Seasonal Modification In The Intestinal Epithelial Cells Of Small Intestine Of Chinese Soft-shelled Turtle,Pelodiscus Sinensis

Intestinal epithelial cells provide the initial line of mucosal host defense in the intestine.The intestine must function first as an organ of absorption and digestion while simultaneously protecting itself from the vast bacterial flora,array of food antigens,and physiochemical stresses caused by digestive and microbial products.Thus,the intestinal epithelium must be permeable to nutrients and fluids.However,remain impermeable to potentially harmful chemicals and pathogens.The intestine also must differentiate between commensal organisms whose growth is encouraged and enteric pathogens that need to be suppressed or eliminated.The single layer of cells comprising the intestinal epithelium is critical for the maintenance of dynamic barrier.For instance,the increased epithelial permeability of intestinal barriers may initiate and maintain persistent inflammation in intestinal inflammatory diseases.It is well known that the pathological conditions disturb the integrity of intestinal epithelial barriers.However,seasonal variation affects the morphology and functionality of intestinal epithelial cells of the small intestine of reptiles is largely unknown.Unlike mammals,there has been few studies on the intestinal epithelial cells in non-mammalian species.The Chinese soft-shelled turtle(Pelodiscus sinensis),belongs to the reptiles,is one of the most important commercially cultured aquaculture species in Asian countries,especially in Chin.This species is well adapted to an aquatic environment and,is famous for its high nutritional and pharmacological values.Although very little information available regarding,seasonal variation affects the morphology and functional activity of intestinal epithelial cells within the small intestine of reptiles.Furthermore,in many studies autophagy responds in the intestinal epithelial cells provide defense mechanism against harmful pathogens.The role of autophagic pathway in the small intestine during hibernation remains obscure.A wide range of physiological and pathological functions are ascribed to exosomes present in the gastrointestinal tract.But there is no published record the intestinal epithelial cells secrete multivesicular bodies and exosome in small intestine of reptiles during hibernation period.The small intestine consists of different types of intestinal epithelial cells nevertheless,mitochondria-rich cells in small intestine are still covered.Intestinal epithelial cells secrete ion transport proteins during hibernation period,but this mechanism is still remain unclear.The present study clearly demonstrates the seasonal effect on the intestinal epithelial barriers and associated innate immune response of intestinal cells of small intestine during hibernation period of Chinese soft-shelled turtle.Besides that,we have investigated role of autophagy in enterocytes of small intestine during hibernation period.We also explore the biological process of multivesicular bodies and their exosomes in intestinal epithelial cells of small intestine during hibernation.Furthermore,we demonstrate for the first time mitochondriarich cells:a novel type of concealed cell in small intestine of turtle.Finally,current study demonstrates the seasonal exploration of mitochondria-rich cells and its ion transport proteins in small intestine of Chinese soft-shelled turtle.Thus the current study has provided the comprehensive knowledge about seasonal variation affects the morphology and functionality of intestinal epithelial cells of small intestine.Further studies will be required to explore these mechanism in detail in vitro at molecular level.Experiment-1 Effect of seasonal variance on intestinal epithelial barriers and the associated innate immune response of the small intestine of the Chinese soft-shelled turtlesIt is conceivable that pathological conditions can cause intestinal barrier disruption and innate immune dysfunction.However,very limited information has been reported on the effect of seasonal variance on intestinal barriers and innate immunity.The present study was designed to investigate the seasonal variance in intestinal epithelial barriers and the associated innate immune response of turtle intestines during hibernation and nonhibernation periods.Goblet cells(GCs)demonstrated dynamic actions of the mucosal barrier with strong Muc2 protein expression during