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1 대한안과학회지 2018 년제 59 권제 2 호 J Korean Ophthalmol Soc 2018;59(2): ISSN (Print) ISSN (Online) Original rticle 미세먼지노출이안구조직과경부림프절에서 NLRP3 인플라마좀발현에미치는효과 Effect of Particulate Matter on the NLRP3 Inflammasomes in Ocular Tissues and Cervical Lymph Nodes 강수연 강보람 김효명 송종석 Su-Yeon Kang, MD, PhD, oram Kang, MS, Hyo Myung Kim, MD, PhD, Jong-Suk Song, MD, PhD 고려대학교의과대학안과학교실 Department of Ophthalmology, Korea University College of Medicine, Seoul, Korea Purpose: To evaluate changes of the NLRP3 inflammasome complex and proinflammatory cytokine interleukin (IL)-1β in ocular tissues and cervical lymph nodes when the ocular surface was exposed to particulate matter. Methods: Five-week-old Sprague Dawley male rats, each weighing g, were used in this study. The rats were randomly divided into two groups involving the TiO 2-exposed group (n = 12) and control group (n = 12). Rats in the TiO 2-exposed group were exposed to airborne TiO 2 microparticles in the exposure chamber twice daily for 2 hours for 5 days. The mean particulate matter (PM) 10 concentration in the exposure chamber was 331 ± 83 µg/m 3. The score of corneal fluorescein staining for evaluating ocular surface damage and the size of cervical lymph nodes for evaluating the immune response in the lymphatic drainage pathway were measured. The expression of the nucleotide-binding domain leucine-rich repeats, pyrin domain-containing 3 (NLRP3) inflammasome complex (NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CRD) domain [SC], and caspase-1) and IL-1β was quantitatively evaluated. Results: In the TiO 2-exposed group, the mean corneal stain score was significantly increased compared to that of the control group (p = 0.002), and the size of the cervical lymph nodes in the TiO 2-exposed group was greater than that of the control group (p = 0.004). The expression of NLRP3 and SC was significantly increased in the corneo-conjunctival tissue and cervical lymph nodes after TiO 2 exposure (both, p = 0.037). Inactive precursor forms of caspase-1 and IL-1β were decreased, but active forms of caspase-1 and IL-1β were increased after TiO 2 exposure (both, p = 0.037). Conclusions: When the ocular surface was exposed to ambient particulate matter, the signal pathway of the NLRP3 inflammasome complex was activated, resulting in increased expression of the proinflammatory cytokine, IL-1β. The results suggested that the NLRP3 inflammasome complex was involved in the pathogenesis of ocular surface inflammation caused by exposure to particulate matter. J Korean Ophthalmol Soc 2018;59(2): Keywords: Dry eye, IL-1β, NLRP3 inflammasome, Particulate matter Received: Revised: ccepted: ddress reprint requests to Jong-Suk Song, MD, PhD Department of Ophthalmology, Korea University Guro Hospital, #148 Gurodong-ro, Guro-gu, Seoul 08308, Korea Tel: , Fax: crisim@korea.ac.kr * This study was presented as a narration at the 115th nnual Meeting of the Korea Ophthalmological Society * This study was supported in part by lumni of Department of Ophthalmology, Korea University College of Medicine. * Conflicts of Interest: The authors have no conflicts to disclose. 