceeb rau inhibit zus tau tej cov pro-inflammatory cytokines los suppress thiab tiv thaiv inflammatory kev tshwm sim thiab kev loj hlob ntawm inflammatory kab mob. Hauv kev tshawb fawb tam sim no, kuwanon T thiab sanggenon A inhibited LPS-induced zus tau tej cov IL-6 thiab TNF- nyob rau hauv BV2 thiab RAW264.7 hlwb.

NF-KB yog qhov tseem ceeb tshaj plaws ntawm kev hloov pauv [47], thiab NF-kB signaling plays lub luag haujlwm tseem ceeb hauv kev qhia cov noob muaj feem xyuam rau cov lus teb, suav nrog cov encoding iNOS, COX-2, thiab pro-inflammatory cytokines [ 48] ib. Nyob rau hauv lub xeev inactivated, NF-kB subunits muaj nyob rau hauv lub cytoplasm khi rau nws inhibitor protein, inhibitor kappa B (kB) - . Ntau qhov stimuli, suav nrog LPS thiab cytokines, ua rau phosphorylation thiab degradation ntawm IkB-, tso cai rau kev ywj pheej thiab hloov pauv ntawm NF-kB subunits mus rau hauv lub nucleus. Cov subunits translocated khi rau kB qhov chaw ntawm lub hom phiaj noob hauv DNA, uas ua rau induction ntawm transcription ntawm noob encoding pro-inflammatory mediators [49]. Yog li, txoj kev tsis ua haujlwm ntawm NF-kB txoj hauv kev yuav yog lub hom phiaj kho mob rau cov kab mob inflammatory. Nyob rau hauv txoj kev tshawb no, pretreatment nrog kuwanon T thiab sanggenon A inhibited LPS-induced activation ntawm NF-kB signaling los ntawm inhibiting lub DNA-binding kev ua si thiab nuclear translocation ntawm NF-kB subunits (Daim duab 6). Cov txiaj ntsig no qhia tau hais tias cov tshuaj tiv thaiv kab mob ntawm kuwanon T thiab sanggenon A tau dhau los ntawm kev tswj hwm ntawm NF-kB kev taw qhia txoj hauv kev.

Raws li ib txwm muaj, Nrf2 khi rau Kelch-zoo li ECH-koom nrog cov protein 1 (Keap1) hauv cytoplasm. Txawm li cas los xij, nws dissociates los ntawm Keapl, translocates mus rau hauv lub nucleus, thiab khi rau antioxidant teb lub ntsiab (ARE) qhov chaw ntawm DNA, ua rau cov kev qhia ntawm ntau yam antioxidant noob, nrog rau cov encoding HO-1, NAD(P)H. : quinone oxidoreductase 1(NQO1), peroxiredoxin (Prx), and thioredoxin (Trx)[50]. Tshwj xeeb, HO-1 paub tias muaj feem cuam tshuam nrog cov tshuaj tiv thaiv kab mob, thiab nws cov haujlwm yog sawv cev los ntawm qib CO, ib qho ntawm peb cov khoom lag luam tsim tawm los ntawm heme los ntawm kev ua haujlwm enzymatic ntawm HO{{12. }} [51] ib. Nyob rau hauv txoj kev tshawb no, peb pom tias kuwanon T thiab sanggenon A-induced HO-1 qhia los ntawm activating Nrf2 (Figures 7 thiab 8). Tsis tas li ntawd, peb tau txheeb xyuas qhov sib txheeb ntawm cov tshuaj tiv thaiv kab mob ntawm kuwanon T thiab sanggenon A thiab HO-1 qhia siv SNPP, ib qho kev xaiv inhibitor ntawm HO-1 kev ua. Cov teebmeem inhibitory ntawm kuwanon T thiab sanggenon A ntawm NOand TNF- ntau lawm thiab NF-kB ua kom tau ib nrab rov qab los ntawm kev kho nrog SNPP (Daim duab 9). Cov txiaj ntsig no qhia tias cov tshuaj tiv thaiv kab mob ntawm kuwanon T thiab sanggenon A tau tswj hwm los ntawm HO-1 qhia.

effects of cistanche improve immunity (11)

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Khoom siv

Roswell Park Memorial Institute 1640 (RPMI 1640) thiab fetal bovine serum tau yuav los ntawm Gibco BRL Co.(Grand Island, NY, USA). Tag nrho cov tshuaj tau txais los ntawm Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Thawj cov tshuaj tiv thaiv anti-iNOS, anti- -actin, anti-p65, thiab anti-HO-1 tau yuav los ntawm Santa Cruz Biotechnology (Santa Cruz, CA, USA); thiab tiv thaiv luav thiab tiv thaiv nas thib ob cov tshuaj tiv thaiv los ntawm Millipore

(Billerica, MA, USA). Enzyme-linked immunosorbent assay (ELISA) cov khoom siv rau PGE,, IL-6, thiab TNF- tau yuav los ntawm R&DSystems Inc.(Minneapolis, MN, USA). Kev sib cais thiab kev txiav txim siab ntawm 11 lub tebchaw los ntawm Morus alba tau piav qhia lwm qhov [19-21].

4.2. Cell Culture thiab Viability Assay

BV2 thiab RAW264.7 hlwb tau cog rau ntawm qhov ceev ntawm 5 × 105 hlwb / mL hauv RPMI 1640 ntxiv nrog 1 feem pua ​​​​cov tshuaj tua kab mob (penicillin-streptomycin) thiab 10 feem pua ​​​​ntawm cov cua sov-inactivated FBS. Cov hlwb raug coj los ntawm 37 degree hauv qhov av noo 5 feem pua ​​​​CO, nrog rau 95 feem pua ​​​​ntawm huab cua, raws li txoj kev tau piav qhia yav dhau los [52].

