Acta Pharmacologica Sinica (2018) 39: 1582–1589 © 2018 CPS and SIMM All rights reserved 1671-4083/18 www.nature.com/aps
Article
Prolonged DADLE exposure epigenetically promotes Bcl-2 expression and elicits neuroprotection in SULPDU\UDWFRUWLFDOQHXURQVYLDWKH3,.$NW1)Ʃ% pathway
Min ZHU1, 2, #, Ming LIU1, 3, #, Qi-lin GUO1, Cui-qing ZHU1, Jing-chun GUO1, *
1Department of Translational Neuroscience, Jing’an District Centre Hospital of Shanghai, State Key Laboratory of Medical Neurobiology & Institutes of Brain Science, Fudan University, Shanghai 200032, China; 2Shanghai Key Laboratory of Visual Impairment and Restoration, Eye & ENT Hospital, Fudan University, Shanghai 200032, China; 3Shanghai High School, International Division, Shanghai 200231, China
Abstract Both in vivo and in vitroVWXGLHVKDYHVKRZQWKHEHQHÀFLDOHIIHFWVRIWKHGHOWDRSLRLGUHFHSWRU '25 RQQHXURGHJHQHUDWLRQLQK\SR[LD LVFKHPLD:HSUHYLRXVO\UHSRUWHGWKDW'25VWLPXODWLRQZLWK> '$OD'/HX HQNHSKDOLQ@ '$'/( DSRWHQW'25DJRQLVWIRUERWK
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Introduction After the neuroprotective effects of DOR activation were Stroke is the leading cause of death and serious, long-term recognized, subsequent studies focused on the underlying disability. To date, clinical strategies that help to stop the neu- mechanism of DORs, and great progress has been made so
rodegeneration following anoxic/ischemic insults remain lim- far. The DOR is a Gi protein-coupled receptor that functions ited. [(D-Ala2, D-Leu5) enkephalin] (DADLE) is a synthetic through the cAMP signaling pathway[6]. Although the details opioid peptide and a highly selective delta opioid receptor remain unclear, it is generally accepted that the mechanisms of (DOR) agonist[1, 2]. Since the neuroprotective effects of DOR DOR neuroprotection fall into two categories: DORs can trig- DFWLYDWLRQZHUHÀUVWGHVFULEHG[1], it has attracted a great deal ger endogenous protective pathways[4, 7-9] and maintain ionic of attention from researchers worldwide. Accumulating evi- homeostasis[10-13],QSDUWLFXODULWKDVEHHQFRQÀUPHGE\PXO- dence has suggested that stimulating DOR with DADLE elicits tiple studies that the PKC/MAPK pathway plays a crucial role potent neuroprotection against anoxic/ischemic injury[1, 3-5]. in the neuroprotective functions of DORs[4, 7, 8, 14]. Moreover, Therefore, DADLE may be a promising novel therapeutic can- DOR activation might elicit neuroprotection via the PI3K/Akt didate for treating neurodegeneration in stroke. signaling pathway[15, 16]. Our previous studies have investigated the neuroprotec-
tive effects of DADLE using a NaN3-induced mitochondrial # 7KHVHDXWKRUVFRQWULEXWHGHTXDOO\WRWKLVZRUN dysfunction model. Much to our surprise, we found that *7RZKRPFRUUHVSRQGHQFHVKRXOGEHDGGUHVVHG (PDLOMLQJFKXQJXR#VKPXHGXFQ stimulating DOR with DADLE for both a short (minutes) and 5HFHLYHG$FFHSWHG long (days) time showed notable protective effects on pri- www.chinaphar.com Zhu M et al
mary rat cortical neuronal cultures[8, 17]. Further investigation extraction kit (Chemicon, Temecula, CA, USA) according to revealed that short-term DADLE stimulation increases neu- WKHPDQXIDFWXUHU·VSURWRFRO%ULHÁ\QHXURQVZHUHFROOHFWHG ronal survival through the PKC-mitochondrial ERK pathway after drug exposure and incubated in cytoplasmic lysis buf- [8] in the NaN3 model . However, the mechanism underlying fer for 15 min. The cells were pelleted by centrifugation at the protective effects of DADLE during prolonged exposure 250×g for 5 min and resuspended in cytoplasmic lysis buffer. remains unclear. In this study, we will explore the possible A syringe with a small gauge needle was used to break up downstream signaling pathways mediating the neuroprotec- the cytoplasmic membrane. The supernatant was collected tive effects of sustained DOR stimulation with DADLE in the after centrifugation at 8000×g for 20 min and used as the cyto-
