Protective effect of Xingnaojia formulation on rats with brain and liver damage caused by chronic alcoholism

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1 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: , 2015 Protective effect of Xingnoji formultion on rts with brin nd liver dmge cused by chronic lcoholism SHUANG LI 1*, SU WANG 2*, ZHI GANG GUO 1*, NING HUANG 3*, FAN RONG ZHAO 3*, MO LI ZHU 3*, LI JUAN MA 3*, JIN YING LIANG 3*, YU LIN ZHANG 3*, ZHONG LIN HUANG 3* nd GUANG RUI WAN 1 1 School of Bsic Medicl Sciences; 2 College of Phrmcy, The Third Affilited Hospitl; 3 College of Phrmcy, Xinxing Medicl University, Xinxing, Henn , P.R. Chin Received My 15, 2014; Accepted July 31, 2015 DOI: /etm Abstrct. The im of this study ws to observe the effect of formultion of trditionl Chinese medicine extrcts known s Xingnoji (XNJ) on the liver function, lerning bility nd memory of rts with chronic lcoholism nd to verify the mechnism by which it protects the brin nd liver. A rt model of chronic lcoholism ws used in the study. The sptil lerning bility nd memory of the rts were tested. The rts were then scrificed nd their brins nd heptic tissues were isolted. The ctivity of superoxide dismutse (SOD) nd levels of glutmte (Glu), N methyl D sprtte receptor subtype 2B (NR2B), cyclin dependent kinse 5 (CDK5) nd cnnbinoid receptor 1 (CB1) in the hippocmpus were nlyzed. The ultrstructure of the heptic tissue ws observed by electron microscopy. In ddition, the ctivities of lcohol dehydrogense (ADH) nd ldehyde dehydrogense (ALDH) in serum were tested nd the levels of low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG) nd totl cholesterol (TCHOL) were nlyzed. XNJ enhnced the lerning nd memory of rts with chronic lcoholism. Tretment with XNJ incresed the ctivity of SOD, nd decresed the expression levels of NR2B mrna nd NR2B, CB1 nd CDK5 proteins in the brin tissues compred with those in the model rts. It lso incresed the ctivity of ALDH in the serum nd liver, decresed the serum levels of LDL, TG nd TCHOL nd incresed the serum level of HDL. These results indicte tht XNJ exhibited protective effect ginst brin nd liver dmge in rts with chronic lcoholism. Correspondence to: Professor Gung Rui Wn, School of Bsic Medicl Sciences, Xinxing Medicl University, 601 Jinsui Rod, Xinxing, Henn , P.R. Chin E mil: gungruiwncn@126.com * Contributed eqully Key words: Xingnoji, chronic lcoholism, cerebrl injury, heptic injury Introduction Alcoholism is common, but serious worldwide socil nd medicl problem. Reduction of lifespn due to lcoholism is more significnt thn tht due to crdiovsculr diseses. The combintion of helth, psychologicl nd socil problems ssocited with lcoholism nd lcohol buse is mjor public helth problem (1). Long term drinking of lcohol cn cuse numerous pthologicl chnges in the body nd is prticulrly hrmful to the brin nd liver. When n individul drinks excessively, the mjority of the lcohol is digested in the liver, which increses the overll burden to the liver. Alcohol dehydrogense (ADH) converts lcohol into cetldehyde, which is hrmful to the body. With the id of ldehyde dehydrogense (ALDH), the cetldehyde is oxidized into cetic cid, which is not hrmful to the body, nd further ctbolized into wter nd CO 2, which re excreted from the body (2). Long term lcohol consumption cn cuse lcoholic liver diseses, including lcoholic heptitis, lcoholic ftty liver nd lcoholic liver cirrhosis (3). According to previous reports, the incidence of lcoholic liver disese hs been rising in recent yers nd lcohol hs become the second most common cuse of heptic lesions fter heptitis virus (4). In the USA, ~111 million people over 12 yers of ge drink lcohol, the mjority of whom re young people (5). Alcoholic liver disese is mjor fctor in the incidence of liver disese nd overll mortlity round the world (6). In the USA, it is estimted tht >2 million people hve lcoholic liver disese (7). Alcoholic cirrhosis is the most common mnifesttion of lcoholic liver disese nd is ssocited with greter number of mortlities thn ll tumors combined (7). Excessive consumption of lcohol cuses n ccumultion of lcohol in the body, which increses the concentrtion of lcohol in the brin where it my be up to 10 fold higher thn the serum concentrtion (7). This ccumultion cn led to mlfunctioning of the nervous system nd cuse n excittion impulse tht my dmge surrounding neurons or be life thretening (8). The nesthetic effect of cetldehyde cn seriously ffect, for exmple, memory, ttention, judgment, self control, eyesight nd blnce. Long term drinking cn led to lcohol ddiction (lcoholism). When n individul becomes ddicted to lcohol, they my experience symptoms such s hnd trembling, confusion, fidgeting nd restlessness. Long term excessive lcohol consumption leds