hibernation.However,weak Muc2 expression during nonhibernation was confirmed by immunohistochemistry,immunofluorescence and immunoblotting.Furthermore,light and transmission electron microscopy revealed that the hypertrophy of GCs resulted in the hypersecretion of mucus granules(MGs)and created a well-developed mucosal layer during hibernation.The absorptive cells(ACs),forming a physical barrier of tight junctions,and desmosomes were firmly anchored during hibernation.Conversely,during nonhibernation,the integrity of tight junctions,adherence junctions and desmosomes was noticeable expanded,causing increased paracellular permeability.As further confirmation,there was strong zonula occluden-1(ZO-1)and connexins 43(Cx43)protein expression during hibernation and weak ZO-1 and Cx43 expression during nonhibernation.Moreover,the expression level of the innate immune response proteins Toll-like receptors 2 and 4(TLR2 and 4)were enhanced during hibernation and were reduced during nonhibernation.These results provide rich information about the seasonal fluctuations that interrupt intestinal epithelial barriers and innate immune response,which might be essential for protection and intestinal homeostasis.Experiment-2 In vivo up-regulation of autophagy in enterocytes of small intestine in Chinese soft-shelled turtle during hibernationMany studies have focused on how autophagy plays an important role in intestinal homeostasis under pathological conditions.However,its role in the intestine during hibernation remains unclear.In the current study,we characterized in vivo up-regulation of autophagy in enterocytes of the small intestine of Chinese soft-shelled turtle during hibernation.Autophagy specific markers were used to confirm the existence of autophagy in enterocytes through immunohistochemistry(IHC),immunofluorescence(IF),and immunoblotting.IHC staining indicated strong positive immunoreactivity of the autophagy related gene(ATG7),microtubule-associated protein light chain(LC3),and lysosomalassociated membrane protein 1(LAMP1)within the mucosal surface during hibernation,and poor expression during non-hibernation.IF staining results showed the opposite tendency for ATG7,LC3 and sequestosome 1(p62).During hibernation ATG7 and LC3 showed strong positive immunosignaling within the mucosal surface,while,p62 showed strong positive immunosignaling during non-hibernation.Similar findings were confirmed by immunoblotting.Moreover,the ultrastructural components of autophagy in enterocytes were revealed by transmission electron microscopy(TEM).During hibernation the cumulative formation of phagophores and autophagosomes were closely associated with well-developed rough endoplasmic reticulum in enterocytes.These autophagosomes were overlapping with lysosomes,multivesicular bodies,and degraded mitochondria to facilitate the formation of autophagolysosome,amphisomes and mitophagy in enterocytes.Immunoblotting showed the expression level of PTEN-induced kinase 1(PINK1)and SOFT-SEHELLED TURTLE,PELODISCUS SINENSIS adenosine monophosphate-activated protein kinase(AMPK)was enhanced during hibernation.Furthermore,the exosome secretion pathway of early-late endosomes and multivesicular bodies were closely linked with autophagosomes in enterocytes during hibernation.These findings suggest that the entrance into hibernation is a main challenge for reptiles to maintain homeostasis and cellular quality control in the intestine.Experiment-3 In vivo multivesicular body and exosome secretion in the intestinal epithelial cells of turtles during hibernationThe present study was designed to investigate the in vivo biological processes of multivesicular bodies(MVBs)and exosomes in mitochondria-rich cells(MRCs),goblet cells(GCs)and absorptive cells(ACs)in turtle intestine during hibernation.The exosome markers,cluster of differentiation 63(CD63)and tumor susceptibility gene 101(TSG101),were positively expressed in intestinal villi during turtle hibernation.The distribution and formation processes of MVBs and exosomes in turtle MRCs,GCs and ACs were further confirmed by transmission electron microscopy(TEM).During hibernation,abundantly secreted early endosomes(ees)were localized in the luminal and basal cytoplasm of the MRCs and ACs,and late endosomes(les)were dispersed with the supranuclear part of the MRCs and ACs.Many "heterogeneous" MVBs were identified throughout the cytoplasm of the MRCs and ACs.Interestingly,the ees,les and MVBs were detected in the cytoplasm of the