NaN3-induced neuronal injury model. solic fraction. The remaining nuclear pellet was resuspended in nuclear extraction buffer for 30 min, and a fresh syringe Materials and methods was used to disrupt the nuclei. Following centrifugation at Primary culture of rat cortical neurons 16 000×g for 5 min, the supernatant was collected and used as Primary cortical neuron cultures were prepared as previously the nuclear extract. Each step was performed on ice at 4 °C. described[8, 17]%ULHÁ\DIWHU6SUDJXH'DZOH\UDWV HPEU\RQLF d17–18; Shanghai Experimental Animal Center, Chinese Acad- Immunoblotting analyses HP\RI6FLHQFH6KDQJKDL&KLQD ZHUHVDFULÀFHGRQJHVWDWLRQ Neurons were harvested after drug exposure, and the cell GïWKHFRUWLFDOQHXURQVZHUHFDUHIXOO\FROOHFWHG7KHQ lysates were prepared as described before[8]. Protein samples the cells were resuspended in neurobasal medium (Gibco- ZHUHHOHFWURSKRUHVHGRQï6'63$*(JHOV7KH BRL, Life Technologies, Rockville, MD, USA) supplemented antibodies used were as follows: rabbit polyclonal antibod- with B-27, glutamine (0.5 mmol/L), penicillin (100 U/mL) and ies against pp38, p38, pERK, ERK, pJNK, JNK, pAkt, Akt, VWUHSWRP\FLQ ǍJP/ DQGSODWHGRQWRSRO\L-lysine (100 and H3 (1:1000; all from Cell Signaling Technology, Danvers, ǍJP/6LJPD6W/RXLV0286$ SUHFRDWHGGLVKHVDWDGHQ- MA, USA), rabbit antibodies against pp85, p85, p65, and Bcl-2 sity of 1×106 cells/mL. The cells were ready for drug admin- (1:1000; all from Chemicon, Temecula, CA, USA), mouse anti- istration on day 8. Animal experiment protocols complied ERG\DJDLQVWǃDFWLQ 6LJPD6W/RXLV0286$ DQG with the guidelines of the Animal Care and Use Committee of sheep antibody against Cyto c (1:1000; Chemicon, Temecula, Fudan University. All efforts were made to minimize the suf- CA, USA). fering of the animals. RNA extraction, RT-PCR and real-time PCR Drug administration Total RNA was extracted by using Trizol reagent
Freshly prepared NaN3 ǍPRO/ ZDVDSSOLHGWRSULPDU\ (Invitrogen, Carlsbad, CA, USA) according to the cultured neurons to induce mitochondrial dysfunction. PDQXIDFWXUHU VLQVWUXFWLRQV51$ ǍJ ZDVUHYHUVH '$'/( ǍPRO/ RUQDOWULQGROH 17,ǍPRO/ ZDV transcribed by using the PrimeScript RT reagent kit administered to selectively activate or inhibit DOR activity[8, 17]. (Takara, Japan), and single stranded cDNA was amplified /< ǍPRO/ LVDVHOHFWLYH3,.LQKLELWRUDQG$NW by quantitative real-time PCR with the SYBR Premix Ex LQKLELWRU9,,, ǍPRO/ LVDVSHFLILF$NWLQKLELWRU[18]. All Taq Kit (Takara, Japan) on a Mastercycler ep realplex real- drugs were purchased from Sigma (St Louis, MO, USA). time PCR system (Eppendorf, Hamburg, Germany). The IROGFKDQJHZDVFDOFXODWHGXVLQJWKHƦƦ&WPHWKRG ƦƦ&W) Lactate dehydrogenase (LDH) assay with GAPDH as the internal control. The following primers The activities of LDH in the cultivated medium were measured were used: Bcl-2, 5’-CCCTGTGGATGACTGAGTAC-3’ and with an LDH kit (Sigma, St Louis, MO, USA) and detected 5’-GCATGTTGACTTCACTTGTG-3’; GAPDH, 5’-CCA- with a DU-640 spectrophotometer (Beckman Instruments, CTCCTCCACCTTTGAC-3’ and 5’-ACCCTGT TGCTGT- Inc., Fullerton, CA, USA) as previously described[8, 17]. The AGCCA-3’. Data are presented as the means±SEM of 4 absorbance data were