2 1644 LI et l: PROTECTIVE EFFECT OF XNJ AGAINST BRAIN AND LIVER DAMAGE DUE TO CHRONIC ALCOHOLISM to diseses including lcoholic brin disese, brin trophy nd lcoholic dementi (9). Xingnoji (XNJ) is prescription formulted by Professor Gung Rui Wn of Xinxing Medicl College (Xinxing, Chin). By combining the known properties of Chinese herbl medicines, 12 edible trditionl Chinese mterils were selected, nd extrcts contining their min functionl ingredients were used to crete the XNJ formultion. XNJ hs been predicted to protect the stomch, liver nd brin. This study focuses on the effect of XNJ on the liver function, s well s the lerning nd memory, of rts with chronic lcoholism, nd lso explores the mechnism through which XNJ protects the liver nd brin. Mterils nd methods Animls nd grouping. Clen mle Sprgue Dwley rts (weight 140±20 g) were provided by the Animl Center of Zhengzhou University [Zhengzhou, Chin; License No. scxk (yu) ]. The rts were rndomly divided into five test groups: A, norml control group; B, model group of lcoholic rts; C, rts given lcohol nd low dose of XNJ; D rts given lcohol nd high dose of XNJ; nd E, positive control group (rts given lcohol nd King Drink). Ech group comprised 10 rts. The rts were kept t n mbient temperture of 20±2 C. Animl mnipultions were mde ccording to the Guide to Experimentl Animl Tretment (Sept 30, 2006) drfted by the Ministry of Science nd Technology of the People's Republic of Chin. This study ws crried out in strict ccordnce with the recommendtions in the Guide for the Cre nd Use of Lbortory Animls of the Ntionl Institutes of Helth (8th edition, 2011). The niml use protocol ws reviewed nd pproved by the Institutionl Animl Cre nd Use Committee (IACUC) of Xinxing Medicl University (Xinxing, Chin). Estblishment nd tretment of rts with chronic lcoholism. XNJ ws prepred t Xinxing Medicl College. King Drink tblets (1.0 g per tblet; Btch No ; Chin ptent, ppliction No A) contining primrily pueririn, Hoveni dulcis flvonoids, bmboo lef flvonoids nd gstrodin were provided by Shenzhen Neptunus Group Co., Ltd. (Shenzhen, Chin). Rts in groups B, C, D nd E were fed with n lcohol wter mixture s their only supply of drink. The lcohol concentrtion ws kept t 6% for 4 weeks nd the rts te freely. Rts in group A te nd drnk wter freely. During this 4 week period, rts in groups C nd D were given XNJ in norml sline (2 ml/kg) t 9:00.m. ech dy by gvge, where the totl flvonoid content of the XNJ ws 260 mg nd 780 mg, respectively; nd rts in group E were given norml sline (2 ml/mg) combined with King Drink (1.5 tblets/kg) by gvge. Rts in groups A nd B were given the sme quntity of norml sline s the other three groups by gvge. Lerning nd memory test. This test ws crried out following the 4 week period of lcohol/wter drinking. Testing ws conducted using Y mze. The bottom of the instrument continer ws ble to generte electric shocks crrying 0.4 ma current. Signl lmps were plced t the top of the three rms of the mze. The bility of the rts to void electric shocks by following the signl lmps ws evluted. The lights were rndomly turned on. Rts were plced in the mze nd ll lmps were turned on to enble the rts to fmilirize themselves with the environment. After 3 min, ll the lmps were turned off. One lmp ws then turned on, nd in the two remining rms, the electric current ws switched on 5 sec lter to shock the rts until they rn to the sfe re, designted by the signl lmp. Then, ll the lmps were turned on for 15 sec. Norml rection ws defined s rts reching the sfe re in 10 sec with only one electric shock. Rts tht hd norml rection were described s hving pssed the lerning test, while rts requiring longer rection times or dditionl electric shocks to rech the sfe re were described s hving filed the lerning test. If rt ws ble to pss 9/10 tests, it ws considered to hve lerned the skill. The number of times tht it took for rt to lern to pss the tests ws tken s