Diabetik sıçan lens ve kan dokusunda oluşan oksidatif stres ve antioksidan enzim düzeylerinin incelenmesi
Investigation on oxidative stress and antioxidant enzymes activitiy in diabetic rat lens and blood tissue
Yazar: EREL SÜTPAK
Danışman: PROF. DR. NİHAT DİLSİZ
Harran Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2007
Konu: Biyokimya = Biochemistry ; Biyoloji = Biology ; Moleküler Tıp = Molecular Medicine

Özet:
Almanya'dan getirilen deney hayvanları çoğaltılarak yedi grup oluşturuldu.Birinci grup kontrol grubu olarak kabul edildi ve deney sonuna kadar bu gruba hiçbirişlem yapılmadı. İkinci gruba 65 mg/kg Streptozotosin uygulanarak (i.p) diabetoluşturuldu. Üçüncü gruba STZ uygulanıp diabet olduğu görüldükten sonra günaşırı20 mg/sıçan Ginkgo biloba (mabet ağacı) ekstraktı oral gastrik gavaj yardımıylaverildi. Dördüncü gruba STZ uygulanıp diabet olduğu görüldükten sonra günaşırıolarak 21.5 mg/sıçan olacak şekilde Tagetes patula (kadife çiçeği) ekstraktı verildi.Beşinci gruba günaşırı 26.6 mg/sıçan olacak şekilde Gentiana olivieri (afat) ekstraktıverildi. Altıncı gruba, diabet oluştuğu anlaşıldıktan sonra 22.5 mg/sıçan Oleaeuropaea (zeytin) ekstraktı uygulandı. Yedinci gruba STZ uygulandıktan beş günsonra 20 mg/sıçan Aristolochia maurorum (loğusa otu) ekstraktı uygulandı.Deney süresi boyuca grupların ortalama ağırlık değişimi ve kan glukoz miktarıdeğişimi ölçülerek kaydedildi. Lens dokusunda malondialdehid (MDA) ve glutatyonGSH) ölçümleri yapıldı. Plazmada MDA, GSH, glukoz, kolesterol, yüksek dansitelilipoprotein-kolesterol (HDL-C), çok düşük dansiteli lipoprotein-kolesterol (VLDLC),trigliserid (TG), üre, kan üre azotu (BUN), kreatinin (CREA), aspartataminotransferaz (AST), alanin aminotransferaz (ALT) ve ?-glutamil transferaz(GGT) parametreleri ölçüldü. Hazırlanan eritrosit paketlerinden, eritrosit katalaz(CAT), glutatyon S transferaz (GST) ve glutatyon redüktaz (GR) enzim aktiviteleriölçüldü. Çıkan sonuçlar istatistiksel olarak Mann-Whitney testine göre yorumlandı.Oluşturduğumuz kontrol grubunda (K) deney sonuna kadar %5.2 oranında birağırlık artışı gözlemlenirken, farklı bitki ekstraktları uygulanan ama aynı koşul veyemlerle beslenen gruplardan Ginkgo biloba grubunda (D/G) % 26.6, Tagetes patulagrubunda (D/KA) %11.3, Gentiana olivieri grubunda (D/A) %5.3, Olea europaeagrubunda (D/Z) %23.6, Aristolochia maurorum grubunda (D/L) %26.3 ve diabetgrubunda (D) %22.3 oranında ağırlık azalması kaydedildi.K grubunun kolesterol değeri 48 mg/dl ölçülürken, D grubu 77 mg/dl, D/Ggrubu 71 mg/dl, D/KA 63 mg/dl, D/A 63 mg/dl, D/Z 100 mg/dl ve D/L 48 mg/dlolarak ölçüldü. D/L grubu hariç tüm gruplarda bu değerin yükseldiği görüldü. Kgrubu TG değeri 67 mg/dl olarak ölçülürken, D grubu 170 mg/dl, D/G 179 mg/dl,D/KA 180 mg/dl, D/A 183 mg/dl, D/Z 310 mg/dl ve D/L 131 mg/dl ölçüldü. Tümgruplarda kontrol grubuna göre anlamlı (p<0.05) bir artışın olduğu görüldü. Kontrolgrubu HDL-C değeri 50 mg/dl olarak ölçülürken, D grubu 34 mg/dl, D/G 56 mg/dl,D/KA 47 mg/dl, D/A 37 mg/dl, D/Z 39 mg/dl ve D/L 34 mg/dl olarak ölçüldü. Dgrubunda K grubuna göre anlamlı bir düşüş gözlenirken, D/G ve D/KA gruplarındaD grubuna göre anlamlı bir artış gözlemlendi. Kontrol grubu VLDL-C sonucu 15mg/dl