Hepatoprotective effect of manual acupuncture at acupoint GB34 against CCl4-induced chronic liver damage in rats.

Transcription

1 PO Box 234, Beijing 123, Chin World J Gstroenterol 26 April 14; 12(14): World Journl of Gstroenterology ISSN wjg@wjgnet.com 26 The WJG Press. All rights reserved. RAPID COMMUNICATION Heptoprotective effect of mnul cupuncture t cupoint ginst CCl4-induced chronic liver dmge in rts Yun-Kyoung Yim, Hyun Lee, Kwon-Eui Hong, Young-Il Kim, Byung-Ryul Lee, Te-Hn Kim, Ji-Young Yi Yun-Kyoung Yim, Te-Hn Kim, Ji-Young Yi, Deprtment of Meridin & Acupoint, College of Orientl Medicine, Dejeon University, #1247, 96-3, Yongun-dong, Dong-gu, Dejeon 3-716, South Kore Hyun Lee, Kwon-Eui Hong, Young-Il Kim, Byung-Ryul Lee, Deprtment of Acupuncture & Moxiustion, Dunsn Orientl Hospitl of Dejeon University, 1136 Dunsn-dong, Seo-gu, Dejeon , South Kore Correspondence to: Professor. Yun-Kyoung Yim, OMD, PhD, Deprtment of Meridin d Acupoint, College of Orientl Medicine, Dejeon University, #1247, 96-3, Yongun-dong, Dong-gu, Dejeon 3-716, South Kore. docwindy@dju.c.kr Telephone: Fx: Received: Accepted: Astrct AIM: To investigte the heptoprotective effect of mnul cupuncture t Ynglingqun () on CCl4- induced chronic liver dmge in rts. METHODS: Rts were injected intrperitonelly with CCl4 (1 ml/kg) nd treted with mnul cupuncture using reinforcing mnipultion techniques t left (Ynglingqun) 3 times week for 1 wk. A noncupoint in left glutel re ws selected s shm point. To estimte the heptoprotective effect of mnul cupuncture t, mesurement of liver index, iochemicl ssys including serum ALT, AST, ALP nd totl cholesterol, histologicl nlysis nd lood cell counts were conducted. RESULTS: Mnul cupuncture t reduced the liver index, serum ALT, AST, ALP nd totl cholesterol levels s compred with the control group nd the shm cupuncture group. It lso incresed nd normlized the popultions of WBC nd lymphocytes. CONCLUSION: Mnul cupuncture with reinforcing mnipultion techniques t left reduces liver toxicity, protects liver function nd liver tissue, nd normlizes immune ctivity in CCl4-intoxicted rts. 26 The WJG Press. All rights reserved. Key words: Mnul cupuncture; Ynglingqun (); CCl4-induced liver dmge; Heptoprotective effect Yim YK, Lee H, Hong KE, Kim YI, Lee BR, Kim TH, Yi JY. Heptoprotective effect of mnul cupuncture t cupoint ginst CCl4-induced chronic liver dmge in rts. World J Gstroenterol 26; 12(14): INTRODUCTION Herl medicine nd cupuncture re the two min methods to tret disese in orientl medicine. Becuse of chemicl residue contmintion, there is recent gining suspicion tht hers my e hrmful to the liver. Accordingly, cupuncture is getting more interest these dys for the tretment of liver diseses in orientl medicl clinics. In the present study, we tried to investigte the effects of mnul cupuncture on long-term liver dmge. To investigte the effects of mnul cupuncture t on liver dmge, we used CCl4-intoxicted rt model nd chose s n cupoint to protect liver nd tret liver dmge induced y CCl4 dministrtion. is n cupoint locted on the gll ldder meridin. In orientl medicl theory, the liver nd gll ldder corresponds to ech other nd their meridins re lso closely relted with ech other in the interior-exterior reltionship [1,2]. Gll ldder meridin pertins to the gll ldder orgn nd connects with the liver orgn. The liver meridin pertins to the liver orgn nd connects with the gll ldder orgn. Therefore, the cupoints on the liver meridin re used to tret gll ldder orgn diseses s well s liver orgn diseses. Consequently, the ucpoints on the gll ldder meridin re used to tret liver orgn diseses s well s gll ldder orgn diseses. Hence, is closely relted with the liver s well s the gll ldder. It explins why is used to tret liver disese so often. Moreover, is the He (mening se ) point of gll ldder. In orientl medicl theory, se He point is considered to e the entrnce of the meridin energy to the corresponding orgn [3]. Therefore, influences the liver nd gll ldder more strongly thn other upoints. The functions of this point re regulting nd tonifying the liver, regulting the gllldder, spreding liver Qi (orientl medicl term for vitl energy ), suduing liver Yng, drining liver pthogens, etc. hs een cliniclly used for hypochondric pin, jundice, heptitis, cute iliry trct diseses, cirrhosis of the liver nd hypertension due to liver Yng excess, etc [1,4]. We presume tht neuronl ctivity is involved in the trnsmission of cupuncture stimultion, so the nimls were not nesthetized during the cupuncture dministr-