normalized to those of the control independent experiments with 3 real-time PCR replicates. group. Chromatin immunoprecipitation (ChIP) assay Cell viability assay ChIP assays were performed using a ChIP assay kit (Mil- Cell viability assays were performed by adding cell counting lipore, Billerica, MA, USA) according to the manufacturer’s kit-8 reagent (CCK-8, Dojindo Laboratories, Japan) directly protocol. In brief, after drug treatment, neuronal cells were to the cell cultures. After incubation at 37 °C for 1 h, the cross-linked with formaldehyde, lysed and sonicated. Immu- absorbance was read by a spectrophotometer (Thermo Fisher QRSUHFLSLWDWLRQZDVSHUIRUPHGZLWKDQWLERGLHVWR1)Nj% &HOO Scientific, Waltham, MA, USA) at a wavelength of 450 nm Signaling Technology, Danvers, MA, USA), acetyl-H3 and and was proportional to the number of living cells. Each acetyl-H4 (both from Chemicon, Temecula, CA, USA). Addi- experiment was repeated three times. tionally, samples were immunoprecipitated with rabbit IgG as a negative control. Immunoprecipitated DNA was subjected Cytosolic and nuclear protein preparation to semi-quantitative and quantitative real-time PCR analy- Cytosolic and nuclear proteins were extracted using a nuclear ses using primers specific for the Bcl-2 promoter (5’-TGTA-
Acta Pharmacologica Sinica ZZZQDWXUHFRPDSV Zhu M et al
ACTTTCAATGGACGC-3’ and 5’-ATCTACTTCCTCCG- neuronal damage was determined by measuring LDH release &$$7*· $IWHUF\FOHVRIDPSOLÀFDWLRQǍ/RIWKH3&5 into the culture medium after drug exposure, and cell viability product was analyzed on 2% agarose gels. Real-time PCR was was detected by using a CCK-8 kit. DADLE completely
performed as described above. suppressed the increase in LDH release induced by NaN3 and restored cell viability; these effects were abolished by Statistical analysis LY296004 and VIII (Figure 2). These data imply that sustained
The data are shown as the mean±SEM and were subjected to DOR stimulation reduces NaN3-induced neuronal injury via statistical analysis using one-way ANOVA. The level of statis- the PI3K/Akt pathway. WLFDOVLJQLÀFDQFHZDVVHWDWP<0.05. 3URORQJHG'$'/(H[SRVXUHHQKDQFHV%FOH[SUHVVLRQDQG
Results attenuates Cyto c release in NaN3-treated neurons through the Prolonged DADLE exposure selectively activates the PI3K/Akt/ PI3K/Akt pathway
1)Ʃ%SDWKZD\LQ1D13 -treated neurons NaN3LVDVSHFLÀFFRPSOH[,9LQKLELWRUZLGHO\XVHGWRLQGXFH To examine the influence of prolonged DADLE stimulation mitochondrial dysfunction and Cyto c release[19, 20]. To explore
on MAPK and Akt activity in NaN3-treated neurons, primary the effect of sustained DADLE stimulation on mitochondrial
UDWFRUWLFDOQHXURQFXOWXUHVZHUHWUHDWHGZLWKǍPRO/ Cyto c release in NaN3-treated neurons, the levels of cytosolic
NaN3 and DADLE or NTI for 2 d. The phosphorylation levels Cyto c were tested by immunoblotting after drug exposure. and the total levels of ERK, JNK, p38 and Akt were detected As seen in Figure 3A, Cyto c levels in the cytosolic fraction
by immunoblotting after drug treatment. Neither the phos- were robustly increased by NaN3; this effect was suppressed phorylation levels nor the total levels of ERK, p38 or JNK were by DADLE. The effect of DADLE was totally abolished
affected by NaN3 (Figure 1A); however, the levels of pAkt by NTI, LY296004 and VIII, implying that sustained DOR
were reduced by NaN3 (0.4-fold higher than those of the con- stimulation in NaN3-treated neurons prevents Cyto c release trols), and the total Akt levels were not affected (Figure 1C). through the PI3K/Akt pathway.