ölçülürken, D grubu 32 mg/dl, D/G 45 mg/dl, D/KA 36 mg/dl, D/A 43 mg/dl,D/Z 63 mg/dl ve D/L 25 mg/dl ölçüldü. Tüm grupların kontrol grubuna göre anlamlıbir artış gösterdikleri tespit edildi.Kontrol grubu GGT seviyesi 1.6 U/L ölçülürken, D grubu 7.29 U/L, D/G 7.2U/L, D/KA 8.17 U/L, D/A 6.57 U/L, D/Z 24.17 U/L ve D/L 6.14 U/L ölçüldü. Tümgrupların GGT seviyesi K grubuna göre anlamlı bir artış gösterdi. K grubu ASTdeğeri 120 U/L olarak ölçülürken, D grubu 187 U/L, D/G 113 U/L, D/KA 205 U/L,D/A 469 U/L, D/Z 1183 U/L ve D/L 174 U/L olarak ölçüldü. Tüm grupların Kgrubuna göre anlamlı bir artış gösterdikleri görüldü. K grubu ALT miktarı 53 U/Lolarak ölçülürken, D grubu 132 U/L, D/G 119 U/L, D/KA 147 U/L, D/A 289 U/L,D/Z 386 U/L ve D/L 66 U/L olarak ölçüldü. D/L grubunun ALT seviyesini anlamlıderecede azaltarak K grubuna yaklaştırdığı görülürken, diğer tüm gruplarda budeğerin K grubuna göre yüksek çıktığı görüldü.Kontrol grubunun üre değeri 48 mg/dl ölçülürken, D grubu 100 mg/dl, D/G 85mg/dl, D/KA 101 mg/dl, D/A 122 mg/dl, D/Z 92 mg/dl ve D/L 76 mg/dl ölçüldü.Tüm gruplarda K grubuna göre anlamlı bir artış olurken, D/L grubu bu artışıazaltarak anlamlı oranda K grubuna yaklaştırmıştır. K grubu BUN seviyesi 22 mg/dlölçülürken, D grubu 47 mg/dl, D/G 39 mg/dl, D/KA 47 mg/dl, D/A 57 mg/dl, D/Z 47mg/dl ve D/L 36 mg/dl ölçüldü. Tüm grupların K grubuna göre anlamlı derecedeartış gösterdikleri, D/L grubunun ise bu yüksek değeri anlamlı oranda azaltarak Kgrubuna yaklaştırdığı görüldü. K grubunun CREA değeri 0.51 mg/dl çıkarken, Dgrubu 0.4 mg/dl,D/G 0.43 mg/dl, D/KA 0.43 mg/dl, D/A 0.48 mg/dl,D/Z 0.42 mg/dlve D/L 0.34 mg/dl çıktı. D/A grubu hariç tüm grupların CREA değerinin K grubunagöre istatistiksel olarak azaldığı görüldü.Kontrol grubu Lens MDA miktarının 16.75 (nmol/100 mg lens yaş ağırlığı)olduğu, D grubunun 19.1, D/G grubunun 11.5, D/KA grubunun 22, D/A grubunun22.8, D/Z grubunun 39.55 ve D/L grubunun 21.5 olduğu görüldü. D/G grubundaanlamlı derecede azalmanın olduğu görüldü.Kontrol gurubunun lens GSH miktarı (nmol GSH/100 mg lens yaş ağrılığı)45.85 iken, D grubu 6.3, D/G grubu 8.78, D/KA grubu 8.9, D/A grubu 9.7, D/Zgrubu 7.28 ve D/L grubu 9.75 olarak ölçüldü. Tüm grupların GSH değeri K grubunagöre anlamlı derecede düşük ölçüldü. D/G, D/A ve D/L gruplarının azalan GSHmiktarını anlamlı derecede arttırarak kontrol grubuna yaklaştırdıkları görüldü.Kontrol grubu plazma MDA değeri 35.5 nmol iken, D grubu 32.2, D/G grubu26.7 nmol, D/KA grubu 39.8 nmol, D/A grubu 31.8 nmol, D/Z grubu 41.0 nmol veD/L grubu 21.0 nmol olarak ölçüldü.Kontrol grubu plazma GSH miktarı 988 nmol ölçülürken, D grubu 1122 nmol,D/G grubu 338 nmol, D/KA grubu 531 nmol, D/A grubu 1450 nmol, D/Z grubu 575nmol ve D/L grubu 280 nmol olarak ölçüldü.Tip I diabetes mellitusta oksidatif stresin zamanla tüm dokularda artaraktahribat oluşturduğunu ve antioksidan savunmanın bu hasarı kısmen de olsaengellediği sonucuna vardık. Uyguladığımız bitkilerden özellikle Ginkgo biloba(mabet ağacı), Aristolochia maurorum (lohusa otu) ve Gentiana olivieri (afatotu)'nin diabetik komplikasyonları anlamlı derecede azalttığı, Olea europaea(zeytin) ve Tagetes patula (kadife çiçeği)'nın da bazı parametrelerde olumlu sonuçlarverdiğini gördük.