2 2246 ISSN CN / R World J Gstroenterol April 14, 26 Volume 12 Numer 14 Hold for 1 s nd Restriction within cge 3 s Nine rottions of needles (clockwise with mplitude of 9 ) Nine scrping downwrds Figure 2 Acupuncture scheme. Figure 1 nd shm point for cupuncture. tion. To keep the nimls from moving during the cupuncture dministrtion, they were put in cges with five holes for til nd four lims. To estimte nd exclude the effect of stress from restriction within the cge, the rts in the control group were lso kept in the cges in the sme mnner s the cupuncture group. The ction of cupuncture could e influenced y cupuncture techniques s well s point selection. There re two min ctegories of cupuncture techniques: reinforcing technique nd reducing technique. Clockwise needle rottion, scrping downwrd of needles, nd odd numer of mnipulting opertions re considered s reinforcing techniques. On the other hnd, counterclockwise needle rottion, scrping upwrd of needles, nd even numer of mnipulting opertions re considered s reducing techniques. Reinforcing cupuncture mnipultion techniques re used for chronic nd deficient syndrome, while reducing techniques re used for cute nd excess syndrome [1]. Since the nimls in the present study were injected with CCl4 for long period, we considered it s chronic nd deficient condition, nd therefore, we dministered cupuncture with reinforcing mnipultion techniques. MATERIALS AND METHODS Animls Sprgue-Dwley mle rts (2-2 g) were purchsed from Dehn Biorink Co. The nimls were dpted to the environment of 22 ± 2 room temperture, 12-h light/ drk cycle for 2 wk nd hd free ccess to wter nd food. Our niml experiment hs een conducted in ccordnce with the Use of Lortory Animls s dopted nd promulgted y the U.S. Ntionl Institutes of Helth. Experimentl design Experimentl nimls were rndomly divided into four groups: norml; control (CCl4); (CCl4 + mnul cupuncture t shm point); nd (CCl4 + mnul cupuncture t left ). Ech group consisted of 7 rts. Liver injury ws induced y intrperitonel injections with ml/l CCl4 (Sigm, USA) solution in olive oil (1 ml/kg), twice week for 1 wk. Mnul cupuncture ws dministered 3 times week during the sme period []. Acupuncture Cges with five holes for the til nd four lims were mnufctured for this study. During cupuncture dministrtion, the nimls were kept in the cge with left hind lim fstened to the wll of the cge with tpe. Sterilized disposle stinless steel needles (.2 mm 3 mm, Dongng Acupuncture Inc., Kore) were inserted perpendiculrly s deep s 2-3 mm t left or shm point. Rt ws determined ccording to humn which loctes on gll ldder meridin, in the depression nterior nd inferior to the hed of the fiul [1]. A non-cupoint in the glutel region ws selected s shm point (Figure 1). After the needles were inserted, they were rotted clockwise with mplitude of 9 nine times, nd scrped downwrd nine times. Nine rottions nd nine scrpes constituted one mnipultion unit, nd three mnipultion units constituted one tretment session. After ech mnipultion unit, there ws hold for out 1 s. The full mnipultion took out 3 s. The rts in the control group were kept in cge for 3 s with the left hind lim tied without cupuncture tretment (Figure 2). All cupuncture ws dministered y trined nd experienced orientl medicl doctor who ws unknown out the reserch protocol except the needle mnipulting methods. Liver index mesurement Rt s ody weight ws mesured efore the nimls were scrificed. Rt liver ws removed nd weighed right fter the niml ws scrificed nd the liver index (% of liver weight/ody weight) [6,7] ws estimted. Biochemicl nlysis nd lood counts Forty-eight hours fter the lst dministrtion with CCl4, the rts were nesthetized with ethyl ether nd lood smples were tken from the hert. Blood ws centrifuged t 3 r/min for 1 min nd serum ws tken. Alnine minotrnsferse (ALT), sprtte minotrnsferse (AST), lkline phosphotse (ALP), totl cholesterol in serum nd the popultions of RBC, WBC, lymphocytes in plsm were detected. Histologicl nlysis The rts were scrificed nd the liver tissues were otined individully from ech group nd fixed in 4 g/l formldehyde. After declcifiction in ml/l formic cid, the specimens were processed for prffin emedding. Tissue sections were otined nd stined with hemtoxylin nd eosin (HE) or msson s trichrome (MT). Tissue destruction nd ftty chnges of liver were oserved t 4 mgnifiction.