DADLE not only reversed the NaN3-induced inhibition of Akt Bcl-2 is a well-known anti-apoptosis protein that prevents activation but also further increased the levels of pAkt (1.72- Cyto c release[21]. To investigate the impact of sustained DOR
fold higher than those of the controls); these effects were abol- activation on Bcl-2 expression in NaN3-exposed neurons, ished by NTI (Figure 1C). Bcl-2 levels were determined by immunoblotting after Next, the phosphorylation levels and total levels of the p85 drug exposure. As shown in Figure 3B, Bcl-2 levels were
subunit of PI3K were determined by immunoblotting after considerably reduced by NaN3, and this effect was reversed drug treatment. As shown in Figure 1B, the levels of pp85 by DADLE; furthermore, the effect of DADLE was entirely
were significantly decreased by NaN3, and DADLE not only prevented by NTI, LY296004 and VIII. Next, the mRNA
reversed the NaN3-induced decrease in pp85 levels but also levels of Bcl-2 were determined after drug treatment. Bcl-2
further elevated PI3K activity; these effects were abolished mRNA levels were significantly decreased by NaN3; this by NTI. Moreover, the total levels of p85 were not altered effect was reversed by DADLE (Figure 3C). Again, the effect in any group after drug exposure. LY296004, a selective of DADLE was completely inhibited by NTI, LY296004 and PI3K inhibitor, also prevented Akt activation in the neurons VIII, indicating that sustained DOR stimulation with DADLE
exposed to DADLE and NaN3 (Figure 1C), indicating that in NaN3-treated neurons restores Bcl-2 protein levels by PI3K acts as a downstream signal of DOR stimulation and is attenuating its transcriptional dysfunction via the PI3K/Akt
required for Akt activation in NaN3-treated neurons. pathway. To explore the impact of sustained DADLE stimulation on These results indicate that sustained DOR stimulation with
WKHDFWLYLW\RI1)Nj%LQ1D13-treated neurons, translocation DADLE decreases cell damage in NaN3-treated neurons by RIWKHSVXEXQLWRI1)Nj%LQWRWKHQXFOHXVZDVGHWHFWHG promoting Bcl-2 expression and preventing Cyto c release by immunoblotting after drug exposure. The levels of p65 through the PI3K/Akt pathway.
in the nucleus were remarkably decreased by NaN3, and this effect was attenuated by DADLE. The effect of DADLE was 3URORQJHG'25VWLPXODWLRQZLWK'$'/(HQKDQFHVWKH1)Ʃ% abolished by NTI, LY296004 and Akt inhibitor VIII, implying binding activity and histone acetylation of the Bcl-2 promoter in
that DOR stimulation reverses NaN3LQGXFHG1)Nj%LQKLELWLRQ neurons treated with NaN3 via the PI3K/Akt pathway. 1)Nj%KDVEHHQUHSRUWHGWRSURPRWHWKHWUDQVFULSWLRQDODFWLY- The data above suggest that sustained DOR stimulation with ity of Bcl-2[22]; therefore, we further investigated whether the '$'/(VHOHFWLYHO\DFWLYDWHVWKH3,.$NW1)Nj%SDWKZD\ XSUHJXODWLRQRI%FOWUDQVFULSWLRQLVPRGXODWHGE\1)Nj%
but not MAPKs in the NaN3 model. during sustained DOR activation. According to ChIP assays, RXUUHVXOWVVKRZHGWKDWWKHOHYHOVRI1)Nj%ERXQGWRWKH
Prolonged DADLE treatment reduces cell injury in NaN3-treated Bcl-2 promoter were remarkably reduced by NaN3; this effect neurons via the PI3K/Akt pathway was completely blocked by DADLE (Figure 4). The effect of To determine whether the protective effect of sustained DADLE was abolished by NTI, which indicates that sustained DADLE stimulation is mediated by the PI3K/Akt pathway, '25DFWLYDWLRQHQKDQFHG1)Nj%ELQGLQJWRWKHBcl-2 promoter
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in NaN3-treated neurons. by NaN3; these effects were completely blocked by DADLE. ChIP was also used to determine the effect of DADLE on the The effects of DADLE were abolished by NTI. These data histone acetylation level of the Bcl-2 promoter in NaN3-treated suggest that sustained DOR activation increased the histone neurons. As shown in Figure 5, the levels of acetylated H3 acetylation levels of the Bcl-2 promoter in neurons treated with and H4 for the Bcl-2 promoter were both remarkably reduced NaN3.