Abstract:
Reproducing the test animals that were brought from Germany, seven groupswere assigned. The first group was accepted as control group and no treatment wasapplied to this group until the end of the experiment. The second group was treatedwith 65 mg/kg streptozotocin (i.p) and diabetes was induced. The third group wastreated with STZ and after observing that diabetes was induced, 20 mg/rat Gingkobiloba (maidenhair tree) extract was given through gastric gavage every other day.The fourth group was treated with STZ and after observing that diabetes wasinduced, 21.5 mg/rat Tagetes patula (marigold) extract was given every other day.The fifth group was given26.6 mg/rat Gentiana olivieri (griseb) extract every otherday. The sixth group was treated with 22.5 mg/rat Olea europaea (olive) extract,after observing that diabetes was induced. Five days after treating the seventh groupwith STZ 20 mg/rat Aristolochia maurorum (bithwort) extract was treated.During the course of the experiment, average weight change and blood glucoseamount changes of the groups were measured and recorded. Malondialdehyde(MDA) and glutatione (GSH) measurements were carried out in lens tissue. In theplasma, MDA, GSH, glucose, cholesterol, high density lipoprotein-cholesterol(HDL-C), very low density lipoprotein-cholesterol (VLDL-C), triglyceride (TG),urea, blood urea nitrogen (BUN), creatinin (CREA), aspartate aminotransferase(AST), alanin aminotransferase (ALT) and gamma glutamyl transferase (GGT)parameters were measured. Erythrocyte catalase (CAT), glutathione-S-transferase(GST) and glutatione reduktase (GR) enzyme activities were measured from theprepared erythrocyte packs. The results that came out were statistically interpretedaccording to Mann-Whitney test.In control group (K) that we assigned, while a 5.2% weight increase wasobserved until the end of the experiment, among the groups which were treated withdifferent plant extracts but which were fed by same conditions and food, 26.6 %weight decrease was recorded in Gingko biloba group (D/G); 11.3% weight decreasewas recorded in Tagetes patula group (D/KA); 5.3% weight decrease was recordedin Gentiana olivieri group(D/A); 23.6% weight decrease was recorded in Oleaeuropaea group(D/Z) 26.3% weight decrease was recorded in Aristolochiamaurorum group (D/L); and 22.3% weight decrease was recorded in diabetes group(D).While the cholesterol value of group K was measured as 48 mg/dl, thecholesterol value of group D was measured as 77 mg/dl; cholesterol value of groupD/G was measured as 71 mg/dl; cholesterol value of group D/KA was measured as63 mg/dl; the cholesterol value of group D/A was measured as 63 mg/dl, thecholesterol value of group D/Z was measured as 100 mg/dl and the cholesterol valueof group D/L was measured as 48 mg/dl. It was observed that excluding D/L group,this value increased in all groups. While TG value of group K was measured as 67mg/dl, group D was measured as 170 mg/dl, group D/G was measured as 179 mg/dl,group D/KA was measured as 180 mg/dl, group D/A was measured as 183 mg/dl,group D/Z was measured as 310 mg/dl and group D/L was measured as 131 mg/dl. Asignificant (p<0.05) increase when compared to the control group was observed in allgroups. In control group while HDL-C value was measured as 50 mg/dl, it wasmeasured as 34 mg/dl in group D, 56 mg/dl in group D/G, 47 mg/dl in group D/KA,37 mg/dl in group D/A, 39 mg/dl in group D/Z and 34 mg/dl in group D/L. While asignificant decrease was observed in group D when compared to group K, nosignificant increase was observed in groups D/G and D/KA when compared to groupD. While control group VLDL-C result was measured as 15 mg/dl, group D wasmeasured as 32 mg/dl, group D/G was measured as 45 mg/dl, group D/KA wasmeasured as 36 mg/dl, group D/A was measured as 43 mg/dl, group D/Z wasmeasured as 63 mg/dl and group D/L was measured as 25 mg/dl. It was found that allgroups indicated a significant increase when compared to control group.While GGT level of control group was measured as 1.6 U/L, group D wasmeasured as 7.29 U/L, group D/G was measured as 