3 Yim YK et l. Heptoprotective effect of cupuncture t 2247 % of Liver/Body weight c Norml A ALT level (nk t/l) ALT Figure 3 Effect of mnul cupuncture t BG34 on liver index of CCl4-intoxicted rts. P <., P <.1 vs norml group; c P <. vs control group. Norml B AST 12 Sttisticl nlysis Dt were otined from the rts which survived to the end of the experiment. All in norml group, 3 in control group, 3 in shm group nd in group survived to the end of the experiment. Dt were expressed s men ± SD. Sttisticl significnce of difference etween groups ws determined using ANOVA, followed y t-test. P <. ws considered sttisticlly significnt. AST level (nk t/l) Norml RESULTS Liver index CCl4 injection induced significnt increse in liver index. On the other hnd, mnul cupuncture t lowered it similr to the norml vlue (Figure 3). Serum ALT, AST, ALP nd totl cholesterol ALT, AST, ALP nd totl cholesterol in serum were incresed remrkly y long-term CCl4 dministrtion, indicting dmge to the liver. Mnul cupuncture t significntly reduced serum ALT, AST nd totl cholesterol in comprison with the control group. Serum ALP ws lso reduced y mnul cupuncture t ut no sttisticl significnce ws found (Figure 4). Blood cell counts The numer of RBC ws slightly reduced y CCl4 intoxiction nd restored y mnul cupuncture t, though sttisticl significnce ws not oserved. The numer of WBC nd the percentge of lymphocytes out of WBC ws significntly reduced y CCl4 intoxiction nd restored y mnul cupuncture t close to the norml level (Figure ). Liver histology Histologicl nlysis using HE stin showed tht necrosis of liver tissue nd ftty chnges were viciously induced y CCl4 dministrtion. The group treted with mnul cupuncture t showed reduced feture of heptocyte necrosis nd ftty chnge compred to the control group nd the shm group. In ddition, MT stining showed lower ccumultion of extrcellulr mtrix in the group compred to the control group nd shm group (Figure 6). C ALP level (nk t/l) D Totl cholesterol (nmol/l) ALP Totl cholesterol DISCUSSION Norml Norml Figure 4 Effect of mnul cupuncture t on serum ALT, AST, ALP nd totl cholesterol of CCl4-intoxicted rts. P <.1 vs norml group; P <. vs control group CCl4 hs een widely used to induce experimentl heptic

4 2248 ISSN CN / R World J Gstroenterol April 14, 26 Volume 12 Numer 14 A RBC ( 1 11 /L) WBC ( 1 9 /L) RBC WBC c B Lymphocyte (%/WBC) d Norml Norml Figure Effect of mnul cupuncture t on RBC, WBC nd lymphocytes in lood. P <., P <.1 vs norml group; c P <., d P <.1 vs control group. HE MT Norml Norml Figure 6 Histologicl results of liver tissue. Necrosis nd ftty chnge could e found in the control group (top). group reveled lower ccumultion of extrcellulr mtrix compred to the control group nd shm group (ottom) ( 4 mgnifiction) dmge [8,9]. It induces liver cell necrosis nd poptosis, nd cn e used to induce heptic firosis or cirrhosis y repetitive dministrtion [1-12]. Liu et l [13] investigted the effect of mnul cupuncture t ST36 nd LR3 on CCl4- induced cute liver dmge. In the present study, we investigted the effect of mnul cupuncture t on chronic liver dmge induced y long-term CCl4 dministrtion. ALT nd AST re the specific mrkers to ssess heptocellulr dmge leding to liver cell necrosis [14]. Slight to moderte increses in ALP (1-2 times norml) occurred in liver disorders [1]. Serum cholesterol is one of the generl indictions of the synthetic nd generl metolic cpcity of the liver [16]. In the present study, CCl4 injection significntly incresed serum ALT, AST, ALP nd cholesterol levels, indicting induction of heptic dmge. Mnul cupuncture t inhiited the increses of these prmeters, indicting tht mnul cupuncture t protected liver nd reduced liver toxicity. Histologicl nlysis lso showed tht the cupuncture t protected liver tissue ginst CCl4-intoxiction. Our histologicl nlysis in this study ws only qulittive. Quntittive ssessment of nti-firotic effect of cupuncture, such s determintion of heptic hydroxyproline content, would e meningful in the next study. In the