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Discussion So far, there are no effective treatments to stop or reverse neu- rodegeneration after a stroke. Despite the encouraging results seen in preclinical studies, most cytoprotective drugs have failed in clinical trials. One of the main reasons for this is that those drugs showed protection at only the very early stage of neurodegeneration, and their effects did not last as the disease Figure 3.6XVWDLQHG'25DFWLYDWLRQZLWK'$'/(DWWHQXDWHV&\WRcUHOHDVH developed. DQGHQKDQFHV%FOH[SUHVVLRQLQ1D13WUHDWHGQHXURQVWKURXJKWKH Our earlier work found that stimulating DOR with DADLE 3,.$NWSDWKZD\1HXURQFXOWXUHVZHUHWUHDWHGZLWK1D13DQG'$'/( for both a short (minutes) and long (days) time resulted in sig- 17,/< RU$NWLQKLELWRU9,,,IRUG7KHSURWHLQOHYHOVRIF\WRVROLF [8, 17] QLÀFDQWQHXURSURWHFWLYHHIIHFWVLQWKH1D13 model . These &\WRc $ DQG%FO % LQQHXURQVZHUHPHDVXUHGE\LPPXQREORWWLQJ results imply that DADLE provides immediate protection at DIWHUGUXJH[SRVXUHDQGWKHP51$OHYHOVRIBcl-2ZHUHDOVRGHWHUPLQHG &
the early stage of neuronal injury and that its protective effects &\WRVROLF&\WRcOHYHOVZHUHUREXVWO\LQFUHDVHGLQ1D13WUHDWHGQHXURQV would last for days if the toxin is not removed. Our further ZKHUHDVWKHP51$OHYHOVDQGSURWHLQOHYHOVRI%FOZHUHERWKUHPDUNDEO\ investigations of the underlying mechanisms revealed that GHFUHDVHG'$'/(VXSSUHVVHGWKHGRZQUHJXODWLRQRI%FODQGWKH at least two downstream signaling pathways may be sequen- LQFUHDVHLQF\WRVROLF&\WRcOHYHOV WKHVHHIIHFWVZHUHDEROLVKHGE\WKHFR * ** tially activated and responsible for the neuroprotective effects DGPLQLVWUDWLRQRI17,/< RU$NWLQKLELWRU9,,, P P FRPSDUHGWR&WUO #P FRPSDUHGWRWKH1D1 JURXSn of DADLE against mitochondrial injury. While short-term 3 DADLE stimulation increases neuronal survival through the [8] PKC-mitochondrial ERK pathway , sustained DOR activation are not yet well-established, several endogenous neuroprotective with DADLE in the present study had little effect on MAPK pathways have been reported to be involved. Activating DOR activity in NaN3-treated neurons but selectively activated the via hypoxia preconditioning can balance the pERK and pp38 3,.$NW1)Nj%SDWKZD\ WKHVHDFWLRQVPLJKWPHGLDWHWKH levels through the PKC/MAPK pathway; this action elicits neuroprotective effects of prolonged DOR activation. neuroprotective effects in a severe hypoxia model in vitro[4]. Though the intricacies of the role of DOR in neuroprotection Narita et al found that the neurogenesis and anti-apoptosis
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Acta Pharmacologica Sinica ZZZQDWXUHFRPDSV Zhu M et al