우리나라대기오염과관련하여최근가장문제가되는것은미세먼지이다. 미세먼지는일반적으로미세먼지입자의크기에따라분류하는데 10 μm 이하의미세먼지를 PM10으로그보다적은 2.5 μm 이하의미세먼지는 PM2.5 로정의하고있다. 이러한미세먼지에노출되는경우호흡기질환은물론심혈관계질환을유발하며사망률도증가시키는것으로보고되고있다. 1-3 미세먼지는호흡기를통해인체로들어오는것만주로생각하지만안구표면도미세먼 c2018 The Korean Ophthalmological Society This is an Open ccess article distributed under the terms of the Creative Commons ttribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 130

2 - 강수연외 : 티타늄디옥사이드노출과인플라마좀 - 지에노출되는점막조직으로반복해서노출되는경우안구표면의손상과함께염증반응을유발하게된다. 4-6 Nucleotide-binding domain leucine-rich repeats, pyrin domain-containing 3 (NLRP3) 는유형인식수용체 (pattern recognition receptor) 와연관된신호전달체계에서중요한역할을하는물질로선천면역반응에관여한다. 7 감염이나손상에의해 NLRP3가활성화되면 apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CRD) domain (SC) 과결합하게되고, 그다음불활성상태의 caspase-1 전구체와결합하여 NLRP3 인플라마좀을형성하게된다. 그후 caspase-1 전구체는활성화되어불활성상태의 IL-1β 전구체를분해하여활성화시키며세포에서분비된 IL-1β는급성염증반응을일으키게된다. 8,9 건성안동물모델에서 NLRP3 인플라마좀발현이증가하고활성화된 IL-1β가증가하는것으로보고되었고건성안환자의눈물에서도 IL-1β의농도가증가하는것으로알려져있다. 10,11 따라서본연구에서는안구표면이미세먼지에노출되었을때 NLRP3 인플라마좀복합체가염증반응에관여하는지알아보고자안구조직과함께경부림프절에서 NLRP3 인플라마좀복합체와염증사이토카인인 IL-1β의발현이어떻게변화하는지알아보고자하였다. 대상과방법 5주된 Sprague-Dawley 수컷쥐 ( 무게, g) 24마리를대상으로하였다. 쥐는무작위로티타늄디옥사이드마이크로입자에노출시킨실험군 (n=12) 과정상대조군 (n=12) 으로나누어진행하였다. 모든실험과정은 the ssociation for Research in Vision and Ophthalmology (RVO) 동물실험규정을준수하였다. 전신마취는염산싸이라진 (Rompun 2%; ayer, Leverkusen, Germany) 과졸레틸 (Virbac Laboratories, Carros, France) 을근육주사하여각막염색이나안락사전시행하였다. 티타늄디옥사이드마이크로입자노출미세먼지를대기에서노출할수있도록특수제작된챔버를이용하여티타늄디옥사이드마이크로입자를안구표면에노출시켰다. 티타늄디옥사이드마이크로입자 ( 평균직경, µm) 는독일의 Johnson Matthey 회사에서구입하였고노출농도를조절하여 MetOne831 erosol Mass Monitor (Met One, Grants Pass, OR, US) 로측정한 PM10 의농도가평균 331 ± 83 µg/m 3 가되는환경에서쥐를 5일간하루 2시간씩 2회노출시켰다. PM10 농도가 331 µg/m 3 인환경에하루에 4시간노출시키는것은하루평균 PM10 농도 가대략 55 µg/m 3 로이수치는 World Health Organization (WHO) 에서하루평균권고기준인 50 µg/m 3 와유사한수치이며, 실제로우리나라에서안내하는미세먼지의나쁨상태는미세먼지 PM10 기준으로 80 µg/m 3 이상일때로실제우리가일상생활에서노출될수있는정도의미세먼지농도이다. 각막형광염색마지막티타늄디옥사이드노출 24시간후전신마취를시킨상태에서각막형광염색점수를양쪽각막에서 National Eye Institute (NEI) 평가기준에따라점수를매기고양쪽눈의평균값을이용하여통계분석을시행하였다. 시행방법은상품화된형광염색종이 (Haag-Streit, ern, Switzerland) 에 5 µl의생리식염수를묻힌후쥐의위눈꺼풀을젖히고형광염색액을안구결막에묻힌뒤쥐의눈을 5회손으로깜박거리게한후휴대용세극등현미경을이용하여각막염색점수를평가하였다. 경부림프절적출및크기측정실험군 6마리와대조군 6마리를마취한후이산화탄소챔버에서안락사시킨후경부절개를통해경부림부절을노출시켰다. 좌우경부림프절중가장큰림프절을절제하여사진을찍은후 ImageJ 프로그램 (1.4u; provided in the public domain by the National Institutes of Health, ethesda, MD, US) 을이용하여림프절면적을측정한후대조군에비해실험군에서림프절의크기가얼마나증가했는지평가하였다. NLRP3 인플라마좀복합체의발현각결막안구조직에서 NLRP3 인플라마좀복합체의발현을정량적으로평가하기위해쥐의양쪽안구의전안부를절제한후단백질분해효소억제제가들어있는 T-per tissue protein extraction reagent (Thermo Scientific, Rockford, IL, US) 에넣어균질화시킨후추출액을이용하여 (n=3 for each group) NLRP3, SC, pre-caspase-1/caspase-1, preil-1 β/il-1β 에대한 western blot 분석을시행하였다. 