Çölyak hastaları ve sağlıklı kişilerden ratlara yapılan fekal mikrobiyota transferi sonucu oluşan immünolojik ve histopatolojik değişimlerin araştırılması
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Tarih
2024
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Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Çölyak hastalığı, genetik yatkınlığı olan kişilerde glüten içeren tahıllara (buğday, arpa, çavdar) maruziyet sonucu kalıcı intolerans olarak gelişen, proksimal ince bağırsağın kronik inflamasyonu ile karakterize edilen sistemik otoimmün bir hastalıktır. Son zamanlarda intestinal mikrobiyotanın da hastalığın etyolojisinde rol oynayabileceği gösterilmiştir. İntestinal mikrobiyotanın glüten ile tetiklenen immün patolojiyi, mikrobiyal ortama bağlı olarak değiştirebildiğini gösteren araştırmalar mevcuttur. Bu çalışmanın amacı çölyak hastalığının etyopatogenezinde mikrobiyotanın rolünü aydınlatmak için ratlara fekal mikrobiyota transferi yapılarak nakil sonrası gelişen immünolojik ve histopatolojik değişimlerin araştırılmasıdır. Selçuk Üniversitesi Tıp Fakültesi Hastanesi, Gastroenteroloji birimine başvurup yeni tanı almış (serolojik ve histopatolojik olarak doğrulanmış, glütensiz diyete başlamamış) çölyak hastalarının ve sağlıklı donör grubunun dışkı örnekleri çalışma için alınmıştır. Selçuk Üniversitesi Deneysel Tıp ve Uygulama Merkezinde yetiştirilmiş on iki haftalık 26 tane Wistar Albino erkek rat üç gruba;1) Hasta grubu; çölyak grubu,10 adet rat, 2) Sağlıklı donör grubu, 10 adet rat ve 3) Kontrol grubu; fosfat tamponlu salin grubu, 6 adet rat ayrılmıştır. Ratlara fekal mikrobiyota transfer öncesi mikrobiyota temizliği için 3 hafta süresince (21 gün) oral yolla (oral gavajla ve sularına katılarak) antibiyotik tedavisi (ampisilin, neomisin, metronidazol, vankomisin) uygulanmıştır. Antibiyotik tedavisi sona erdikten sonra, fekal mikrobiyota transferi; hasta grubu ratlarına glütensiz diyete başlamamış çölyak hastalarından hazırlanmış dışkı süspansiyonları, sağlıklı rat grubuna sağlıklı donörlerden alınmış hazırlanmış dışkı süspansiyonları ve kontrol grubu ratlarına ise sadece fosfat tamponlu salin günde 200 mikrolitre olacak şekilde 3 hafta süresince (her hafta 5 gün ve iki günlük periyodik arayla) 16 mm'lik oral gastrik gavajla yapılmıştır. Deneyin 50. gününde 26 rata genel anestezi altında servikal dislokasyon ile ötenazisi yapılmıştır. Genel anestezi altındaki ratlardan ilk önce kan alınmıştır. Daha sonra sakrifiye edilen ratların duodenum (D1-Bulbus ve D2-Distal), ileum ve kolonundan kesitler alınarak hazırlanmış ve Hematoksilen-Eozin boyama ile boyanarak histopatolojik olarak incelenmiştir. Duodenum kesitlerinde önemli rol oynayan bazı proinflamatuar sitokinlerin (IL-15, IL- 21, TNF-α ve IFN-α) mRNA ekspresyon düzeylerine bakılmıştır. Ayrıca, ratlardan alınmış kan örneklerinden hastalığın etiyolojisinde rol olan bazı proinflamatuar sitokinlerin (IFN-γ, IL-17) ve β-aktinin de serum düzeyleri ELISA yöntemiyle incelenmiştir. Sonuçlara göre fekal mikrobiyota transferi yapıldıktan bir hafta sonra, sağlıklı donörlerden gaita transferi yapılan ratlara ve sadece fosfat tamponlu salin transferi yapılmış kontrol grubu ratlarına kıyasla hasta grubu ratlarının zayıfladığı ve ağırlık kaybettiği gözlemlenmiştir. Çölyak hastalarından gaita transferi yapılan ratların duodenum kesitlerinde yapılmış histopatolojik incelemelerde, sağlıklı donörlerden gaita transferi yapılan ratlara ve sadece fosfat tamponlu salin transferi yapılmış kontrol grubu ratlarına göre intraepitelyal lenfosit sayısında artış (>20), değişen şiddetlerde villus-kript oranında azalma ve villöz kütleşme saptanmıştır. Hastalardan dışkı transfer yapılan ratlarda proinflamatuar sitokinlerin (IL-15, IL-21, TNF-α ve IFN-α) mRNA ekspresyon seviyeleri, sağlıklı dönerlerden dışkı transferi yapılan ratlara ve fosfat tamponlu salin transferi yapılmış kontrol grubu ratlara kıyasla anlamlı şekilde yüksek bulunmuştur (p<001). Ayrıca, çölyak hastalarından dışkı transferi yapılmış ratların bağırsaklarında saptanan patalojik değişikliklerle proinflamatuar sitokinlerin (IL-15, IL-21, TNF-α ve IFN-α) mRNA düzeyleri arasındaki pozitif korelasyon istatistiksel olarak anlamlı