1 Trky University Fculty of Medicine Aville t BALKAN MEDICAL JOURNAL THE OFFICIAL JOURNAL OF TRAKYA UNIVERSITY FACULTY OF MEDICINE Blkn Med J DOI: /lknmedj Originl Article 309 Protective Effects of Alph-Lipoic Acid on Oleic Acid-Induced Acute Lung Injury in Rts Fund Gülcü Bulmuş 1, Mehmet Ferit Gürsu 2, Mehmet Hmdi Muz 3, İhsn Ymn 4, Özgür Bulmuş 5, Ftih Skin 6 1 Voctionl School of Helth Services, Fırt University, Elzığ, Turkey 2 Deprtment of Biochemistry, Fırt University Fculty of Medicine, Elzığ, Turkey 3 Deprtment of Pulmonry Diseses, Fırt University Fculty of Medicine, Elzığ, Turkey 4 Sivrice Voctionl School, Fırt University, Elzığ, Turkey 5 Veterinry Control Institute, Ministry of Agriculture, Elzığ, Turkey 6 Deprtment of Phrmcology nd Toxicology, Mustf Keml University Fculty of Veterinry Medicine, Hty, Turkey ABSTRACT Bckground: Oxidtive stress is elieved to e n importnt fctor in the pthogenesis of cute lung injury (ALI). Aims: The im of this study ws to investigte the possile protective role of lph-lipoic cid (α-la) on oleic cid (OA)-induced ALI in rts. Study Design: Animl experiment. Methods: A totl of thirty-five rts were divided into five groups in the study. Group 1 served s control group. Rts in Group 2 (α-la) were dministered α-la intrperitonelly t dose of 100 mg/kg ody weight (BW). Rts in Group 3 (OA) were dministered OA intrvenously t dose of 100 mg/kg BW. In Group 4 (pre-oa-α-la), α-la ws given 15 minutes prior to OA infusion, nd in Group 5 (post-oa-α-la), α-la ws given two hours fter OA infusion. Four hours fter the OA infusion, rts were decpitted. Blood smples were collected to mesure serum levels of mlondildehyde (MDA) nd glutthione (GSH), nd the levels of ctivity for superoxide dismutse (SOD), ctlse (CAT) nd glutthione peroxidse (GSH-Px). Lung tissue smples were tken for histopthologicl exmintion. Results: Exposure to OA resulted in increses in serum MDA levels (p<0.001), s well s histopthologicl lesions in lung tissue, nd decreses in CAT (p<0.05), GSH-Px (p<0.05) ctivities nd GSH (p<0.05) levels. On the other hnd, MDA levels were decresed significntly (p<0.001), while CAT (p<0.05), GSH-Px (p<0.01) ctivities nd GSH (p<0.05) levels were incresed significntly in the pre-oa-α-la group compred with the OA group. Conclusion: α-la ws found to lessen oxidtive stress nd to hve positive effects on ntioxidnts in cses of OA-induced ALI. In conclusion, α-la ppers to hve protective effects ginst ALI nd potentil for the prevention of ALI. Key Words: Acute lung injury, oleic cid, α-lipoic cid, oxidtive stress Received: Accepted: Introduction Acute lung injury (ALI) nd cute respirtory distress syndrome (ARDS) re chrcterised y hypoxemi tht is resistnt to oxygen therpy, decresed lung complince, incresed microvsculr permeility, nd the presence of diffuse lveolr dmge nd lveolr oedem (1, 2). ALI nd ARDS re diffuse responses of the lung to vrious clinicl conditions including trum, spirtion, sepsis, endotoxemi, nd pneumoni. Despite the introduction of new tretment techniques nd recent dvncements in intensive cre technology, the mortlity rte of ptients with ARDS remins high (3, 4). The min nomly in ARDS is the disruption of the lveolr-cpillry rrier. The common chrcteristics of this syndrome re diffuse interstitil nd lveolr oedem, incresed microvsculr permeility, persistent hypoxemi, nd diffuse inflmmtion (4, 5). ARDS is considered n exggerted inflmmtory rection of the lung to vrious stimuli, such s pneumoni, spirtion, inhltion, emoli, sepsis, nd disseminted intrvsculr cogultion. In these clinicl conditions leding to ARDS, incresed neutrophil migrtion to the lungs nd ctivtion of inflmmtory meditors re oserved. Neutrophils cuse cell dmge y secreting free rdicls, inflmmtory meditors, proteses (elstse nd collgense), nd cytokines such s tumor necrosis fctor-α (TNF-α) (6). The pthogenesis of ALI nd ARDS involves multiple mechnisms including oxidnt-induced inflmmtory dmge to the lveolr