또한경부림프절에서의변화를평가하기위해제거한경부림프절을균질화시킨후 NLRP3 인플라마좀복합체각각에대한 western blot 분석을시행하였다. 일차항체는상품화되어있는제품을구입하였으며각각의희석배수와구입처는다음과같다 : NLRP3 (1:1,000, NP , Novus iologicals, Littleton, CO, US), SC (1:1,000, sc r, Santa Cruz iotechnology, Dallas, TX, US), Caspase-1 (1:1,000, sc-514, Santa Cruz iotechnology, Dallas, TX, US), IL-1β (1:1,000, sc-7884, Santa Cruz iotechnology, Dallas, TX, US), β-actin 131

3 - 대한안과학회지 2018 년제 59 권제 2 호 - Figure 1. The mean corneal staining scores of the control and TiO 2-exposed groups were compared. () Representative photographs of rat eyes after fluorescein staining in both groups. () comparison of the mean corneal staining scores between the two groups. n asterisk (*) indicates a p-value < 0.05 by the Mann-Whitney U test (p = 0.002). Standard deviations are indicated by error bars. Figure 2. The mean relative size of cervical lymph nodes in the control and TiO 2-exposed groups were compared. () Representative photographs of cervical lymph nodes in both groups. () comparison of cervical lymph node size between the two groups. n asterisk (*) indicates a p-value < 0.05 by the Mann-Whitney U test (n = 6, each group). Standard deviations are indicated by error bars. LN = lymph node. (1:20,000, #5125, Cell Signal Technology, oston, M, US). 2차항체는 horseradish peroxidase (HRP) linked anti-rabbit immunoglobulin G (IgG) (#7074, Cell Signal Technology, oston, M, US) 를이용하였다. 통계분석은 SPSS version 12.0 (IM Corp., rmonk, NY, US) 의 Mann-Whitney U-test를이용하였고, western blot에서는 β-actin에대한비율로표시하였다. 통계결과는 p값이 0.05 미만인경우유의하다고정의하였다. 씩 5일간노출한후실험군 (n=6) 의형광염색점수는중앙값 3.25 [ ] 로대조군 (n=6) 의중앙값 0.75 [ ] 보다유의하게높게평가되었다 (p=0.002) (Fig. 1). 경부림프절의크기경부림프절크기는대조군 (n=6) 과비교하여티타늄디옥사이드에노출된실험군 (n=6) 에서약 1.56배크기가증가하였고통계적으로도유의한차이를보였다 (p=0.004) (Fig. 2). 결과 각막형광염색티타늄디옥사이드마이크로입자에하루에 2시간씩 2회 NLRP3 인플라마좀복합체의발현각결막조직에서시행한 western blot 검사에서 NLRP3 와 SC의발현은대조군 (n=3) 에비해티타늄디옥사이드에노출된실험군 (n=3) 에서통계적으로유의하게증가하였다 132

4 - 강수연외 : 티타늄디옥사이드노출과인플라마좀 - Figure 3. The expression of nucleotide-binding domain leucine-rich repeats, pyrin domain-containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CRD) domain (SC) in the corneo-conjunctival tissues in the control and TiO 2-exposed groups (n = 3, each group). () representative example of a Western blot gel image and densitometry analysis for NLRP3. () representative example of a Western blot gel image and densitometry analysis for SC. β-ctin was used as an internal loading control for the Wetern blot. n asterisk (*) indicates a significant difference (p-value < 0.05) in the expression of NLRP3 and SC in the TiO 2-exposed group compared with the control group by the Mann-Whitney