saptanmıştır (p<001). Serolojik sonuçlara göre ise hasta grubu ratlarının proinflamatuar sitokinlerin (IL-17, IFN-γ) ve β-aktin düzeylerinin sağlıklı dönerlerden dışkı transferi yapılan ratlara ve fosfat tamponlu salin transferi yapılmış kontrol grubu ratlara göre anlamlı şekilde yüksek saptanmıştır (p<001). Bundan başka, hasta grubu ratlarında saptanan proinflamatuar sitokinlerin (IL-17, IFN-γ) ve β-aktin düzeylerinin aynı grup ratların bağırsaklarında saptanan patolojik değişikliklerle arasındaki uyum istatistiksel olarak anlamlı bulunmuştur (p<001). Çölyak hastalarının patolojik sonuçları ile hasta grubu ratlarının histopatolojik sonuçları karşılaştırıldığın da orta düzeyde (% 66,7) pozitif bir korelasyon görülmüştür. Sonuç olarak çölyak hastalarından fekal transfer yapılmış ratlarda, sağlıklı dönerlerden gaita transferi yapılan ratlara ve fosfat tamponlu salin transferi yapılmış kontrol grubu ratlarına göre hem bağırsaklarında histopatalojik hasar (intraepitelyal lenfosit sayısında artış, değişen şiddetlerde villus-kript oranında azalma ve villöz kütleşme) hem de proinflamatuar sitokinlerin (IL-15, IL-21, IL-17, TNF-α, IFN-α, IFN-γ)ve β-aktinin düzeylerinde artış saptanmıştır. Bu nedenle bu çalışmanın sonuçları tıbbi literatürde deney hayvanı modelinde in vivo olarak çölyak hastalığının etyopatogonezinde mikrobiyotanın önemimi fekal mikrobiyota transferiyle gösteren ilk özgün verilerden biridir. Ayrıca, bu çalışmadan elde edilen veriler, hem çölyakla mikrobiyota arasındaki patofizyolojik mekanizmaları anlamak hem de hastalığın seyrine etki edebilecek alternatif tedavilerin araştırılacağı bilimsel araştırmaların yapılabilmesine katkı sağlayacaktır.
Celiac disease is a systemic autoimmune disease characterized by chronic inflammation of the proximal small intestine, which develops as a permanent intolerance as a result of exposure to gluten-containing cereals (wheat, barley, rye) in genetically predisposed individuals. Recently, it has been shown that intestinal microbiota may also play a role in the etiology of the disease. There are studies showing that intestinal microbiota can change gluten-triggered immune pathology depending on the microbial environment. The aim of this study was to investigate the immunological and histopathological changes that developed after fecal microbiota transfer to rats in order to elucidate the role of microbiota in the etiopathogenesis of celiac disease. Stool samples of newly diagnosed celiac patients (serologically and histopathologically confirmed, not on a gluten-free diet) who applied to the Gastroenterology Unit of Selçuk University Medical Faculty Hospital and a healthy donor group were taken for the study. Twelve-week-old 26 Wistar Albino male rats, raised in Selcuk University Experimental Medicine and Application Center, were divided into three groups; 1) Patient group; celiac group, 10 rats, 2) Healthy donor group, 10 rats and 3) Control group; phosphate buffered saline group, 6 rats. Before transferring the fecal microbiota to the rats, antibiotic treatment (ampicillin, neomycin, metronidazole, vancomycin) was administered orally (by oral gavage and by adding to their water) for 3 weeks (21 days) for microbiota cleansing. After the antibiotic treatment ended, fecal microbiota transfer was performed by 16 mm oral gastric gavage for 3 weeks (5 days a week with two-day periodic intervals) with fecal suspensions prepared from celiac patients who had not started a gluten-free diet for the patient group rats, with fecal suspensions prepared from healthy donors for the healthy group rats, and with only phosphate-buffered saline at 200 microliters per day for the control group rats. Blood was first taken from rats under general anesthesia. Then, sections were taken from the duodenum (D1-Bulbus and D2-Distal), ileum and colon of the sacrificed rats and were prepared and stained with Hematoxylin-Eosin and examined histopathologically. The mRNA expression levels of proinflammatory cytokines, IL-15, IL-21, TNF-α and IFN-α, which play an important role in the pathogenesis of the disease, were examined in tissue sections (duodenum). In addition, serum levels of some proinflammatory cytokines (IFN-γ, IL-17) and β-aktin, which play a role in the etiology of the disease, were examined using the ELISA method in blood samples taken from rats.In addition, serum levels of some proinflammatory cytokines (IFN-γ, IL-17) and β-aktin, which play a role in the etiology of the disease, were examined using the ELISA method in blood samples taken from rats. On the 50th day of the experiment, 26 rats were euthanized by cervical dislocation under general anesthesia. Blood was first taken from rats under general anesthesia. Then, sections were taken from the duodenum (D1-Bulbus and D2-Distal), ileum and colon of the sacrificed rats and were prepared and stained with Hematoxylin-Eosin and examined histopathologically. The mRNA expression levels of some proinflammatory cytokines (IL-15, IL-21, TNF-α and IFN-α) that play an important role in the pathogenesis of the disease were examined in duodenum sections. In addition, serum levels of some proinflammatory cytokines (IFN-γ, IL-17) and β-actin, which play a role in the etiology of the disease, were examined using the ELISA method in blood samples taken from rats. According to the results, one week after the fecal microbiota transfer, it was observed that the patient group rats became thinner and lost weight compared to the rats that received stool transfer from healthy donors and the control group rats that received only phosphate buffered saline transfer. Histopathological examinations of duodenum sections of rats that received stool transfer from celiac patients revealed an increase in the number of intraepithelial lymphocytes (>20), a decrease in the villus-crypt ratio of varying severity, and villous massing compared to rats that received stool transfer from healthy donors and the control group rats that received only phosphate-buffered saline transfer. The mRNA expression levels of proinflammatory cytokines (IL-15, IL-21, TNF-α and IFN-α) in rats that underwent feces transfer from patients were found to be significantly higher compared to rats that underwent feces transfer from healthy donors and the control group rats that underwent phosphate buffered saline transfer (p<001). In addition, the positive correlation between the pathological changes detected in the intestines of rats that received fecal transfer from celiac patients and the mRNA levels of proinflammatory cytokines (IL-15, IL-21, TNF-α and IFN-α) was found to be statistically significant (p<001). According to serological results, proinflammatory cytokines (IL-17, IFN-γ) and β-actin levels were found to be significantly higher in the patient group rats than in the rats that underwent feces transfer from healthy donors and in the control group rats that underwent phosphate buffered saline transfer (p<001). Furthermore, the consistency between the proinflammatory cytokines (IL-17, IFN-γ) and β-actin levels detected in the patient group rats and the pathological changes detected in the intestines of the same group rats was found to be statistically significant (p<0.001). When the pathological results of celiac patients were compared with the histopathological results of the patient group rats, a moderate (66.7%) positive correlation was observed. In conclusion, in rats that underwent fecal transfer from celiac patients, histopathological damage in the intestines (increased intraepithelial lymphocyte count, decreased villus-crypt ratio of varying severity and villous massing) and increased levels of proinflammatory cytokines (IL-15, IL-21, IL-17, TNF-α, IFN-α, IFN-γ) and β-actinin were detected compared to rats that underwent fecal transfer from healthy donors and to control rats that underwent phosphate buffered saline transfer. Therefore, the results of this study are one of the first original data in the medical literature showing the importance of microbiota in the etiopathogenesis of celiac disease in an in vivo experimental animal model via fecal microbiota transfer. In addition, the data obtained from this study will contribute to scientific research to understand the pathophysiological mechanisms between celiac and microbiota and to investigate alternative treatments that may affect the course of the disease.