wll. Recent studies hve imed to prevent or reduce the progression of ALI (7, 8). Oleic cid (OA; cis-9-octdecnoic cid) is ftty cid present in niml- nd plnt-derived oils. When dministered intrvenously, OA induces morphologicl nd cellulr chnges similr to those produced y ALI nd ARDS in humns. Thus, OA dministrtion is commonly used to generte n experimentl model of ALI for the ssessment of potentil therpeutic gents ginst ARDS (9-11). Following OA dministrtion, n initil pulmonry vsculr endothelil cell injury gives rise to susequent inflmmtion nd ALI. Numerous studies hve reported the following indictors of this oxido-inflmmtory stte: degenertion of the lveocpillry Address for Correspondence: Dr. Fund Gülcü Bulmuş, Voctionl School of Helth Services, Firt University, Elzığ, Turkey. Phone: e-mil:
2 310 Blkn Med J memrne structure, incresed lveocpillry permeility, uild-up of intr-lveolr nd interstitil fluid, hemorrhge, infiltrtion of polymorphonucler leukocytes, ctivtion of oxidnt enzymes, secretion of cytokines, nd the production of dhesion molecules, rective oxygen species (ROS), nd rective nitrogen species (9-12). Alph-lipoic cid (α-la; 1,2-dithiolne-3-pentnoic cid, 6,8-dithio-octnoic cid, thioctic cid) is strong ntioxidnt tht plys n essentil role s cofctor in the metolism of ll orgnisms, from microorgnisms to humns (13, 14). Becuse α-la hs een found to e effective in the tretment of vrious models of oxidtive stress, it represents potentil therpeutic possiility for mny diseses ssocited with oxidtive stress, such s ischemi-perfusion injury, dietes, ctrct formtion, HIV ctivtion, neurodegenertion, nd rdition dmge (14). The im of this study ws to exmine the possile protective effects of α-la tretment on oxidnt-ntioxidnt sttus nd histopthologicl chnges in commonly used, OA-induced experimentl model of ALI. We lso imed to evlute the effectiveness of α-la, which my e useful in mny oxidtive stress models, in the tretment of ALI nd ARDS. Mteril nd Methods Chemicls OA ws purchsed from Sigm-Aldrich (St. Louis, MO, USA). α-la ws otined from MP Biomedicls Inc. (Solon, OH, USA). Glutthione (GSH) nd GSH reductse, thiorituric cid (TBA), phosphotungstic cid, hydrogen peroxide (H 2 O 2 ), nitrolue tetrzolium (NBT), nicotinmide denine dinucleotide phosphte (NADPH), nd other regents were supplied y Sigm-Aldrich (St. Louis, MO, USA) or Merck (Drmstdt, Germny). OA ws dissolved in ethnol nd then diluted to 25 mg/ ml s finl concentrtion using 0.9% NCl (The finl rtio of ethnol:sline ws 1:9). The prepred solution ws infused intrvenously (i.v.) into the til vein for 5 minutes through 24 G rnul (15, 16). α-la ws dissolved in physiologicl sline solution contining 0.5% NOH, nd the ph of the solution ws djusted to 7.4 with HCl. A dose of 100 mg/kg α-la ws dministered intrperitonelly (i.p.) to the rts (17). Animls nd experimentl design The protocol for niml use ws pproved y the Institutionl Review Bord of the Ntionl Institute of Helth nd the Locl Committee on Animl Reserch. The nimls were otined from the Experimentl Reserch Centre of Firt University, Elzığ, Turkey, nd mintined under stndrd lortory conditions (24±3 C, 40-60% humidity, 12/12 h light/drk cycle). A commercil pellet diet (Elzig Food Compny, Elzig, Turkey) nd fresh drinking wter were provided d liitum. Thirty-five femle Wistr lino rts weighing etween 140 nd 160 g were used in the study. The rts were rndomly divided into five groups, with seven rts in ech group. The experimentl groups were rrnged s follows: Group 1 (control group) received control solution of physiologicl sline/ethnol t dose of 100 mg/kg BW vi i.v. Group 2 (α-la group) ws given α-la t dose of 100 mg/ kg BW vi i.p. Group 3 (OA group) ws given OA t dose of 100 mg/ kg BW vi i.v. Group 4 (pre-oa-α-la group) ws dministered α-la 15 minutes efore OA infusion. Group 5 (post-oa-α-la group) ws dministered α-la two hours fter OA infusion. Smple preprtion Four hours fter OA infusion, the rts in