quntittive evlution of its bility to lern nd remember sptil detils. The fewer ttempts rt required to pss the test, the stronger ws its lerning bility. All the tests were crried out t night in quiet environment nd under dim light. Rts tht were slow to rect to the electronic shock were eliminted. Detection of superoxide dismutse (SOD) in rt brin tissue. Surgicl procedures were crried out ccording to the instructions provided with the SOD detection kit (Nnjing Jincheng Bioengineering Institute, Nnjing, Chin). Following the memory test, the rts were nesthetized by n intrperitonel injection of sodium pentobrbitl (30 mg/kg), then scrificed by decpitted. The brins were excised immeditely nd put in n ice bth contining norml sline prior to homogeniztion. The hydroxylmine oxidtion method ws used to detect the ctivity of SOD. One unit of SOD ctivity ws defined s tht hving n SOD inhibition rte of 50% for 1 g tissue protein in 1 ml rection liquid. Anlysis of glutmte (Glu) levels by high performnce liquid chromtogrphy (HPLC). Glutmic cid stndrd ws bought from Shnghi Kngd Amino Acid Fctory (Shnghi, Chin). Following the memory test nd under nesthesi, rts were decpitted nd the hippocmpus ws peeled off nd weighed. Then 1 ml methnol wter mixture (1:1) ws dded to hlf of the whole hippocmpl tissue to prepre cryogenic homogente. Following centrifugtion (4 C, 10,000 x g) for 15 min, the superntnt ws removed nd kept t 80 C following membrne filtrtion for subsequent nlysis. Tissue components were mesured in units of µg/g. Smples were derived with o phthlldehyde. HPLC conditions were s follows: ProStr/Dynmx System control system (including Prostr 210 pump, Prostr 363 progrmmble fluorescence detector, 800 nlog digitl converter, ProStr 500 column nd temperture box; Agilent Technologies, Snt Clr, CA, USA); mobile phse A, potssium cette 0.1 M; nd mobile phse B, crbinol, for processing by bi grdient elution. The elution procedure ws s follows [time (min), percentge B]: (0, 0%), (1, 5%), (10, 20%), (17, 40%), (20, 60%), (22, 55%), (40, 55%) nd (45, 100%). The mobile phses were filtered using 0.45 µm microporous filter membrnes, degssed by ultrsound, nd run t flow rte of 1.0 ml/min. The excittion wvelength ws 250 nm nd the trnsmission wvelength

3 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: , ws 410 nm. The levels of Glu were determined from the pek re. Anlysis of N methyl D sprtte receptor subtype 2B (NR2B) by reverse trnscription polymerse chin rection (RT PCR). TRIzol regent nd PrimeScript RT PCR kit were provided by Tkr (Dlin) Biotechnology Co., Ltd. (Dlin, Chin), nd primers were synthesized by Sngon Biotech Co., Ltd. (Shnghi, Chin). SYBR Premix Ex Tq II kits were purchsed from Tkr (Dlin) Biotechnology Co., Ltd. Following the memory test, the rts were quickly decpitted. A mg smple of brin tissue from the hippocmpus ws plced on ice nd immeditely put into TRIzol regent for extrction of RNA. The RNA ws quntified nd ssessed for purity by mesuring the bsorbnce t 260 nm nd 280 nm. Reverse trnscription ws performed by following the directions provided with the kit. NR2B primers were generted using Primer 5.0 softwre (Premier Biosoft, Plo Alto, CA, USA) nd their sequences were: Upstrem, 5' CTT ACT GAA GGC AAT CCT CG 3' nd downstrem, 5' TCC TCA GAA CAC CTT CGC TT 3'. For β ctin, the primer sequences were: Upstrem, 5' ATG GAT GAC GAT ATC GCT GCG 3' nd downstrem, 5' TCG TCC CAG TTG GTG ACA ATG 3'. A PCR mixture ws prepred comprising: 10X PCR Buffer II 2.5 µl, dntp mixture 1 µl, NR2B/β ctin forwrd primer (10 µmol/l) 1 µl, NR2B/β ctin reverse primer (10 µmol/l) 1 µl, Tkr Ex Tq HS DNA polymerse 1 µl, Stencil cdna 2 µl nd ddh 2 O 17.5 µl (totl volume 25 µl). The PCR mixture ws plced in PCR rection tubes nd cycled using M MyCycler PCR instrument (Bio Rd Lbortories, Inc., Hercules, CA, USA). Amplifiction conditions were s follows: i) One cycle of denturing t 94 C for 1 min; nd ii) 30 cycles of denturing t 94 C for 30 sec; nneling t 60 C for 30 sec; nd elongtion t 72 C for 1 min. PCR results were nlyzed using BndScn gel nlysis softwre (Glyko, Hywrd, CA, USA) nd compred with β ctin expression in ech group. The rtio of the integrted opticl density (IOD) of the NR2B gry scle vlue to the β ctin reference ws nlyzed. Detection of NR2B protein expression in rt hippocmpus by n immunofluorescence method. The brin tissue in the hippocmpus ws cut into 20 µm sections using thermosttic freezing microtome nd ffixed to polylysine coted slide. The smple ws microwve repired for 15 min, nd then blocked with got serum for 20 min. Rbbit nti mouse NR2B polyclonl ntibody (1:100 dilution; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, Chin) ws dded prior to incubtion t 4 C overnight. Cy3 lbeled got nti rbbit IgG (1:500; Beijing Biosynthesis Biotechnology Co., Ltd.) ws dded nd the smple ws incubted t room temperture for 2 h. Nucler stining with 4',6 dimidino 2 phenylindole [DAPI; Tkr Biotechnology (Dlin) Co., Ltd.] ws performed for 3 min. Anti fluorescence quencher ws dded to the slide, which ws then plced under BX61 fluorescence microscope (Olympus Corportion, Tokyo, Jpn) for observtion nd imging. Serologicl detection. Abdominl ortic blood ws smpled from the rts. The serum ws then isolted for the determintion of the levels of low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG) nd totl cholesterol (TCHOL), using BS 800 utomtic biochemicl nlyzer (Shenzhen Mindry Bio Medicl Electronics Co., Ltd., Shenzhen, Chin) ccording to the mnufcturer's instructions. Detection ctivity of ADH nd ALDH in serum nd heptic tissue. Using kits from BioVision Inc. (Milpits, CA, USA), colorimetry ws used to test the ctivity of ADH nd ALDH in serum nd heptic tissue smples from the rts in ech group. The experiments were crried out following the instructions provided by the mnufcturer. Detection of cnnbinoid receptor 1 (CB1) expression nd cyclin dependent kinse 5 (CDK5) in rt brin hippocmpus by western blot nlysis. The totl protein content ws extrcted from the hippocmpl tissue using T PER (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA) nd quntified using BCA Assy kit (Pik Dy Biotechnology, Himen, Chin). The proteins were trnsferred to nitrocellulose membrne (Sigm Aldrich, St. Louis, MO, USA) nd then blocked following electrophoresis. Primry nti CB1 (rbbit nti rt; 1:300; sc 10066; Snt Cruz Biotechnology, Inc., Dlls, TX, USA) nd nti CDK5 ntibodies (rbbit nti mouse; 1:300; bs 0559R; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, Chin) overnight t 4 C. Membrnes were wshed three times for 5 min in PBS, then secondry got nti rbbit IgG Cy3 lbeled ntibodies (1:300; A 0521; Beijing Biosynthesis Biotechnology, Co., Ltd.) were dded for hybridiztion for 2 h. Electrochemiluminescence nlysis ws conducted using SuperSignl West Dur Extended Durtion Substrte (Pierce, Thermo Fisher Scientific, Inc.). The opticl density of the film ws mesured using gel scnner, nd β ctin ws used s the internl reference. The IOD ws clculted to determine the reltive mount of test protein. The experiment ws repeted three times, nd the men ws clculted. Sttisticl nlysis. Results re shown s the men ± stndrd devition. SPSS softwre pckge, version 12.0 (SPSS, Inc., Chicgo, IL, USA) ws used to conduct the sttisticl nlysis. Comprisons mong groups were performed using one wy nlysis of vrince nd lest significnt difference detection. Results Effect of XNJ on the lerning nd memory of rts with chronic lcoholism. According to the Y mze behvior test, the lerning bility nd memory of rts with chronic lcoholism ws significntly wekened compred with tht of norml control rts (P<0.01). However, high nd low doses of XNJ hd protective effect on rts with chronic lcoholism (Tble I). Effect of XNJ on SOD ctivity in brin homogente. the ctivity of SOD in the brin homogente of the model group ws significntly reduced compred with tht in the control group (P<0.01). In the groups treted with high or low dose of XNJ, the ctivity of SOD in the brin homogente incresed significntly (P<0.05 nd P<0.01, respectively; Tble II). Effect of XNJ on NR2B mrna expression. Imge nlysis results following RT PCR demonstrted tht the expression

4 1646 LI et l: PROTECTIVE EFFECT OF XNJ AGAINST BRAIN AND LIVER DAMAGE DUE TO CHRONIC ALCOHOLISM Tble I. Effect of XNJ formultion on lerning nd memory. Group No. of trining times for n ccurte response Control 37.38±11.61 Model ±12.98 Positive control 97.00±7.71 Low dose 93.25±7.63 b High dose 46.00±5.78 c Results re from Y mze test. If the rt escped to the sfe re directly within 10 sec, it ws considered correct response; otherwise, it ws considered n incorrect response. Nine correct responses per 10 trils indicted tht the rt hd lernt to escpe. Trining times tken in lerning re mesure the performnce of sptil lerning nd memory. One wy nlysis of vrince nd lest significnt difference multiple rnge tests were used to determine the differences of mens. P<0.01 vs. control; b P<0.05, c P<0.01 vs. model. Dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). XNJ, Xingnoji. Tble III. Comprison of NR2B mrna levels (gry vlues). Group NR2B mrna Control 0.62±0.05 Model 1.27±0.06 Positive control 1.23±0.09 Low dose 1.08±0.07 b High dose 0.83±0.08 b P<0.01 vs. control; b P<0.01 vs. model. Gry scle vlue nlysis of NR2B mrna bnds. mrna ws reverse trnscribed nd mplified with β ctin s the internl control. All dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. NR2B, N methyl D sprtte receptor subtype 2B. Tble II. Effect of XNJ formultion on brin levels of SOD (x10 3 U/gprot). Group SOD level Control ± Model ± Positive control ± Low dose ± b High dose ± c P<0.01 vs. control; b P<0.05, c P<0.01 vs. model. All dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. XNJ, Xingnoji; SOD, superoxide dismutse. Figure 1. Expression of NR2B mrna. Lne A, control; lne B, model; lne C, positive control; lne D, low dose XNJ; lne E, high dose XNJ. NR2B, N methyl D sprtte receptor subtype 2B; XNJ, Xingnoji. level of NR2B mrna ws highest in the model group, nd lowest in the control group. In the groups treted with high or low doses of XNJ, the NR2B mrna levels were intermedite between those of the control nd model groups, but were notbly lower thn the levels in the model group (P<0.01; Tble III, Fig. 1). Effect of XNJ on NR2B protein expression in the hippocmpus of rts with chronic lcoholism. The expression of NR2B protein in the hippocmpus is shown in Fig. 2. NR2B positive cell cytoplsm is red. In the model group, compred with the control group, there ws lrge number of fluorescent stined cells, which were densely distributed. The fluorescence intensity ws incresed in the rts with chronic lcoholism. The dministrtion of XNJ reduced the number of NR2B protein positive cells. The number of NR2B protein positive cells ws reduced significntly s the dose of XNJ ws incresed, in comprison with the number in the model group. Effect of XNJ on Glu levels in the hippocmpus of rts with lcoholism. HPLC dt hve previously shown tht the tretment of chronic lcoholism cn reduce the Glu level in the rt hippocmpus. The results of the present study revel tht high or low doses of XNJ hd no effect on the levels of Glu in the hippocmpl tissue of rts with lcoholism (Tble IV, Fig. 3). Effect of XNJ on the expression of CB1 in the hippocmpus of rts with chronic lcoholism. As shown in Fig. 4, the expression level of CB1 in the hippocmpl tissue of rts in the model group incresed significntly compred with tht in the control group, wheres CB1 expression ws mrkedly reduced in rts treted with high nd low doses of XNJ compred with tht in the model group. Effect of XNJ on the expression of CDK5 in the hippocmpus of rts with chronic lcoholism. As shown in Fig. 4, the expression level of CDK5 ws incresed in the rts with

5 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: , A B C D E F G H I J K L M N O Figure 2. Representtive photomicrogrphs of NR2B (Cy3 lbeled, red fluorescence; DAPI lbeled, blue fluorescence; mgnifiction, x400) with immunofluorescent stining in the rt hippocmpus. A lrge number of neurons were fluorescently stined in the hippocmpus in the rts from the model group, with dense distribution nd incresed fluorescence intensity in comprison with the control group. By contrst, the number nd density of fluorescently stined neurons nd the fluorescence intensity were decresed in the rts from the drug treted groups compred with those in the model group. (A) Positive NR2B expression, (B) DAPI stining nd (C) merged imge of stining in the norml group. (D) Positive NR2B expression, (E) DAPI stining nd (F) merged imge of stining in the model group; (G) Positive NR2B expression, (H) DAPI stining nd (I) merged imge of stining in the positive control group. (J) Positive NR2B expression, (K) DAPI stining nd (L) merged imge of stining in the group treted with low dose of XNJ. (M) Positive NR2B expression, (N) DAPI stining nd (O) merged imge of stining in the group treted with high dose of XNJ. NR2B, N methyl D sprtte receptor subtype 2B; DAPI, 4',6 dimidino 2 phenylindole; XNJ, Xingnoji.

6 1648 LI et l: PROTECTIVE EFFECT OF XNJ AGAINST BRAIN AND LIVER DAMAGE DUE TO CHRONIC ALCOHOLISM A Tble IV. Effect of XNJ formultion on brin levels of glutmte in rts with chronicl lcoholism (µmol). Group Glutmte level B Control 3.49±0.70 Model 0.24±0.06 Positive control 0.27±0.07 Low dose 0.29±0.07 High dose 0.28±0.06 P<0.01 vs. control. All dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. XNJ, Xingnoji. C D E Figure 4. Expression of CB1 nd CDK5 in the rt hippocmpus s demonstrted by western blotting. Lne A, control; lne B, model; lne C, positive control; lne D, low dose XNJ; lne E, high dose XNJ. CB1, cnnbinoid receptor 1; CDK5, cyclin dependent kinse 5; XNJ, Xingnoji. Figure 3. Results of HPLC nlysis of glutmte (Glu) levels in the rt hippocmpus. (A) Control, (B) model, (C) positive control, (D) low dose XNJ nd (E) high dose XNJ groups. HPLC, high performnce liquid chromtogrphy; XNJ, Xingnoji. chronic lcoholism compred with tht in the control group. The expression level of CDK5 ws reduced in the groups receiving high or low dose of XNJ compred with tht in the model group. Heptocyte ultrstructure ltertion. In group A (the control group), rounded or ovl shped nuclei were observed in the heptocytes nd the cell membrnes retined their integrity. Cler nucleoli were lso observed, in ddition to cler nd complete nucler membrnes. Furthermore, the ppernce of the mitochondri ws norml nd the criste structure could be distinctly seen. Occsionlly, tiny lipid droplets were observed in the cells (Fig. 5A). In group B (model group with lcoholism), it ws observed tht certin cells hd n incomplete membrne nd mrginlized heterochromtin in the nucleus. Mitochondri were swollen in these heptocytes nd their criste were segmented nd blurred. There were number of lipid droplets of vrious sizes in the cytoplsm (Fig. 5B). In group C (the positive control group), there were smll mounts of lipid droplets in the heptocytes nd norml number of mitochondri (Fig. 5C). Group D (low dose XNJ group) exhibited lipid droplets of vrious sizes in the heptic cells (Fig. 5D). In group E (high dose XNJ group), the number of mitochondri in heptocytes ws incresed nd lipid droplets were seldom observed (Fig. 5E). Effect of XNJ on levels of LDL, HDL, TG nd TCHOL in the serum of rts with lcoholism. The levels of LDL, TG nd TCHOL in the serum incresed nd those of HDL decresed significntly in the model group compred with those in the control group (P<0.05) nd these chnges were significntly ttenuted in the group treted with XNJ when compred with those in the model group (P<0.05; Tbles V nd VI). Effect of XNJ on the ctivity of ADH in the serum nd liver tissue of rts with lcoholism. Tests of ADH ctivity in the serum nd liver tissue of rts in ech group were conducted using colorimetric ssy. As the results show, the ctivity of ADH incresed significntly in the model group nd the two

7 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: , A B C D E Figure 5. Chnges in the ultrstructure of rt liver cells in ech group. (A) Norml group, (B) model group with lcoholism, (C) positive control group, (D) low dose XNJ nd (E) high dose XNJ groups. XNJ, Xingnoji. groups receiving XNJ tretment, ll of which differed from the control group (P<0.05). However, there ws no significnt difference in the ctivity of ADH mong groups B, C, D nd E (Tble VII). Effect of XNJ on the ctivity of ALDH in the serum nd liver tissue of rts with lcoholism. The ctivity of ALDH in the rt serum nd liver tissue ws detected by colorimetry. According to the results, the ctivity of ALDH in the model group nd group receiving low dose CNJ exhibited no significnt differences compred with tht in the control group. However, the ALDH ctivity ws mrkedly incresed in the high dose XNJ group, nd ws significntly different from tht detected in the model group (P<0.05; Tble VIII). Discussion Bsed on the properties nd function of trditionl Chinese medicines nd knowledge of modern phrmcology, 12 trditionl Chinese medicines were selected nd their primry functionl ingredients were extrcted to crete the XNJ formultion. The essentil ingredients include isoflvones from kudzu root nd risin tree seeds, s well s flvones from bmboo leves, Gstrodi tuber nd resistnt strch. Previous studies hve demonstrted tht XNJ cn significntly improve lerning nd memory in humns, llevite neurologicl dmge nd protect the liver (10,11). In order to verify the neutrlizing effect of XNJ on the physiologicl effects of lcoholic drinks, King Drink, sobering preprtion tht hs been pplied cliniclly for mny yers, ws used s positive control. The mjor dmging effect of lcoholism on the nervous system is toxic effect on neurons nd dmging effects on lerning nd memory (12). The results of the present study demonstrte tht lerning nd memory in the model rt group ws much worse thn tht of the control group (P<0.01), wheres lerning nd memory ws improved in the groups receiving high or low dose XNJ (P<0.05 or P<0.01). There ws no pprent distinction between the positive control nd model groups. This indictes tht the XNJ formultion my hve protective effect ginst the neurologicl dmge cused by chronic lcoholism in rts. In order to investigte the mechnism by which XNJ protects the nervous system, the ctivity of SOD nd the levels of NR2B, Glu, CDK5 nd CB1 in rt brin tissue were ssyed. The experimentl results revel tht the SOD ctivity of rts in the model group ws significntly reduced when compred with tht in the control group, while SOD ctivity incresed mrkedly in the XNJ treted groups compred with those in the model group. This shows tht the ctivity of the ntioxi-

8 1650 LI et l: PROTECTIVE EFFECT OF XNJ AGAINST BRAIN AND LIVER DAMAGE DUE TO CHRONIC ALCOHOLISM Tble V. Effect of XNJ on LDL nd HDL in the serum of rts with lcoholism (mmol/l). Group LDL HDL Control 2.29± ±0.16 Model 2.89± ±0.04 Positive control 2.59±0.12 b 0.52±0.07 b Low dose 2.46±0.21 b 0.46±0.15 b High dose 2.57±0.07 b 0.40±0.08 b P<0.05 vs. control; b P<0.05 vs. model. All dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. LDL, low density lipoprotein; HDL, high density lipoprotein; XNJ, Xingnoji. Tble VIII. Effect of XNJ on the ctivity of ALDH in the serum nd heptic tissue of rts with lcoholism (U/ul). Group Serum Liver Control ± ± Model ± ± Positive control ± ± Low dose ± ± High dose ± ± P<0.05 vs. model. All dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. ALDH, ldehyde dehydrogense; XNJ, Xingnoji. Tble VI. Effect of XNJ on TCHOL nd TG in the serum of rts with lcoholism (mmol/l). Group TCHOL TG Control 1.72± ±0.16 Model 2.04± ±0.11 Positive control 1.75±0.50 b 0.69±0.06 b Low dose 1.75±0.12 b 0.79±0.10 b High dose 1.77±0.24 b 0.76±0.08 b P<0.05 vs. control; b P<0.05 vs. model. All dt re expressed s men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. TCHOL, totl cholesterol; TG, triglycerides; XNJ, Xingnoji. Tble VII. Effect of XNJ on the ctivity of ADH in the serum nd heptic tissue of rts with lcoholism (mu/ml). Group Serum Liver Control 66,855.05± ,667.41± Model 166,160.25± ,414.59± Positive control 164,725.47± ,917.94± Low dose 167,004.97± ,895.20± High dose 164,899.20± ,956.57± P<0.05 vs. control. All dt re expressed s the men ± stndrd devition (n=10 rts per group; experiment repeted three times). One wy nlysis of vrince nd lest significnt difference multiple rnge test were used to determine the differences of mens. ADH, lcohol dehydrogense; XNJ, Xingnoji. dnt enzyme SOD in the brin tissue ws incresed with the use of XNJ, which in turn suggests tht tht XNJ my help to neutrlize free oxygen rdicls nd mitigte their dmging effects on brin tissue. Incresing the ctivity of ntioxidnt enzymes my be mechnism by which XNJ protects the brin from the effects of lcohol. The min ingredients of XNJ re isoflvones nd flvones, which hve lipid peroxidtion nd free rdicl mitigting effects. The hippocmpus is n importnt region for neuron plsticity nd is closely ssocited with lerning bility nd memory in mmmls (13). However, NMDA receptors, which re the min regultors of synptic plsticity nd long term potentition (LTD), re richly expressed in brin tissue (14). Studies hve shown tht LTD in the hippocmpus is medited by Glu nd NMDA receptors (15). The NMDA receptor NR2B plys n importnt role in neurl plsticity (16). A previous study demonstrted tht lcohol is ble to ffect the NR2B receptor, leding to neuronl dmge nd chnges in lerning nd memory (17). The present study hs shown tht with high dose XNJ dministrtion, the expression of NR2B mrna in the hippocmpus of rts with chronic lcoholism ws much lower thn tht in the model group (P<0.01). This dt suggests tht the XNJ formultion hs protective effect ginst brin dmge in rts with chronic lcoholism nd tht this effect is probbly due to regultion of the expression of NR2B protein. However, the moleculr mechnism of this protective effect is unknown. Reserchers hve shown tht the excessive ctivtion of NMDA receptors increses the concentrtion of C 2+ continuously, which leds to C 2+ overlod nd cn ctivte series of enzymes relted to cytotoxicity, such s protein kinse C (PKC) nd nitric oxide synthse (NOS). These enzymes cn dmge the min components of the cellulr lipid membrne nd cytoskeletl proteins of nerve cells nd cuse grdul necrosis of neurons (18). Vnillin nd p hydroxybenzldehyde, which re components of Gstrodi tuber, hve been shown to significntly suppress the Glu stimulted intrcellulr increse of C 2+ in IMR 32 neuroblstom cells nd poptosis (19). Gstrodin cn lso suppress the levels of C 2+ in PC12 cells stimulted by Glu, which suggests tht the clcium chnnel could be the trget of gstrodin components tht suppress the effects of excitotoxicity (19). It is hypothesized tht XNJ downregultes NR2B receptors nd suppresses C 2+ mobiliztion, which in turn protects ginst neurologicl dmge in rts

9 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: , with chronic lcoholism. In this mnner, XNJ my improve rt lerning nd memory. After ssying the levels of Glu in the rt hippocmpus, it ws found tht there ws no significnt chnge in Glu levels in the rts receiving XNJ. This indictes tht the function of XNJ is not ssocited with Glu regultion. In order to determine the specific mechnism of this process, two fctors tht re closely connected with lerning, memory nd neurologicl dmge, nmely CB1 nd CDK5, were exmined. Humns hve used mrijun throughout history. Cnnbinoids re the ctive ingredients in mrijun nd ct on CB1 in the brin to bring feelings of euphori nd rewrd. CB1 is mostly expressed in the centrl nervous system (CNS) nd belongs to the clss of G protein coupled receptors. CB1 is closely ssocited with neurogenesis, neurl development, synpse formtion, lerning nd memory, food intke nd energy metbolism (20,21). CB1 is the most bundnt receptor in mmmlin brins. By binding to its cognte lignd, CB1 trnsfers signls between neurons nd regultes wide rnge of signling mechnisms. Previous studies hve shown tht there is close correltion between CB1 nd the neurotoxic effects of lcoholism (21). In the CNS, endogenous cnnbinoids function s reverse neurotrnsmitters following their relese from post synptic neurons nd ct on the pre synptic membrnes of neurons bering CB1. When CB1 receptors re ctivted nd coupled with the voltge dependent C 2+ chnnel, the chnnel closes nd the influx of C 2+ is reduced. Through this mechnism, neurotrnsmitters such s γ minobutyric cid (GABA) nd Glu re relesed in lesser mounts in the presynptic membrne of neurons (22). CDK5 is member of the cyclin dependent kinse fmily with moleculr weight of 35 kd. It is proline directed serine/threonine protein kinse tht phosphoryltes serine/threonine residue with C terminlly djcent proline residue. CDK5 is bundntly expressed in the nervous system nd is regulted by the ctivtor proteins P35 nd P39 (23). A previous study hs shown tht CDK5 is involved in synptic plsticity in the nervous system s well s lerning bility nd memory (24). Moreover, it is implicted in drug ddiction. Erlier studies hve shown tht P35 nd P39, ctivtor proteins of CDK5, re regulted by intrcellulr C 2+ nd hydrolyzed to P25 nd P29. P35, P39, P25 nd P29 cn form heterodimer with, nd thereby ctivte CDK5; the hlf lives of P25 nd P29 re long, which would result in the excessive ctivtion of CDK5, thereby cusing neurotoxicity, such s neuronl poptosis nd cytoskeleton dmge (24). It hs been reported tht certin components of trditionl Chinese medicines cn function s clcium chnnel blockers tht re ble to suppress the overctivtion of CDK5 nd protect the brin from the dmge cused by clcium overlod in neurons to certin degree (25). The results of the present study demonstrte tht XNJ clerly reduced the expression of CB1 nd CDK5 in brin tissue. Bsed these results, it is considered tht the effects of XNJ on the clcium signling pthwy require further investigtion. In order to investigte the protective effect of XNJ on the liver nd the brin, the ultrstructure of heptic tissue in rts with chronic lcoholism ws observed under n electron microscope. In ddition, the ctivity of ADH nd ALDH in rt serum nd heptic tissue, s well s the levels of LDL, HDL, TG nd in serum were mesured. The effects of XNJ were found to include significnt improvement of the dmged ultrstructure of the heptic tissue in rts with chronic lcoholism, nd reductions of the levels of LDL, TCHOL nd TG in the serum of rts with chronic lcoholism ccompnied by n increse in the level of HDL. This indictes tht XNJ is ble to regulte the lipid metbolic disorder cused by lcohol. ADH nd ALDH together constitute the oxidtive pthwy by which lcohol is metbolized into cetic cid in the liver (26). ADH is crucil enzyme tht enbles liver cells to metbolize short chin lcohols nd is responsible for oxidizing lcohol into cetldehyde. ALDH is locted in the mitochondri of liver cells nd is the enzyme responsible for metbolizing cetldehyde into cetic cid, which is hrmless to the body. Chronic lcoholism cn led to incresed ctivity of ADH but not ALDH, which leds to buildup of cetldehyde nd chronic cetldehyde intoxiction with time. Rts with chronic lcoholism re very similr to humns with this condition; the quntity of ADH is bundnt nd similr to tht found in the norml humn body. However, there is significnt distinction mong individuls nd their levels of ALDH. A significnt proportion of people lck ALDH. As result, in these individuls, lcohol is metbolized into cetldehyde by ADH, but cnnot be converted to cetic cid by ALDH. This leds to the ccumultion of cetldehyde in the body, which in turn cuses symptoms of drunkenness, including sickness, inrticultion, stggering nd unconsciousness (27). Certin individuls hve ALDH with reduced enzymtic ctivity (27); therefore, if they drink too much or too quickly, t rte beyond the enzymtic ctivity of ALDH, they cn become inebrited. XNJ cn significntly increse the ctivity of ALDH in rts with chronic lcoholism nd ccelerte the brekdown of cetldehyde into H 2 O nd CO 2, thereby protecting the liver. In conclusion, XNJ ws ble to significntly neutrlize the dverse effects of lcohol, improve memory, llevite neurl injuries nd protect liver function. The formultion comprises nturl herbs, providing wide ppliction prospects. Although the mechnisms underlying the protective effects re discussed in the present study, the exct moleculr mechnism nd pthwy remin uncler nd require further study. 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