genes, and some of these genes are involved in the process Author contribution of apoptosis[31]. Several studies showed that translocation of Jing-chun GUO and Cui-qing ZHU designed the study; Min WKH1)Nj%SVXEXQLWLQWRWKHQXFOHXVLVDFWLYDWHGYLDWKH ZHU and Ming LIU performed the research; Min ZHU and PI3K/Akt pathway; there, it participates in regulating anti- Qi-lin GUO analyzed the data; and Min ZHU, Ming LIU and apoptotic gene expression[32, 33], possibly through enhancing Jing-chun GUO wrote the paper. acetylation efficiency in the p65 binding region[34]. In the present study, we revealed that DOR stimulation reversed References
NaN3-induced p65 inhibition via the PI3K/Akt pathway =KDQJ-+DGGDG**;LD<GHOWDEXWQRWPXDQGNDSSDRSLRLG and promoted Bcl-2 transcription by increasing the histone UHFHSWRUDFWLYDWLRQSURWHFWVQHRFRUWLFDOQHXURQVIURPJOXWDPDWH acetylation of the Bcl-2 promoter. These results are supported LQGXFHGH[FLWRWR[LFLQMXU\%UDLQ5HV ² E\RWKHUVWXGLHVVKRZLQJWKDW1)Nj%PLJKWGLUHFWO\ELQG /DZ3<:RQJ<+/RK++0ROHFXODUPHFKDQLVPVDQGUHJXODWLRQ RIRSLRLGUHFHSWRUVLJQDOLQJ$QQX5HY3KDUPDFRO7R[LFRO to the Bcl-2 promoter and promote its transcription[35, 36], as ² well as the fact that sustained DOR activation enhanced p65 =KDQJ-4LDQ+=KDR3+RQJ66;LD<5DSLGK\SR[LD binding to the Bcl-2 promoter in neurons treated with NaN3 SUHFRQGLWLRQLQJSURWHFWVFRUWLFDOQHXURQVIURPJOXWDPDWHWR[LFLW\ in this study. Therefore, upon sustained DADLE stimulation WKURXJKGHOWDRSLRLGUHFHSWRU6WURNH ² in NaN3-induced mitochondrial injury, the epigenetic 0D0&4LDQ+*KDVVHPL)=KDR3;LD<2[\JHQVHQVLWLYH^GHOWD` upregulation of Bcl-2 expression might play an important RSLRLGUHFHSWRUUHJXODWHGVXUYLYDODQGGHDWKVLJQDOV QRYHOLQVLJKWV role in the downstream signaling of DOR neuroprotection. LQWRQHXURQDOSUHFRQGLWLRQLQJDQGSURWHFWLRQ-%LRO&KHP Since Bcl-2 levels could be affected by both the expression and ² =KDQJ-*LEQH\*7=KDR3;LD<1HXURSURWHFWLYHUROHRIGHOWDRSLRLG degradation processes, whether NaN3 or DADLE affect Bcl-2 protein degradation still needs further investigation. UHFHSWRUVLQFRUWLFDOQHXURQV$P-3K\VLRO&HOO3K\VLRO In conclusion, we showed here that the mechanism under- &² 'UROHW*'XPRQW(&*RVVHOLQ,.LQNHDG5/DIRUHVW67URWWLHU-) lying sustained DOR activation by DADLE is very different 5ROHRIHQGRJHQRXVRSLRLGV\VWHPLQWKHUHJXODWLRQRIWKHVWUHVV from the mechanism underlying its short-term stimulation in UHVSRQVH3URJ1HXURSV\FKRSKDUPDFRO%LRO3V\FKLDWU\ NaN3-treated neurons. A different signaling pathway (PI3K/ ² $NW1)Nj% LVDFWLYDWHGDQGLWIXUWKHUSURPRWHV%FOH[SUHV- 3HQJ3++XDQJ+6/HH<-&KHQ<60D0&1RYHOUROHIRUWKHGHOWD sion through epigenetic regulation. Bcl-2 upregulation sup- RSLRLGUHFHSWRULQK\SR[LFSUHFRQGLWLRQLQJLQUDWUHWLQDV-1HXURFKHP