ech group were decpitted, nd lood smples were collected in regulr iochemistry tues. The tues were centrifuged t 3000 rpm for 10 minutes, nd the serum ws recovered nd kept t -20 C until nlysis. Lung smples for lter histopthologicl exmintion were lso removed from the rts nd provided in 10% formldehyde to the pthology lortory. Biochemicl nlysis Mlondildehyde (MDA), the finl product of lipid peroxidtion, ws mesured using spectrophotometric TBA ssy dpted from Stoh (18) nd Ygi (19). The results re expressed s nmol/ml. Superoxide dismutse (SOD) ctivity in the serum ws ssessed using the method of Durk et l. (20), modified from Sun et l. (21), which mesures the reduction of NBT y superoxide generted with the xnthine/xnthine oxidse system. One unit of SOD ctivity is defined s the mount of enzyme tht inhiits NBT reduction y 50%. The results re expressed s U/mL. Serum ctlse (CAT) ctivity ws determined y the method descried y Aei (22), during which the decomposition of H 2 O 2 is followed using spectrophotometry. The results re expressed s k/ml. Serum GSH peroxidse (GSH-Px) ctivity ws mesured using the method descried y Pgli de Vlentine (23), in which oxidtion of NADPH is spectrophotometriclly mesured s decrese in sornce t 340 nm. The results re expressed s U/mL. Serum GSH levels were determined using the dithionitroenzoic cid recycling method developed y Ellmn (24). The results re expressed s nmol/ml. Histopthologicl exmintion The tissue smples from the rts were fixed in 10% formldehyde solution. Following routine preprtion, smples were emedded in prffin locks using estlished methods. Sections mesuring 5 µm were otined from the locks, stined with hemtoxylin-eosin, nd imged y light microscopy. Sttisticl nlysis All vlues re presented s the men±stndrd devition. The normlity nlysis of the dt ws performed using the Kolmogorov-Smirnov test; nd the Levene s test ws used to nlyse the homogeneity of vrinces. Since ll of the dt were normlly distriuted nd the vrinces were homog-
3 Blkn Med J 311 enous, one-wy nlysis of vrince (one-wy ANOVA) ws used nd followed y post hoc Tukey HSD test, which is more sensitive test thn other post hoc tests, to compre differences etween the mens of mesured iochemicl prmeters mong the experimentl groups. Differences were considered significnt t p<0.05. Results Serum concentrtions of MDA nd GSH, nd ctivity levels of SOD, CAT, nd GSH-Px mesured for the different groups re summrised in Tle 1. The level of MDA in serum smples from the OA group ws incresed compred with tht of the control group (p<0.001). However, the MDA level in pre-oa-α-la smples ws found to e lower thn tht in OA smples (p<0.001). When compred with smples from the control group, smples from the α-la group exhiited incresed CAT nd GSH-Px ctivity levels (p<0.001 nd p<0.05, respectively); in contrst, smples from the OA group exhiited decresed ctivity levels in the serum (p<0.05). Notly, the ctivity levels of CAT nd GSH-Px in pre-oa-α-la smples were greter thn the ctivity levels in OA smples (p<0.05 nd p<0.01, respectively). Compred with control smples, the serum level of GSH ws incresed in α-la smples (p<0.05) ut decresed in OA smples (p<0.05). However, the serum GSH level in pre-oaα-la smples ws greter thn the GSH level in OA smples (p<0.05). No significnt difference in serum SOD ctivity ws oserved mong the groups. Lung smples from the control nd α-la groups were histologiclly norml (Figure 1), nd miniml perivsculr infiltrtion y mononucler cells ws oserved in α-la smples (Figure 1). In contrst, smples from the OA group displyed diffuse lveolr dmge including incresed vsculr permeility, interstitil intr-lveolr oedem nd hemorrhge, perivsculr nd intr-lveolr hyline memrne formtion, nd dense interstitil intr-lveolr infiltrtion y mononucler cells (Figure 2 nd 2). Smples from the pre-oa-α-la group lso displyed the presence of perivsculr interstitil mononucler cell infiltrtion. In ddition, congestion nd mild hemorrhge