GCs during hibernation;however,they were absent during non-hibernation.Furthermore,the exocytosis pathway of exosomes and autophagic vacuoles were observed in the MRCs,GCs and ACs during hibernation.In addition the number of different MVBs with intraluminal vesicles(ILVs)and heterogeneous endosome-MVB-exosome complexes was significantly increased in the MRCs,GCs and ACs during hibernation.All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes,which are essential for mucosal immunity,during hibernation.Experiment-4 Mitochondria-rich cells:A novel type of concealed cell in the small intestine of Chinese soft-shelled turtles(Pelodiscus sinensis)Although some studies have been conducted over the past few decades,the existence of mitochondria-rich cells(MRCs)in reptiles is still obscure.This is the first study to uncover the presence of MRCs in the small intestine of Chinese soft-shelled turtles.In this study,we investigated the ultrastructural characteristics of MRCs and the secretion of different ion transport proteins in the small intestine of Pelodiscus sinensis.Transmission electron microscopy revealed that the ultrastructural features of MRCs are clearly different from those of other cells.The cytoplasmic density of MRCs was higher than absorptive epithelial cells(AECs)and goblet cells(GCs).MRCs possessed abundant heterogeneous mitochondria and an extensive tubular system in the cytoplasm.However,the AECs and GCs completely lacked tubular system.Statistical analysis showed that the diameter and quantification of mitochondria were highly significant in MRCs.Mitochondrial vacuolization and despoiled mitochondria were closely associated with autophagosomes in MRCs.The multivesicular bodies(MVBs)and exosome secretion pathway were observed in MRCs.Immunohistochemical staining of ion transport proteins indicated positive immunoreactivity of Na+K-ATPase(NKA)and Na+/K+/2C1cotransporter(NKCC)at basal region of mucosal surface.Likewise,the immunofluorescence staining results showed the strong positive localization of NKA,NKCC and carbonic anhydrase(CA)at basal and apical region of mucosal surface of small intestine.The above findings suggested that the MRCs provide support and regulate cellular ions for intestinal homeostasis and provide energy for cellular quality control in intestine.Experiment-5 Seasonal exploration of ultrastructure and Na+/K+-ATPase,Na+/K+/2Cl-cotransporter of mitochondria-rich cells in the small intestine of turtlesDespite the exploration of mitochondria-rich cells(MRCs)in different animal classes,very limited information has been documented about MRCs in reptiles.The present study was designed to investigate the effect of seasonal variation on the cell ultrastructure and ion transport protein activity of MRCs during hibernation and non-hibernation of Chinese soft-shelled turtle’s intestine.Transmission electron microscopy revealed that,during hibernation the high-density cytoplasm of MRCs occupied large cross-sectional area and showed heterogeneous abundance of mitochondria and an expanded extensive tubular system as compared to non-hibernation.During hibernation the cytoplasm of MRCs exhibited more mitochondrial vacuolization,autophagosomes,phagophore formation and well-structured endoplasmic reticulum.During hibernation,MRCs connected with absorptive cells through wide interdigitation,and created tight junction and more desmosomes as compared to non-hibernation.Immunohistochemistry and immunofluorescence showed,the strong immunopositive reactions and immunosignaling of Na+/K+-ATPase(NKA)and Na+/K+/2Cl-cotransporter(NKCC)at basolateral region of mucosal surface of intestine during hibernation.However,weak immunopositive reactions and immunosignaling of NKA and NKCC during non-hibernation.The statistical analysis showed that the number and size of MRCs with NKA-associated immunoreactivity were significantly increased during hibernation.NKA and NKCC mRNA expression was significantly increased during hibernation via qPCR.Further confirmed,the intensity of NKA and NKCC proteins was more elevated during hibernation than non-hibernation shown by immunobloting.However,the concentrations of the plasma ions Na+ and Clwere significantly higher during hibernation;conversely,K+concentration was significantly higher during non-hibernation.The findings suggest that the potential role of MRCs is affected by seasonal fluctuations,during which intestinal homeostasis and hydromineral balance are essential for turtles.