presses Cyto c release and rescues neuronal cells (Figure 6). ² The present work is an essential supplement to our earlier =KX0/L0 HIIHFWVRIVXVWDLQHG'25VWLPXODWLRQZLWK'$'/(DJDLQVW1D13 insult. PRGHOV%LRPHG3KDUPDFRWKHU ² Acta Pharmacologica Sinica www.chinaphar.com Zhu M et al =KX0/L0:7LDQ;62X;0=KX&4*XR-&1HXURSURWHFWLYH .HVDUDMX61D\DN*3UHQWLFH+00LOWRQ6/8SUHJXODWLRQRI+VS UROHRIGHOWDRSLRLGUHFHSWRUVDJDLQVWPLWRFKRQGULDOUHVSLUDWRU\FKDLQ PHGLDWHVDQR[LDUHR[\JHQDWLRQQHXURSURWHFWLRQLQWKHIUHVKZDWHU LQMXU\%UDLQ5HV ² WXUWOHYLDPRGXODWLRQRI526%UDLQ5HV ² =KX/'HULMDUG%&KDNUDEDQGKX.:DQJ%6&KHQ+=+XHEHU$2 :DQJ4&KDR'&KHQ76DQGKX+;LD<GHOWD2SLRLGUHFHSWRUVDQG 6\QHUJLVPRI3,.$NWLQKLELWLRQDQG)DVDFWLYDWLRQRQFRORQFDQFHU LQIODPPDWRU\F\WRNLQHVLQK\SR[LD GLIIHUHQWLDOUHJXODWLRQEHWZHHQ FHOOGHDWK&DQFHU/HWW ² JOLDODQGQHXURQOLNHFHOOV7UDQVO6WURNH5HV ² 6DÀXOLQD'9HNVOHU9=KDUNRYVN\$.DDVLN$/RVVRIPLWRFKRQGULDO %DVVLN0&6FRUUDQR/2DNHV6$3R]]DQ7.RUVPH\HU6- PHPEUDQHSRWHQWLDOLVDVVRFLDWHGZLWKLQFUHDVHLQPLWRFKRQGULDO 3KRVSKRU\ODWLRQRI%&/UHJXODWHV(5&D homeostasis and YROXPH SK\VLRORJLFDOUROHLQQHXURQHV-&HOO3K\VLRO DSRSWRVLV(0%2- ² ² ,WR7'HQJ;&DUU%0D\:6%FOSKRVSKRU\ODWLRQUHTXLUHGIRUDQWL 0DULQR60DUDQL/1D]]DUR&%HDQL/6LQLVFDOFKL$0HFKDQLVPVRI DSRSWRVLVIXQFWLRQ-%LRO&KHP ² VRGLXPD]LGHLQGXFHGFKDQJHVLQLQWUDFHOOXODUFDOFLXPFRQFHQWUDWLRQ %DHXHUOH3$+HQNHO7)XQFWLRQDQGDFWLYDWLRQRI1)NDSSD%LQWKH LQUDWSULPDU\FRUWLFDOQHXURQV1HXURWR[LFRORJ\ ² LPPXQHV\VWHP$QQX5HY,PPXQRO ² -LDQJ;:DQJ;&\WRFKURPHcPHGLDWHGDSRSWRVLV$QQX5HY /L*.DODELV-;X;0HLHU)2ND0%RJHQULHGHU7, et al5HFLSURFDO %LRFKHP ² UHJXODWLRQRI0HO&$0DQG$.7LQKXPDQPHODQRPD2QFRJHQH %DUNHWW0*LOPRUH7'&RQWURORIDSRSWRVLVE\5HO1)NDSSD% ² WUDQVFULSWLRQIDFWRUV2QFRJHQH ² 'KDZDQ36LQJK$%(OOLV'/5LFKPRQG$&RQVWLWXWLYHDFWLYDWLRQRI 23 Jeong S, Kim SJ, Jeong C, Lee S, Jeong H, Lee J, et al1HXURSURWHFWLYH $NWSURWHLQNLQDVH%LQPHODQRPDOHDGVWRXSUHJXODWLRQRIQXFOHDU HIIHFWVRIUHPLIHQWDQLODJDLQVWWUDQVLHQWIRFDOFHUHEUDOLVFKHPLDLQ IDFWRUNDSSD%DQGWXPRUSURJUHVVLRQ&DQFHU5HV ² UDWV-1HXURVXUJ$QHVWKHVLRO ² 7LDQ-*X<6XQ.:DQJ%&KHQ-:DQJ;, et al>'$OD'/HX@ /DQ]LOORWWD$6DUQLFR,,QJUDVVLD5%RURQL)%UDQFD&%HQDUHVH HQFHSKDOLQ '$'/( UHYHUVLEO\LQKLELWVFHOOXODUWUDQVFULSWLRQLQQHXURQV M, et al7KHDFHW\ODWLRQRI5HO$LQ/\VGLFWDWHVWKH1)NDSSD% ZLWKRXWFDXVLQJFHOOLQMXU\%UDLQ5HV ² GHSHQGHQWUHVSRQVHLQSRVWLVFKHPLFLQMXU\&HOO'HDWK'LV :DQJ=7DQJ%7DQJ)/L<=KDQJ*=KRQJ/, et al3URWHFWLRQRIUDW H LQWHVWLQDOHSLWKHOLDOFHOOVIURPLVFKHPLDUHSHUIXVLRQLQMXU\E\ '$OD +XDQJ;4LQ-/X6.DQJODLWHVWLPXODWHVDQWLFDQFHULPPXQH '/HX HQNHSKDOLQWKURXJKLQKLELWLRQRIWKH0..-1.VLJQDOLQJ UHVSRQVHVDQGLQKLELWV+HS*FHOOWUDQVSODQWDWLRQLQGXFHGWXPRU SDWKZD\0RO0HG5HS ² JURZWK0RO0HG5HS ² 6HQ'+XFKLWDO0&KHQ Acta Pharmacologica Sinica