were oserved in some of the vessels. However, the impirments nd mononucler cell infiltrtion present in pre-oa-α-la smples were minor compred with those in OA smples. Additionlly, wheres incresed vsculr permeility, oedem, nd hyline memrne formtion were common in OA smples, these injuries were not present in pre-oa-α-la smples (Figure 3 nd 3). Tle 1. Serum MDA nd GSH levels nd SOD, CAT, GSH-Px ctivities of the groups (n=7) Group 1 Group 2 Group 3 Group 4 Group 5 (Control) (α-la) (OA) (pre-oa-α-la) (post-oa-α-la) MDA (nmol/ml) 0.86± ± ±0.09*** 0.94± ±0.04 SOD (U/mL) 9.33± ± ± ± ±0.68 CAT (k/ml) 2.17± ±0.56*** 1.66±0.23* 2.23± ±0.25 GSH-Px (U/mL) 1.96± ±0.18* 1.75±0.06* 2.01± ±0.11 GSH (nmol/ml) 11.37± ±1.66* 9.21±0.54* 11.24± ±1.11 * p<0.05, compred with control group; ** p<0.01, compred with control group; *** p<0.001, compred with control group. p<0.05 compred with OA group; p<0.01, compred with OA group; p<0.001, compred with OA group. MDA: mlondildehyde; SOD: superoxide dismutse; CAT: ctlse; GSH: glutthione; GSH-Px: glutthione peroxidse; α-la: lph-lipoic cid; OA: oleic cid. Figure 1.,. Histopthologicl HE stining of smples from the control nd α-la groups. Norml lung histology in control smples (). Miniml perivsculr infiltrtion y mononucler cells α-la smples ().
4 312 Blkn Med J Figure 2.,. Histopthologicl HE stining of smples from the OA group. Interstitil intr-lveolr oedem, hemorrhge, nd diffuse hyline memrne formtion (). Perivsculr oedem nd interstitil intr-lveolr infiltrtion y mononucler cells (). Figure 3.,. Histopthologicl HE stining of smples from the pre-oa-α-la group. Perivsculr interstitil infiltrtion y mononucler cells (). Perivsculr interstitil infiltrtion y mononucler cells, with mild hemorrhge (). Figure 4.,. Histopthologicl HE stining of smples from the post-oa-α-la group. Vsculr permeility increse, perivsculr oedem, nd hyline memrne formtion (). Interstitil intr-lveolr infiltrtion y mononucler cells ().
5 Blkn Med J 313 Smples from the post-oa-α-la group developed severe increse in vsculr permeility. Diffuse lveolr dmge including interstitil intr-lveolr mononucler cell infiltrtion ws lso present in these smples (Figure 4 nd 4). Discussion Previous reserch hs estlished tht ARDS increses oxidtive stress nd secretion of ROS from the neutrophils of ptients (25-30). Studies involving the experimentl induction of ALI s model for ARDS hve reported incresed MDA concentrtion, indicting incresed oxidtive stress nd ltered ctivity of ntioxidnt enzymes (15, 31-33). Additionl reserch hs reveled tht the plsm of ptients with ARDS contins reduced levels of ntioxidnts nd tht neutrophil infiltrtion nd ROS ccumultion cn exhust the totl ntioxidnt cpility of the lungs (26, 30). α-la is strong ntioxidnt nd potentil therpeutic for mny diseses, prticulrly those ssocited with oxidtive stress. The ntioxidnt ctivity of α-la my e the result of its ilities to oth scvenge ROS nd restore the cpility of endogenous ntioxidnts, such s vitmin C, vitmin E, nd GSH (13, 14). α-la hs een shown y mny studies, oth in vivo nd in vitro, to decrese lipid peroxidtion levels. Reduced lipid peroxidtion following α-la dministrtion hs een oserved to e ssocited with protective effect ginst drimycin-induced nephrotoxicity nd crdiotoxicity in rts (34, 35). Additionl studies hve found tht α-la tretment results in decresed MDA level in the plsm (36), liver, kidney (17) nd different regions of the rin (37). Mny studies hve investigted the effects of α-la on ntioxidnt enzymes (35, 36). Mritim et l. (38) reported tht α-la dministrtion reduced oxidtive stress nd ltered SOD, CAT, nd GSH-Px enzymtic ctivity levels in the liver, kidney, nd hert in rts. Similrly, Shil et l. (39) showed tht tretment with α-la resulted in decresed oxidnt production nd lipid peroxidtion in the rin, wheres it led to incresed levels of enzymtic ctivity for SOD, CAT, nd GSH-Px in ll rin regions. In this study, serum smples from the OA group