Celiac disease is a systemic autoimmune disease characterized by chronic inflammation of the proximal small intestine, which develops as a permanent intolerance as a result of exposure to gluten-containing cereals (wheat, barley, rye) in genetically predisposed individuals. Recently, it has been shown that intestinal microbiota may also play a role in the etiology of the disease. There are studies showing that intestinal microbiota can change gluten-triggered immune pathology depending on the microbial environment. The aim of this study was to investigate the immunological and histopathological changes that developed after fecal microbiota transfer to rats in order to elucidate the role of microbiota in the etiopathogenesis of celiac disease. Stool samples of newly diagnosed celiac patients (serologically and histopathologically confirmed, not on a gluten-free diet) who applied to the Gastroenterology Unit of Selçuk University Medical Faculty Hospital and a healthy donor group were taken for the study. Twelve-week-old 26 Wistar Albino male rats, raised in Selcuk University Experimental Medicine and Application Center, were divided into three groups; 1) Patient group; celiac group, 10 rats, 2) Healthy donor group, 10 rats and 3) Control group; phosphate buffered saline group, 6 rats. Before transferring the fecal microbiota to the rats, antibiotic treatment (ampicillin, neomycin, metronidazole, vancomycin) was administered orally (by oral gavage and by adding to their water) for 3 weeks (21 days) for microbiota cleansing. After the antibiotic treatment ended, fecal microbiota transfer was performed by 16 mm oral gastric gavage for 3 weeks (5 days a week with two-day periodic intervals) with fecal suspensions prepared from celiac patients who had not started a gluten-free diet for the patient group rats, with fecal suspensions prepared from healthy donors for the healthy group rats, and with only phosphate-buffered saline at 200 microliters per day for the control group rats. Blood was first taken from rats under general anesthesia. Then, sections were taken from the duodenum (D1-Bulbus and D2-Distal), ileum and colon of the sacrificed rats and were prepared and stained with Hematoxylin-Eosin and examined histopathologically. The mRNA expression levels of proinflammatory cytokines, IL-15, IL-21, TNF-α and IFN-α, which play an important role in the pathogenesis of the disease, were examined in tissue sections (duodenum). In addition, serum levels of some proinflammatory cytokines (IFN-γ, IL-17) and β-aktin, which play a role in the etiology of the disease, were examined using the ELISA method in blood samples taken from rats.In addition, serum levels of some proinflammatory cytokines (IFN-γ, IL-17) and β-aktin, which play a role in the etiology of the disease, were examined using the ELISA method in blood samples taken from rats. On the 50th day of the experiment, 26 rats were euthanized by cervical dislocation under general anesthesia. Blood was first taken from rats under general anesthesia. Then, sections were taken from the duodenum (D1-Bulbus and D2-Distal), ileum and colon of the sacrificed rats and were prepared and stained with Hematoxylin-Eosin and examined histopathologically. The mRNA expression levels of some proinflammatory cytokines (IL-15, IL-21, TNF-α and IFN-α) that play an important role in the pathogenesis of the disease were examined in duodenum sections. In addition, serum levels of some proinflammatory cytokines (IFN-γ, IL-17) and β-actin, which play a role in the etiology of the disease, were examined using the ELISA method in blood samples taken from rats. According to the results, one week after the fecal microbiota transfer, it was observed that the patient group rats became thinner and lost weight compared to the rats that received stool transfer from healthy donors and the control group rats that received only phosphate buffered saline transfer. Histopathological examinations of duodenum sections of rats that received stool transfer from celiac patients revealed an increase in the number of intraepithelial lymphocytes (>20), a decrease in the villus-crypt ratio of varying severity, and villous massing compared to rats that received stool transfer from healthy donors and the control group rats that received only phosphate-buffered saline transfer. The mRNA expression levels of proinflammatory cytokines (IL-15, IL-21, TNF-α and IFN-α) in rats that underwent feces transfer from patients were found to be significantly higher compared to rats that underwent feces transfer from healthy donors and the control group rats that underwent phosphate buffered saline transfer (p<001). In addition, the positive correlation between the pathological changes detected in the intestines of rats that received fecal transfer from celiac patients and the mRNA levels of proinflammatory cytokines (IL-15, IL-21, TNF-α and IFN-α) was found to be statistically significant (p<001). According to serological results, proinflammatory cytokines (IL-17, IFN-γ) and β-actin levels were found to be significantly higher in the patient group rats than in the rats that underwent feces transfer from healthy donors and in the control group rats that underwent phosphate buffered saline transfer (p<001). Furthermore, the consistency between the proinflammatory cytokines (IL-17, IFN-γ) and β-actin levels detected in the patient group rats and the pathological changes detected in the intestines of the same group rats was found to be statistically significant (p<0.001). When the pathological results of celiac patients were compared with the histopathological results of the patient group rats, a moderate (66.7%) positive correlation was observed. In conclusion, in rats that underwent fecal transfer from celiac patients, histopathological damage in the intestines (increased intraepithelial lymphocyte count, decreased villus-crypt ratio of varying severity and villous massing) and increased levels of proinflammatory cytokines (IL-15, IL-21, IL-17, TNF-α, IFN-α, IFN-γ) and β-actinin were detected compared to rats that underwent fecal transfer from healthy donors and to control rats that underwent phosphate buffered saline transfer. Therefore, the results of this study are one of the first original data in the medical literature showing the importance of microbiota in the etiopathogenesis of celiac disease in an in vivo experimental animal model via fecal microbiota transfer. In addition, the data obtained from this study will contribute to scientific research to understand the pathophysiological mechanisms between celiac and microbiota and to investigate alternative treatments that may affect the course of the disease.
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Samadzade, R. (2024). Çölyak hastaları ve sağlıklı kişilerden ratlara yapılan fekal mikrobiyota transferi sonucu oluşan immünolojik ve histopatolojik değişimlerin araştırılması. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