U test. Standard deviations are indicated by error bars. Figure 4. The expression of inactive precursor and active forms of caspase-1 and interleukin (IL)-1β in the corneo-conjunctival tissues in the control and TiO 2-exposed groups (n = 3, each group). () representative example of a Western blot gel image and densitometry analysis for pro-caspase-1 and caspase-1. () representative example of a Western blot gel image and densitometry analysis for pro-il-1β and IL-1β. β-ctin was used as an internal loading control for the Wetern blot. n asterisk (*) indicates a significant difference (p-value < 0.05) in the expression of inactive precursor and active forms of caspase-1 and IL-1β in the TiO 2-exposed group compared with the control group by the Mann-Whitney U test. Standard deviations are indicated by error bars. 133

5 - 대한안과학회지 2018 년제 59 권제 2 호 - Figure 5. The expression of nucleotide-binding domain leucine-rich repeats, pyrin domain-containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CRD) domain (SC) in the cervical lymph nodes in the control and TiO 2-exposed groups (n = 3, each group). () representative example of a Western blot gel image and densitometry analysis for NLRP3. () representative example of a Western blot gel image and densitometry analysis for SC. β -ctin was used as an internal loading control for the Wetern blot. n asterisk (*) indicates a significant difference (p-value < 0.05) in the expression of NLRP3 and SC in the TiO 2-exposed group compared with the control group by the Mann-Whitney U test. Standard deviations are indicated by error bars. Figure 6. The expression of inactive precursor and active forms of caspase-1 and interleukin (IL)-1β in the cervical lymph nodes in the control and TiO 2-exposed groups (n = 3, each group). () representative example of a Western blot gel image and densitometry analysis for pro-caspase-1 and caspase-1. () representative example of a Western blot gel image and densitometry analysis for pro-il-1βand IL-1β. β-ctin was used as an internal loading control for the Wetern blot. n asterisk (*) indicates a significant difference (p-value < 0.05) in the expression of inactive precursor and active forms of caspase-1 and IL-1β in the TiO 2-exposed group compared with the control group by the Mann-Whitney U test. Standard deviations are indicated by error bars. 134

6 - 강수연외 : 티타늄디옥사이드노출과인플라마좀 - (both, p=0.037) (Fig. 3). Caspase-1의경우대조군 (n=3) 에비해티타늄디옥사이드에노출된실험군 (n=3) 에서 45 kda 의 caspase-1 전구체의발현은감소하였으나 10 kda인활성화된 caspase-1의발현은증가하였다 (p=0.037) (Fig. 4). NLRP3 인플라마좀의최종산물인 IL-1β의경우도대조군 (n=3) 에비해티타늄디옥사이드에노출된실험군 (n=3) 에서 31 kda의 IL-1β 전구체의발현은감소하였으나 17 kda인활성화 IL-1β의발현은유의하게증가하였다 (p=0.037) (Fig. 4). 경부림프절에서시행한 western blot 검사에서도각결막조직에서와동일하게 NLRP3와 SC의발현은대조군 (n=3) 에비해티타늄디옥사이드에노출된실험군 (n=3) 에서통계적으로유의하게증가하였고 (both, p=0.037) (Fig. 5), caspase-1과 IL-1β도불활성상태인전구체의발현은감소하였으나활성화상태인 caspase-1과 IL-1β의발현은대조군에비해유의하게증가하였다 (both, p=0.037) (Fig. 6). 