contined greter mount of MDA thn smples from the control group (p<0.001). Notly, the serum MDA level ws lower in the pre-oa-α-la smples compred with tht in the OA smples; suggesting oxidtive stress preventive effects of α-la. These results imply tht ALI increses oxidtive stress nd tht α-la exhiits n opposing effect protecting ginst ALI. The sence of significnt difference in the serum MDA level etween the post-oa-α-la nd OA groups suggests tht the dministered dose of α-la is insufficient to produce n injuryreversing effect. As result of the exmintion of the α-la smples, it ws determined tht the incresed ctivity levels of CAT nd GSH-Px nd elevted serum GSH content re likely cused y the strong ntioxidnt properties of α-la nd its restortive effect on endogenous ntioxidnts. Conversely, with the OA smples, the decresed enzymtic ctivity levels nd GSH concentrtion indicte pro-oxidnt shift in the oxidntntioxidnt lnce during OA-induced ALI. The conclusions drwn from comprison of pre-oa-α-la smples with OA smples suggest tht the OA-induced increse in oxidtive potentil ws countercted y α-la; in fct, ntioxidnt cpility ppered to recover until it pproximted tht in control smples. However, in post-oa-α-la smples, the increse in oxidtive stress resulting from injury ws not neutrlised y α-la. Tken together, these findings suggest tht α-la exerts preventive effects only if it is dministered prior to the induction of n injury. OA is commonly used to induce ALI in lortory nimls. Exposure to OA hs een previously shown to result in lveolr oedem, congestion, neutrophil infiltrtion, nd deteriortion of the pulmonry structure in the lung tissue of rts (33, 15). Similrly, neutrophil infiltrtion, lveolr oedem, nd expnsion nd thickening of the lveolr wll hve een reported in n experimentl lung injury model for cute pncretitis (40). In the current study, rts exposed to OA experienced ALI chrcterised y similr prolems: perivsculr oedem, interstitil nd intr-lveolr oedem, mononucler cell infiltrtion, hemorrhge, hyline memrne formtion, nd diffuse lveolr dmge. Prominent histopthologicl chnges oserved in smples from the OA group, such s incresed vsculr permeility, oedem, nd hyline memrne formtion, were less severe or sent in pre-oa-α-la smples. The histopthologicl exmintion nd supporting iochemicl nlyses confirmed the presence of OA-induced ALI nd the protective effects of n α-la infusion prior to exposure to OA. In conclusion, the ppliction of OA to develop n experimentl ALI model resulted in oxidtive stress chrcterised y elevted serum MDA concentrtions nd decresed levels of ctivity for the ntioxidnt enzymes GSH-Px, SOD, nd CAT. Exposure to OA lso produced histopthologicl dmge in the lung tissue of rts. However, if α-la ws dministered 15 minutes efore OA infusion, oxidtive stress ws reduced, which ws indicted y lower serum MDA content, incresed levels of ntioxidnt enzymtic ctivity, nd meliortion of histopthologicl dmge to the lungs of rts. In summry, we conclude tht α-la protects ginst nd my prevent ALI. Ethics Committee Approvl: Ethics committee pprovl ws received for this study from the locl ethics committee of Fırt University Medicl Fculty. Informed Consent: N/A. Peer-review: Externlly peer-reviewed. Author contriutions: Concept M.F.G., M.H.M.; Design M.F.G., M.H.M., F.G.B.; Supervision M.F.G., F.G.B.; Resource M.F.G., F.G.B.; Mterils F.G.B., İ.Y., Ö.B., F.S.; Dt Collection&/or Processing F.G.B., İ.Y., Ö.B., F.S.; Anlysis&/or Interprettion F.G.B., İ.Y., Ö.B., F.S.; Literture Serch F.G.B., F.S.; Writing F.G.B., F.S.; Criticl Reviews M.F.G., M.H.M., F.G.B. Conflict of Interest: No conflict of interest ws declred y the uthors. Finncil Disclosure: This study ws supported y the Fırt University Scientific Reserch Projects Unit (FUBAP), project numer References 1. Repine JE. Scientific perspectives on dult respirtory distress syndrome. Lncet 1992;339: [CrossRef]
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