present study, long-term CCl4 dministrtion reduced the popultion of leucocytes nd lymphocytes in lood. We infer tht this reduction ws due to the decline of immune ctivity y long-term liver dmge. Mnul cupuncture t recovered the popultion of leucocytes nd lymphocytes in lood. Therefore, we presume tht mnul cupuncture t restored the immune ctivity, which is lso in greement with Hu s report on the white cell increse y cupuncture [17]. However, the effect of cupuncture on inflmmtion in liver is not cler yet. It is known tht inflmmtory cells re infiltrted in CCl4- intoxicted liver, nd there exists n normlity of cytokines in liver tissue. For instnce, TNF-α nd IL-1 re incresed, nd IL-2 nd IFN-γ re reduced in CCl4-intoxicted liver tissue [18,19]. More profound studies re required on how cupuncture influences the chronic inflmmtory conditions of the liver. Bsed on the results of the present study, we speculte tht mnul cupuncture t is eneficil to protect liver function nd tissue, reduce heptic toxicity nd normlize immune ctivity ginst CCl4-intoxiction in rts. The heptoprotective effect of mnul cupuncture t in this study my e relted to the immune reinforcing effect of cupuncture or neuro-immune interction on the pthwy of the trnsmission of cupuncture stimultion. REFERENCES 1 Deng L, Gn Y, He S, Ji X, Li Y, Wng R, Wng W, Wng X, Xu H, Xue X, Yun J. Chinese cupuncture nd moxiustion. Beijing: Foreign Lnguges Press, 1997: 4, 71-74, 28, Cptchuk TJ. The we tht hs no wever: understnding Chinese medicine. NY: Contemporry ooks, 2: 8 3 Choe YT, Yu CH, Kim CH, Kim DH. Orientl medicine series. Vol 2; Acupuncture & moxiustion. Seoul: Res Inst of Orientl Med Inc, 1987: 16 4 Lde A. Acupuncture points imges & functions. Wshington: Estlnd Press, 1998: 23 Ulicn O, Greksk M, Vncov O, Zltos L, Glvy S, Bozek P, Nkno M. Heptoprotective effect of rooios te (Asplthus lineris) on CCl4-induced liver dmge in rts. Physiol Res 23; 2: Yng Q, Xie RJ, Geng XX, Luo XH, Hn B, Cheng ML. Effect of

5 Yim YK et l. Heptoprotective effect of cupuncture t 2249 Dnsho Huxin cpsule on expression of mtrix metlloproteinse-1 nd tissue inhiitor of metlloproteinse-1 in firotic liver of rts. World J Gstroenterol 2; 11: Lin WC, Lin WL. Ameliortive effect of Gnoderm lucidum on cron tetrchloride-induced liver firosis in rts. World J Gstroenterol 26; 12: Tsukmoto H, Mtsuok M, French SW. Experimentl models of heptic firosis: review. Semin Liver Dis 199; 1: Yo XX, Jing SL, Tng YW, Yo DM, Yo X. Efficcy of Chinese medicine Yi-gn-kng grnule in prophylxis nd tretment of liver firosis in rts. World J Gstroenterol 2; 11: Constndinou C, Henderson N, Iredle JP. Modeling liver firosis in rodents. Methods Mol Med 2; 117: Chng ML, Yeh CT, Chng PY, Chen JC. Comprison of murine cirrhosis models induced y heptotoxin dministrtion nd common ile duct ligtion. World J Gstroenterol 2; 11: Shi GF, Li Q. Effects of oxymtrine on experimentl heptic firosis nd its mechnism in vivo. World J Gstroenterol 2; 11: Liu HJ, Hsu SF, Hsieh CC, Ho TY, Hsieh CL, Tsi CC, Lin JG. The effectiveness of Tsu-Sn-Li (St-36) nd Ti-Chung (Li-3) cupoints for tretment of cute liver dmge in rts. Am J Chin Med 21; 29: Amcher DE. Serum trnsminse elevtions s indictors of heptic injury following the dministrtion of drugs. Regul Toxicol Phrmcol 1998; 27: Isselcher KJ, Brunwld E, Wilson JD, Mrtin JB, Fuci AS, Ksper DL. Hrrison s principles of internl medicine, 13 th ed. NY: McGrw-Hill, 1994: Fregi A, Jensen DM. Evlution of norml liver tests. Compr Ther 1994; 2: Hu DM. Effects of electrocupuncture on leukocytes nd plsm protein in the X-irrdited rts. Am J Chin Med 1984; 12: Jeong DH, Lee GP, Jeong WI, Do SH, Yng HJ, Yun DW, Prk HY, Kim KJ, Jeong KS. Altertions of mst cells nd TGFet1 on the silymrin tretment for CCl(4)-induced heptic firosis. World J Gstroenterol 2; 11: Yu XH, Zhu JS, Yu HF, Zhu L. Immunomodultory effect of oxymtrine on induced CCl4-heptic firosis in rts. Chin Med J (Engl) 24; 117: S- Editors Pn BR nd Wng J L- Editor Kumr M E- Editor Bi SH