7.2 U/L, group D/KA wasmeasured as 8.17 U/L, group D/A was measured as 6.57 U/L, group D/Z wasmeasured as 24.17 U/L and group D/L was measured as 6.14 U/L. GGT level of allgroups indicated a significant increase when compared to group K. While ASTvalues of group K was measured as 120 U/L, group D was measured as 187 U/L,group D/G was measured as 113 U/L, group D/KA was measured as 205 U/L, groupD/A was measured as 469 U/L, group D/Z was measured as 1183 U/L and groupD/L was measured as 174 U/L. It was found that all groups indicated a significantincrease when compared to group K. While ALT amount of group K was measuredas 53 U/L, group D was measured as 132 U/L, group D/G was measured as 119 U/L,group D/KA was measured as 147 U/L, group D/A was measured as 289 U/L, groupD/Z was measured as 386 U/L and group D/L was measured as 66 U/L. While it wasobserved that ALT level of group D/L significantly decreased and approximated togroup K, it was found that in all other groups this value was higher than group K.While urea value was measured as 48 mg/dl in control group, in group D it wasmeasured as 100 mg/dl, in group D/G it was measured as 85 mg/dl, in group D/KA itwas measured as 101 mg/dl, in group D/A it was measured as 122 mg/dl, in groupD/Z it was measured as 92 mg/dl and in group D/L it was measured as 76 mg/dl.While there was a significant increase in all groups when compared to group K,group D/L reduced this increase and significantly approximated to group K. WhileBUN level of group K was measured as 22 mg/dl, group D was measured as 47mg/dl, group D/G was measured as 39 mg/dl, group D/KA was measured as 47mg/dl, group D/A was measured as 57 mg/dl, group D/Z was measured as 47 mg/dland group D/L was measured as 36 mg/dl. It was found that all groups indicated asignificant increase when compared to group K, and group D/L significantly reducedthis high value and it was observed that approximated it to group K. While CREAvalue of group K came out to be as 0.51 mg/dl, group D came out as 0.4 mg/dl,group D/G came out as 0.43 mg/dl, group D/KA came out as 0.43 mg/dl, group D/Acame out as 0.48 mg/dl, group D/Z came out as 0.42 mg/dl and group D/L came outas 0.34 mg/dl. It was observed that CREA value of all groups except D/A groupstatistically decreased when compared to group K.It was found that Lens MDA amount of control group was 16.75 (nmol/100 mglens wet weight) group D was 19.1, group D/G was 11.5, group D/KA was 22, groupD/A was 22.8, group D/Z was 39.55 and group D/L was 21.5. It was observed thatthere was a significant decrease in group D/G.While Lens GSH of control group (nmol GSH/100 mg lens wet weight) was45.85, group D was measured as 6.3, group D/G was measured as 8.78, group D/KAwas measured as 8.9, group D/A was measured as 9.7, group D/Z was measured as7.28 and group D/L was measured as 9.75. The GSH value of all groups wasmeasured as were measured as significantly low when compared to group K. It wasfound that D/G, D/A and D/L groups increased decreasing GSH amountsignificantly, and approximated them to control group.While plasma MDA value of control group was 35.5 nmol, group D wasmeasured as 32.2, group D/G was measured as 26.7 nmol, group D/KA wasmeasured as 39.8 nmol, group D/A was measured as 31.8 nmol, group D/Z wasmeasured as 41.0 nmol and group D/L was measured as 21.0 nmol.While plasma GSH amount of control group was measured as 988 nmol, groupD was measured as 1122 nmol, group D/G was measured as 338 nmol, group D/KAwas measured as 531 nmol, group D/A was measured as 1450 nmol, group D/Z wasmeasured as 575 nmol and group D/Lwas measured as 280 nmol.We concluded that oxidative stress increased in Type I diabetes mellitus intime and caused damage in all tissues and antioxidant defense prevented this damageeven in part. Among the plants that we treated, we observed that especially Ginkgobiloba (maidenhair tree), Aristolochia maurorum (birthwort) and Gentiana olivieri(griseb) reduced diabetic complications significantly, and Olea europaea (olive) andTagetes patula (marigold) also had positive results in some parameters.