고찰 티타늄디옥사이드마이크로입자는미세한크리스탈파우더로페이트, 화장품, 직물등에사용되고있으며미세먼지에의한영향을평가하기위해호흡기질환연구에사용되는물질이다. 12 기관지세포가티타늄디옥사이드에노출될경우뮤신을분비하고염증반응을유발하여다양한호흡기질환을유발하거나악화시키는것으로알려져있다 이전저자들의연구에서안구표면에티타늄디옥사이드가반복해서노출되었을경우안구표면의손상과함께눈물내 lactate dehydrogenase (LDH) 의활성도가증가하였고, 이러한현상은건성안유발쥐에서더유의하게증가하였다 이는안구표면의보호작용을하는눈물분비가감소한경우동일한농도의미세먼지에노출되어도안구표면의손상이건성안환자에서더심해질수있다는것을뒷받침한다. 이번실험에서는미세먼지에의해유발되는안구표면의염증반응에 NLRP3 인플라마좀복합체가관여하는지알아보고자하였다. 각결막조직을이용한 western blot 검사에서 NLRP3 신호전달체계에관여하는 NLRP3와 SC의발현은대조군에비해유의하게증가하였으며, caspase-1과 IL-1β의경우불활성상태인전구체의양은감소하였으나활성화형태의발현은대조군에비해유의하게증가하여미세먼지에노출된경우 NLRP3 인플라마좀복합체의신호전달체계에의해주요염증사이토카인인 IL-1β의생성이증가하여안구표면에염증이유발되는것을확인하였다. 이러한 NLRP3 인플라마좀복합체의증가는미세먼지에노출된안구조직뿐아니라크기가증가한경부림프절에서 도동일하게관찰되었다. 건성안유병률연구를보면한국, 일본, 중국, 대만등아시아국가의건성안유병률이서양국가에비해높아아시아인종이건성안의위험인자중하나로분류되고있다. 19,20 이러한연구결과는인종에따른차이일수도있지만아시아국가의대기오염상태가심하고특히황사에영향을받는동북아시아의지정학적위치도영향을끼칠수있다고생각된다. 건성안동물모델에서 NLRP3 인플라마좀신호전달체계가관여하여 IL-1β의분비를증가시키므로염증반응을유발한다는이전연구보고가있다. 10,11 따라서이미건성안으로유발된안구표면의염증반응에미세먼지노출이동반된다면안구표면의염증반응은더심해지고그에따라손상이더심해지리라생각된다. 미세먼지에대한노출은비단호흡기계에서만일어나는것이아니라안구표면도미세먼지노출에따른손상이발생하며안구표면손상뿐아니라경부림프절을통해전신적으로도염증반응을유발할수있으므로마스크를착용하듯미세먼지가심한경우보호안경을착용하여미세먼지노출을최소화하는노력이필요하다고생각된다. 본연구에서는안구표면이티타늄디옥사이드마이크로입자에노출되었을때각결막등안구조직은물론경부림프절에서 NLRP3 인플라마좀복합체의신호전달체계가활성화되어 IL-1β 발현이증가하는것을처음으로확인하였다. 이러한현상을통해유추해볼때미세먼지에안구표면이만성적으로노출될경우염증반응을유발하여건성안을일으키거나악화시킬가능성이있으며이러한상호작용에대해서는추가적인연구가필요하리라생각된다. REFERENCES 1) rook RD, Rajagopalan S, Pope C 3rd, et al. Particulate matterair pollution and cardiovascular disease: an update to the scientific statement from the merican Heart ssociation. Circulation 2010;121: ) tkinson RW, nderson HR, Sunyer J, et al. cute effects of particulate air pollution on respiratory admissions: results from PHE 2 project. ir Pollution and Health: a European pproach. m J Respir Crit Care Med 2001;164(10 Pt 1): ) Samoli E, nalitis, Touloumi G, et al. Estimating the exposure-response relationships between particulate matter and mortality within the PHE multicity project. Environ Health Perspect 2005;113: ) Novaes P, Saldiva PH, Matsuda M, et al. The effects of chronic exposure to traffic derived air pollution on the ocular surface. Environ Res 2010;110: ) Malerbi FK, Martins LC, Saldiva PH, raga L. mbient levels of air pollution induce clinical worsening of blepharitis. Environ Res 2012;112:

7 - 대한안과학회지 2018 년제 59 권제 2 호 - 6) Galor, Kumar N, Feuer W, Lee DJ. Environmental factors affect the risk of dry eye syndrome in a United States veteran population. Ophthalmology 2014;121: ) Eisenbarth SC, Flavell R. Innate instruction of adaptive immunity revisited: the inflammasome. EMO Mol Med 2009;1: ) He Y, Hara H, Núñez G. Mechanism and regulation of NLRP3 inflammasome activation. Trends iochem Sci 2016;41: ) Jo EK, Kim JK, Shin DM, Sasakawa C. Molecular mechanisms regulating NLRP3 inflammasome activation. Cell Mol Immunol 2016;13: ) Zheng Q, Ren Y, Reinach PS, et al. Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye. Exp Eye Res 2014;125: ) Zheng Q, Ren Y, Reinach PS, et al. Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients. Exp Eye Res 2015;134: ) Warheit D, Sayes CM, Reed KL, Swain K. Health effects related to nanoparticle exposures: environmental, health and safety considerations for assessing hazards and risks. Pharmacol Ther 2008;120: ) Sun Q, Tan D, Ze Y, et al. Pulmotoxicological effects caused by long-term titanium dioxide nanoparticles exposure in mice. J Hazard Mater 2012; : ) Chang X, Fu Y, Zhang Y, et al. Effects of Th1 and Th2 cells balance in pulmonary injury induced by nano titanium dioxide. Environ Toxicol Pharmacol 2014;37: ) Hong J, Wang L, Zhao X, et al. Th2 factors may be involved in TiO2 NP-induced hepatic inflammation. J gric Food Chem 2014;62: ) Eom Y, Song JS, Lee DY, et al. Effect of titanium dioxide nanoparticle exposure on the ocular surface: an animal study. Ocul Surf 2016;14: ) Eom Y, Song JS, Lee HK, et al. The effect of ambient titanium dioxide microparticle exposure to the ocular surface on the expression of inflammatory cytokines in the eye and cervical lymph nodes. Invest Ophthalmol Vis Sci 2016;57: ) Han JY, Kang, Eom Y, et al. Comparing the effects of particulate matter on the ocular surfaces of normal eyes and a dry eye rat model. Cornea 2017;36: ) The epidemiology of dry eye disease: report of the Epidemiology Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf 2007;5: ) Stapleton F, lves M, unya VY, et al. TFOS DEWS II Epidemiology Report. Ocul Surf 2017;15: = 국문초록 = 미세먼지노출이안구조직과경부림프절에서 NLRP3 인플라마좀발현에미치는효과 목적 : 안구표면이미세먼지에노출되었을때안구조직과경부림프절에서 nucleotide-binding domain leucine-rich repeats, pyrin domain-containing 3 (NLRP3) 인플라마좀복합체와주요염증사이토카인인 interleukin (IL)-1β 의발현이어떻게변화하는지알아보고자하였다. 대상과방법 : 5 주된 Sprague-Dawley 수컷쥐 ( 무게, g) 를대상으로하였다. 쥐는무작위로공기중에서티타늄디옥사이드마이크로입자에노출시킨실험군 (n=12) 과정상대조군 (n=12) 으로나누어진행하였다. 티타늄디옥사이드마이크로입자에의해지름 10 마이크로미터이하의미세먼지농도 (particulate matter [PM] 10) 가평균 331 ± 83 μg/m 3 가되는환경에서쥐를하루 2 시간씩 2 회, 5 일간노출시켰다. 안구표면의손상을평가하기위해각막형광염색을시행하였고, 염증반응이국소림프절에전달되는지평가하기위해경부림프절크기를측정하였으며각결막조직과경부림프절에서 NLRP3 인플라마좀복합체의발현을정량적으로평가하였다. 결과 : 티타늄디옥사이드마이크로입자에노출시킨실험군에서대조군에비해각막형광염색점수가유의하게증가하였고 (p=0.002) 경부림프절의크기도증가하였다 (p=0.004). 각결막조직과경부림프절에서 NLRP3 신호전달체계에관여하는 NLRP3 와 apoptosisassociated speck-like protein containing a caspase activation and recruitment domain (CRD) domain (SC) 의발현은대조군에비해유의하게증가하였으며 ( 각각, p=0.037), caspase-1 과 IL-1β 의경우불활성상태인전구체의양은감소하였으나활성화형태의발현은대조군에비해유의하게증가하였다 ( 각각, p=0.037). 결론 : 안구표면이미세먼지에노출되었을때 NLRP3 인플라마좀복합체신호전달체계가활성화되어염증사이토카인인 IL-1β 의생성이증가하였으며이러한기전이미세먼지에의한안구표면염증반응에관여하는것으로판단된다. < 대한안과학회지 2018;59(2): > 136

012임수진

012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar