Protective effect of rosuvastatin treatment by regulating oxidized low-density lipoprotein expression in a rat model of liver fibrosis

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1 BIOMEDICAL REPORTS 5: , 2016 Protective effect of rosuvsttin tretment by regulting oxidized low-density lipoprotein expression in rt model of liver fibrosis SHUIPING YU 1, XUELING ZHOU 1, BINGZONG HOU 2, BO TANG 1, JIAN LI 2 nd BAIMENG ZHANG 2 1 Deprtment of Heptobiliry Surgery, The Affilited Hospitl of Guilin Medicl University, Guilin, Gungxi ; 2 Deprtment of Generl Surgery, The Fifth Affilited Hospitl of Sun Yt Sen University, Zhuhi, Gungdong , P.R. Chin Received Februry 12, 2016; Accepted April 27, 2016 DOI: /br Abstrct. The present study imed to evlute the protective effect of rosuvsttin tretment on the mechnism of oxidized low density lipoprotein (Ox LDL) in rts with liver fibrosis. In totl, 72 mle Sprgue Dwley rts were divided into 3 groups: 24 in the control group (A), 24 in the obstructive jundice models group (B) nd 24 in the rosuvsttin group (C). Ech group ws further divided into four subgroups for ssessment t different time-points. The obstructive jundice models were estblished nd rosuvsttin ws dministered by gvge. Liver fibrosis indictors, Ox LDL, mlonldehyde (MDA) nd superoxide dismutse (SOD), were mesured nd liver pthologicl chnges were observed t weeks 1, 2, 3 nd 4 fter model induction. In groups B nd C, the rt models were successfully estblished, nd there were significnt chnges in the expression of Ox LDL nd the three liver fibrosis indictors when compred to group A (P<0.01). However, the expression of Ox LDL nd the three liver fibrosis indictors in group C were decresed compred with group B (P<0.05), while SOD incresed (P<0.05) nd MDA decresed (P<0.05). The three liver fibrosis indictors were different in comprison to group B (P<0.05). Thus, there ppered to be n ssocition between the expression of Ox LDL nd liver fibrosis. Tretment with rosuvsttin could regulte the expression of Ox LDL nd improve liver fibrosis in rt models with obstructive jundice. Introduction Liver fibrosis, primry procedure of cirrhosis, is progressive chronic procedure chrcterized by the ccumultion of Correspondence to: Dr Shuiping Yu, Deprtment of Heptobiliry Surgery, The Affilited Hospitl of Guilin Medicl University, 15 Lequn Rod, Guilin, Gungxi , P.R. Chin E mil: @qq.com Key words: obstructive jundice model, oxidized low density lipoprotein, rosuvsttin, liver fibrosis extrcellulr mtrix (ECM), which leds to serious dmge to humn helth. Numerous studies hve reported tht fibrosis cnnot be reversed; however, if the genertion could be suppressed or degrdtion could be promoted of the ECM, liver fibrosis is reversible (1,2). Therefore, it is importnt to explore the mechnism of delying or improving liver fibrosis. For the purpose of providing criterions for the tretment of liver fibrosis, certin studies re in process to discover the mechnism of liver fibrosis. Specificlly, in obstructive jundice rt models, the level of oxidized low density lipoprotein (Ox LDL) ws previously considered to hve key role in therosclerosis, the presence of the Ox LDL specific receptor in liver endothelil cells nd hd n importnt role in liver fibrosis (3,4). Additionlly, mlonldehyde (MDA) nd superoxide dismutse (SOD) re signling receptors tht hve specific roles in lipid peroxidtion nd functions s liver fibrosis regultors (5,6). The level of Ox LDL is known to increse in the liver nd is correlted with the level of liver fibrosis. These evidences hd conclusion tht Ox LDL, MDA nd SOD my be involved in improving liver fibrosis. Despite considerble evidence, the potentil mechnism of liver fibrosis remins to be elucidted. Erly drug intervention cn dominte the levels of Ox LDL. For instnce, rosuvsttin could reduce the levels of Ox LDL in the plsm of hemodilysis ptients with end stge renl disese (7). Rosuvsttin could control the progress of liver fibrosis by inhibiting the ctivity of 3 hydroxy 3 methylglutryl coenzyme A (HMG CoA), which is key enzyme in the ccumultion of cholesterol (8). It hd been reported tht rosuvsttin tretment could reduce the plsm concentrtions of endogenous peroxidse nd peroxidse ctivity, nd regulte the expression of Ox LDL in Wistr rts in 24 weeks (9). However, the underlying mechnism combined with rosuvsttin on liver fibrosis remins to be elucidted. The present study imed to show the ssocition between rosuvsttin nd liver fibrosis, nd confirm the mechnism of Ox LDL in rt models of obstructive jundice. This could fcilitte the present understnding of the mechnism nd improve clinicl tretment for liver fibrosis. Mterils nd methods Mterils. In totl, 72 mle specific pthogen free Sprgue Dwley rts weighing g were obtined from

2 312 YU et l: ROSUVASTATIN EFFECTS Ox-LDL IN RATS WITH LIVER FIBROSIS Tble I. Liver fibrosis indictors of the rts in the control nd model groups to confirm the estblishment of the model. Group Hyluronic cid, ng/ml Lminin, ng/ml Procollgen III, ng/ml Week 1 A 46.50± ± ±12.36 B 62.67± ± ±17.45 Week 2 A 38.65± ± ±12.20 B 58.33± ± ±9.46 Week 3 A 35.60± ±21.65 b 25.32±13.26 B 90.64± ± ±26.15 Week 4 A 48.06± ±13.42 b 30.63±22.47 b B ± ± ±17.56 P<0.01 or b P<0.05 vs. group B. A, control group; B, model group. Vlues re men ± stndrd devition, n=6. Figure 1. (A) Representtive microgrphs of the norml ppernces of heptocytes in group A. (B) Necrosis nd the emergence of prolifertion of heptocytes in were observed group C. (C) Fibrous septmorphologicl bnormlities of heptocytes in group B were evident (ll mgnifiction, x200). Figure 2. Expression of (A-C) SOD nd (D-F) MDA in the different groups (mgnifiction, x200). SOD stining ws stronger in (C) group A compred to groups (A) B nd (B) C. Group B hd weker expression of SOD compred to group C. MDA stining ws stronger in (F) group B, wheres this ws weker in (D) group A. The stining intensity ws grded s (A nd D) 0, (B nd E) 1, nd (C nd F) 2. SOD, superoxide dismutse; MDA, mlonldehyde. the Experimentl Animl Center of Sun Yt Sen University (Gungzhou, Gungdong, Chin). Six rts were housed in ech cge under stndrd lbortory conditions. Subsequently, the rts were rndomized into 3 groups: Control (group A, 24 rts), model (group B, 24 rts) nd rosuvsttin groups (group C, 24 rts). Rts were fed d libitum with stndrd diet in group A, wheres rts underwent surgery with obstructive jundice models in group B nd rts were gvged by

3 BIOMEDICAL REPORTS 5: , Tble II. SOD nd MDA levels of rts from groups A nd B. Group SOD MDA Week 1 A 5.28± ±0.38 B 4.36± ±0.65 C 5.22± ±0.46 Week 2 A 4.86± ±0.36 B 4.65± ±0.63 C 4.95± ±0.35 Week 3 A 5.66± ±0.32 B 3.86± ±0.66 C 4.62±1.88 b 2.18±0.62 b Week 4 A 5.45± ±0.46 B 3.26± ±2.16 C 3.94±2.66 b 3.82±1.36 b P<0.01 or b P<0.05 vs. group B. SOD, superoxide dismutse; MDA, mlonldehyde; A, control group; B, model group; C, rosuvsttin group. Vlues re men ± stndrd devition, n=6. rosuvsttin in group C. At weeks 1, 2, 3 nd 4 fter the model induction, 6 rts were chosen rndomly from every group nd nesthetized prior to being scrificed. To determine whether the obstructive jundice models were successful, 1 cm length liver tissue smples were obtined through excising nd subjected to hemtoxylin nd eosin (H&E) stining. Blood smples were obtined from ech rt for subsequent nlysis of three liver fibrosis indictors. Following tht, drug intervention ws initited following model induction. In ddition, rts received tretment of rosuvsttin by intrgstric dministrtion in group C (btch no ; AstrZenec, London, UK) (5 mg (kg body mss) 1 dy 1). Anlysis of serum levels by rdioimmunossy. Hyluronic cid (HA), lminin (LN) nd procollgen III (PCIII) (Gungzhou Yingwei Chungjin Biologicl Engineering Co., Gungzhou, Chin) were exmined by n utomtic rdioimmunossy nlyzer ccording to the mnufcturer's protocol. Anlysis of liver fibrosis lesion by opticl microscope. Prffin embedded smples were deprffinized nd hydrted by routine techniques. Therefter, tissue sections were stined by H&E nd observed by light microscope. Immunohistochemicl nlysis of SOD nd MDA. The expression levels of SOD nd MDA (Shnghi Institute of Biologicl Regents Sles Co., Shnghi, Chin) in liver tissue were detected by immunohistochemistry. Smples were incubted with polyclonl nti SOD nd nti MDA ntibodies t dilution of 1:50. Phosphte buffered solution ws used s the negtive control. Brown stining ws considered positive stining of SOD nd MDA. Imges were nlyzed by Imge Pro plus 6.0 imge nlysis softwre (Medi Cybernetics, Rockville, MD, USA). The intensity of the stining ws grded s 0 (no color), 1 (yellow) nd 2 (brown). In ddition, the percentge of positive cells ws grded s 0 (<5%), 1 (5 25%), 2 (25 50%), 3 (51 75%) nd 4 (>75%). The sum of the 2 grdes served s the score for ech smple. Immunofluorescence nlysis of Ox LDL. The expression levels of Ox LDL (Gungzhou Yingwei Chungjin Biologicl Engineering Co., Gungzhou, Chin) in liver tissue were detected by immunofluorescence. Smples were incubted with monoclonl nti Ox LDL ntibody t dilution of 1:100 ccording to the mnufcturer's protocol. The expression levels of Ox LDL were observed by fluorescence microscopy ccording to the positive rtio nd intensity score of fluorescence, which hd totl score of 10 points. Sttisticl nlysis. All dt were sttisticlly nlyzed using SPSS 17.0 softwre (SPSS, Inc., Chicgo, IL, USA). Different groups of dt were nlyzed with nlysis of vrince to detect significnt differences. The significnce level ws set s α=0.05. P 0.05 ws considered to indicte sttisticlly significnt difference. Results Estblishment of niml model. Rts in group B exhibited poor ppetite, cloudy yellow urine nd less ctivity compred to rts in group A. To evlute whether liver fibrosis ws successful, HA, LN nd PCIII were tested in the blood smples of three groups every week. As shown in Tble I, group A hd lower levels of HA, LN nd PCIII compred to group B t weeks 3 nd 4 (P<0.01). In ddition, the liver tissues were observed by light microscope. As shown in Fig. 1, heptocytes were morphologiclly norml nd orderly in group A when liver tissues exhibited degenertion, edem, necrosis, prolifertion nd fibrous sept morphologicl bnormlities t week 4 in group B. These results demonstrted tht the rt model ws estblished. Results of indictors combining with MDA nd SOD. MDA nd SOD were detected by immunohistochemicl nlysis (Fig. 2). Tble II showed the levels of MDA nd SOD in group A compred with groups B nd C. MDA levels were elevted (P<0.01) nd SOD were declined (P<0.01) compred to group A t weeks 3 nd 4. Furthermore, there were reduced levels of MDA in group C compred with tht in group B (P<0.05), but elevted levels of SOD (P<0.05). These findings reveled tht rosuvsttin enhnced decrements in MDA nd increments in SOD. Expression of Ox LDL. The expression level of Ox LDL ws observed by immunofluorescence. As shown in Fig. 3, Ox LDL ws locted in the heptocyte membrne. Tble III showed tht the expression levels of Ox LDL were considerbly elevted in groups B (P<0.01) nd C (P<0.01) compred with group A t weeks 3 nd 4. However, the expression levels of Ox LDL were decresed in group C compred with group B (P<0.05). Anlysis of liver fibrosis. HA, LN nd PCIII were tested ech week in the blood smples of the rts in groups B nd C. As

4 314 YU et l: ROSUVASTATIN EFFECTS Ox-LDL IN RATS WITH LIVER FIBROSIS Tble III. Oxidized low-density lipoprotein expression level of the rts from the different groups. Group Week 1 Week 2 Week 3 Week 4 A 1.17± ± ± ±0.41 B 1.00± ± ± ±0.70 C 0.83± ± ±0.63 b 2.33±1.21 b P<0.01 nd b P<0.05 vs. group B. A, control group; B, model group; C, rosuvsttin group. Vlues re men ± stndrd devition, n=24. Tble IV. Liver fibrosis indictors of the rts in the model nd following rosuvsttin tretment. Group Hyluronic cid, ng/ml Lminin, ng/ml Procollgen III, ng/ml Week 1 B 62.67± ± ±17.45 C 52.67± ± ±15.58 Week 2 B 58.33± ± ±9.46 C 48.36± ± ±8.50 Week 3 B 90.64± ± ±26.15 C 70.18± ± ±18.64 Week 4 B ± ± ±17.56 C ± b 65.24± ±21.36 P<0.05 nd b P<0.01 vs. group B. B, model group; C, rosuvsttin group. Vlues re men ± stndrd devition, n=6. Figure 3. Expression of Ox-LDL in the different groups (mgnifiction, x400). Ox-LDL stining ws weker in (A) group A compred to groups (B) C nd (C) B. Group B hd stronger expression of Ox-LDL compred to group C. The stining score of A ws grded s 1, B ws 3, nd C ws 5. Ox-LDL, oxidized low-density lipoprotein. shown in Tble IV, there were considerbly lower levels of HA, LN nd PCIII in group C (P<0.05) compred to group B t weeks 3 nd 4. Fibrous sept morphologicl bnormlities were evident in groups B nd C, but these were significntly improved in group C. These results demonstrted tht tretment with rosuvsttin in rts with liver fibrosis ws effective. Discussion Liver fibrosis is progressive chronic disese chrcterized with the ccumultion of ECM, which leds to serious dmge to humn helth. However, whether the mechnism works by delying or improving liver fibrosis remins to be elucidted. The present study focused on the effect of rosuvsttin nd its mechnism on liver fibrosis in rt model. The model ws estblished by bile duct ligtion nd gvged dministrtion of rosuvsttin. The study showed tht the liver fibrosis indictors, HA, LN nd PCIII, in group B were incresed compred with tht in group A. Simultneously, the results of H&E stining suggested tht there ws more necrosis, n emergence of prolifertion nd the formtion of fibrous sept morphologicl bnormlities in group B. These results demonstrted tht the rt models were estblished. It hs been demonstrted tht SOD nd MDA hve importnt roles for oxidtive stress in the progression of endothelil injury nd liver fibrosis. MDA is product of

5 BIOMEDICAL REPORTS 5: , oxidtive stress, which cn produce free rdicls nd reflect the degree of dmge of liver cells. SOD cn eliminte oxidtive stress by removing oxygen free rdicls (10,11). Numerous biomolecules re closely combined with the oxidtive stress on liver fibrosis. For instnce, Ox LDL ppered to stimulte liver fibrosis by cusing the ECM disorder, nd subsequently there is more degenertion, edem, necrosis, the emergence of prolifertion nd the formtion of fibrous sept morphologicl bnormlities (12,13), which re similr with the result of the present study s there ws n increse of Ox LDL in group B compred with tht in group A. Furthermore, Turer nd Scherer (14) nd Chen et l (15) reported tht Ox LDL hs role nd is key in the regultion of the metbolism of ftty cid nd glucose, which is ssocited with therosclerosis. Hulthe nd Fgerberg (16) reported tht Ox LDL recruited monocytes into the endothelium nd cused dysfunction of rtery endothelil cells vi the genertion of rective oxygen species. Currently, certin bnormlities of Ox LDL hve been identified in vriety of diseses, such s liver fibrosis, however, the mechnisms remin to be elucidted. Fn et l (17) reported tht Ox LDL could induce inflmmtory fctors by up regultion of utophgy vi AMPK/mTOR signling pthwy. Yo et l (18) nd Xiong et l (19) reported tht Ox LDL could induce cholesterol ccumultion nd poptosis in mcrophges by upregulting CHOP expression nd the poptosis signl regulting kinse 1 c Jun N terminl kinse pthwy. Smrkoon et l (20,21) nd Antus et l (22) reported tht Ox LDL stimulted plsminogen ctivtor inhibitor 1 expression in humn mesngil cells medited by the trnsforming growth fctor β (TGF β)/smd signling pthwy. TGF β could ctivte extrcellulr signl regulted kinse (ERK) in mesngil cells, nd ERK ws involved in the ctivtion of Smd2/3, which incresed the formtion of ECM nd promoted fibrosis. However, further studies re essentil to elucidte the mechnism of liver fibrosis on the expression of Ox LDL. Chnges to the MDA nd SOD expression levels were lso shown in the present study. Antus et l (22) nd Youseff et l (23) reported tht MDA is useful mrker for monitoring excerbtion ssocited oxidtive stress, wheres SOD is n importnt ntioxidnt enzyme for protecting cells ginst oxidtive stress. The present study showed tht the level of Ox LDL ws elevted, but the level of MDA ws reduced in group B compred with group A. Furthermore, rosuvsttin inhibited the elevtions of Ox LDL nd MDA but promoted the levels of SOD in group C. Therefore, the dministrtion of the drug could mrkedly inhibit oxidtive stress ssocited with liver fibrosis. Rosuvsttin suppressed the development of liver fibrosis by inhibiting the ction of HMG CoA reductse. Resch et l (24) reported tht rosuvsttin significntly reduced oxidtive stress with the effect pprent fter tretment for 24 weeks in vivo, which is in ccordnce with the result of the present study, which indictes tht the dministrtion of rosuvsttin could inhibit the decrese of fibrosis. The decrese of Ox LDL in group C compred with tht in group B indictes tht rosuvsttin could exert n nti fibrosis effect vi downregultion of Ox LDL, nd subsequently prohibiting the progression of liver fibrosis. In conclusion, the present study showed tht the level of Ox LDL could be inhibited by the dministrtion of rosuvsttin on inhibiting oxidtive stress, nd rosuvsttin could improve the progression of liver fibrosis. The present study my id in the understnding of the biologicl effect of rosuvsttin nd improve the tretment of liver fibrosis. 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