4.3. Kev ntsuas NO Production

Kev tsim cov nitrite, ib qho khoom ruaj khov kawg ntawm NO oxidation, tau ntsuas raws li qhov ntsuas tsis muaj ntau lawm hauv hlwb. Luv luv, qhov concentration ntawm nitrite nyob rau hauv cov xov xwm txias tau txiav txim siab siv ib txoj kev raws li Griess cov tshuaj tiv thaiv [53]. Cov ntsiab lus ntawm qhov kev ntsuam xyuas tau piav qhia yav dhau los [54].

4.4. PGE2 Ass4y/

Qhov concentration ntawm PGEz nyob rau hauv txhua tus qauv tau ntsuas los ntawm kev siv cov khoom siv ELISA, raws li txoj kev tau piav qhia yav dhau los [55].

4.5.Measurement of IL-6 and TNF-a Levels

Cov kab lis kev cai nruab nrab tau sau los txiav txim siab theem ntawm IL-6 thiab TNF- siv cov khoom siv muag khoom (BioLegend, San Diego, CA, USA). Qhov kev ntsuam xyuas tau ua raws li cov neeg tsim khoom cov lus qhia. Luv luv, BV2 thiab RAW264.7 hlwb tau muab noob rau hauv 24-cov kab lis kev cai zoo ntawm qhov ntom ntawm 20 × 10 cell / zoo. Tom qab incubation, lub supernatant tau sau thiab siv los ntsuas qhov concentrations ntawm IL-6 thiab TNF- nrog ELISA cov khoom siv.

4.6. Western Blot Analysis

Cov pelleted BV2 thiab RAW264.7 hlwb raug ntxuav nrog PBS thiab lysed nyob rau hauv RIPA tsis. Qhov sib npaug ntawm cov proteins tau suav nrog siv cov tshuaj ntsuam xyuas cov tshuaj dye reagent concentrate tau los ntawm Bio-Rad Laboratories (#5000006; Hercules, CA, USA), sib xyaw hauv cov qauv thauj khoom tsis sib xws, thiab sib cais siv SDS-PAGE. Tom qab ntawd cov proteins uas sib cais tau raug xa mus rau ib qho nitrocellulose membrane. Non-specific binding rau daim nyias nyias tau thaiv los ntawm incubation nyob rau hauv ib tug daws ntawm skim mis nyuj. Lub membrane tau incubated nrog thawj cov tshuaj tiv thaiv (tag nrho cov uas tau siv nyob rau hauv ib tug 1: 1000) ntawm 4 degree thaum hmo ntuj thiab ces reacted nrog ib tug horseradish peroxidase-conjugated thib ob antibody (tag nrho cov uas tau siv ntawm ib tug 1: 5000) (Millipore) [ 31] ib.

4.7.NF-xB Localization thiab Immunofluorescence Analysis

Txhawm rau kawm txog qhov chaw ntawm NF-kB, BV2 thiab RAW264.7 hlwb raug coj los ntawm Lab-Tek II chamber slides thiab kho nrog qhov sib txawv ntawm cov tshuaj sib xyaw rau 2h ua ntej LPS stimulation (0.5 ug/mL) rau 1 h. Tom qab ntawd cov hlwb raug kho rau hauv formalin thiab permeabilized nrog txias acetone thiab soj ntsuam nrog anti-p65 antibodies (1:200), tom qab ntawd incubation nrog fluorescein isothiocyanate-labeled secondary antibody (1:1000) (Alexa Fluor 488, Invitrogen). Txhawm rau pom cov nuclei, cov hlwb raug kho nrog 1 ug / mL ntawm 4'6-diamidino-2-phenylindole (DAPI rau 30 min, ntxuav nrog PBS rau 5 feeb, thiab kho nrog 50 μL VectaShield (Vector Laboratories, Burlingame, CA, USA). Cov hlwb stained tau pom thiab cov duab tau txais los ntawm Zeiss fluorescence microscope (Provis AX70; Olympus Optical Co., Tokyo, Nyiv). [31].

4.8. Kev txheeb cais

Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± tus qauv sib txawv ntawm peb qhov kev sim ywj pheej. Ib txoj kev tsom xam ntawm qhov sib txawv, ua raws li Tukey qhov kev sib piv ntau yam, tau siv los sib piv qhov sib txawv ntawm peb pawg. Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism software (version 5.01, GraphPad Software Inc., San Diego, CA, USA),

5. Cov lus xaus

Kuwanon T thiab sanggenon A exerted anti-inflammatory teebmeem hauv LPS-induced BV2 thiab RAW264.7 hlwb los ntawm inhibiting zus tau tej cov NO, PGE2, IL-6, thiab TNF- thiab kev qhia ntawm iNOS thiab COX{{8} }. Peb cov txiaj ntsig tau pom tias cov teebmeem inhibitory no tau kho los ntawm kev tsis ua haujlwm ntawm NF-KB txoj hauv kev los ntawm kev kho nrog kuwanon T thiab sanggenon A. Tsis tas li ntawd, cov tshuaj no tau ntxias cov lus qhia ntawm HO-1 los ntawm kev ua kom hloov pauv ntawm Nrf2 mus rau hauv lub nucleus. . Peb qhov kev tshawb pom tau pom tias HO-1 qhia tawm los ntawm kuwanon T thiab sanggenon A tau cuam tshuam nrog kev cuam tshuam los ntawm LPS-induced o. Ua ke, peb cov txiaj ntsig tau muab pov thawj tias Kuwana T thiab sanggenon A cais los ntawm M. Albu tuaj yeem yog cov neeg sib tw rau kev txhim kho kev kho mob thiab tiv thaiv cov kab mob neuroinflammatory xws li Alzheimer's disease thiab Parkinson's disease.


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