| Starch residue,stems and leaves have higher nutritional value,but there are glycosides in them,as a result,safety evaluation must be performed first for the exploitation of these potato by-productsas non-conventional feedstuffs.In this study,potato skin was oven dried,ground and supplemented to the diet,and the growth,internal organs,small intestinal mucosal morphology and caecum microbial diversity in mice were determined to evaluate the toxic effects of potato glycoalkaloids for better understanding of the relationship between glycoalkaloid toxicity and the dose of potato skin supplementation,which will benefit for the exploitation of potato and its by-products as feedstuffs.To further evaluate effects of potato glycoalkaloids on intestinal health,?-chaconine,the most abundant glycoalkaloids in potato,was used to study its effects on the morphology,cycle,apoptosis and mechanical and oxidant damage of small intestinal epithelium in mice and to investigate its effects on the distribution and relative expression of tight junction proteins of cell membrane for better understanding of the toxicity of ?-chaconine to digestive system and relevant mechanisms.The study aimed to initiate research on potato glycoalkaloids for people to pay attention to and to provide theoretical basis for the future utilization of potato by-products and further study on potato glycoalkaloids.Experiment 1: Effects of potato glycoalkaloids on mouse growth and the development of internal organsTo test effects of potato glycoalkaloids on mouse growth and the development of internal organs,a study with kunming mice as experimental animals was conducted in a complete randomization design.Sixty male mice were randomly allocated to 6 groups with 10 each and fed the basal diet plus dried potato skin at 0,2,4,6,8 and 10% of the basal diet,respectively.To make the diets,the basal diet was ground first and re-pelleted after potato skin was added at designated proportions.Mice were adapted to their diets for a week and then adopted for the formal experimental period for 21 d.The experimental results showed that mice in the experimental groups had lower liveweight than those in the control group with a statistically significant difference detectable between the 10% and the control groups(P<0.05)but not between the 2,4,6 and 8% groups and the control(P>0.05).The average liveweight gain decreased with the supplementation of potato skin into the basal diet and all experimental groups had significant lower liveweight gain than the control group(P<0.05),with the 10% group being the lowest and higher than the 2,4,6 and 8% groups(P<0.05).The average daily feed intake decreased in experimental groups with the 10% group being the lowest and significantly lower than the rest groups(P<0.05).The ratio of feed to liveweight gain increased with the addition of potato skin and the 10% group being the highest.On the day 21 of the experiment,mice were killed in the method of cervical vertebra dislocation and internal organs were measured.The results show that:the spleen index of mice decreased,and there was no significant difference between the groups(P>0.05);the liver index increased first and then decreased with the increase of the potato skin level,and the lowest in the 10% additive group,compared with the 0% groups,the difference was significant(P<0.05);there was no significant change in heart index;the kidney index increased first and then decreased with the addition of potato skin,and there was significant difference between the 8% and 10% adding groups compared with the 0% groups(P<0.05).Experiment 2: Effects of potato glycoalkaloids on mouse small intestinal mucosal morphology and caecum microbial diversityTo study effects of potato on mouse small intestinal mucosal morphology and caecum microbial diversity,all mice from Experiment 1 were killed using dislocating cervical vertebra and dissected.Segments in a length of 2-3 cm from the middle portion of duodenum,jejunum and ileum were obtained and treated for sections for histological and morphological studies and the content of caecum was sampled for the analysis of microbial diversity.The results showed that the height of duodenum fine hairs decreased,the depth of crypts increased and the ratio of hair height to crypt depth decreased in all experimental groups in comparison with the control(P<0.05).The height of jejunum fine hairs decreased and a statistically significant difference reached when supplementation was at 8 or 10% compared to the control group(P<0.05).The height of jejunum crypt increased and the ratio of jejunum fine hairs to crypts decreased with all experimental groups significantly differed to the control group(P<0.05).The height of ileum fine hairs in all experimental groups significantly decreased compared to the control group(P<0.05).The depth of ileum crypt increased and the 10% group significantly differed from the control group(P<0.05).The ratio of ileum fine hair height and crypt depth in all experimental groups decreased compared with the control group(P<0.05).Alpha and Beta diversity analyses indicated that glycoalkaloids in potato skin significantly decreased the diversity of microbes in mouse caecum with the diversity index in the 10% group being statistically significantly lower than the control group(P<0.05).Experiment 3: Effects of ?-chaconine on the cell cycle and apoptosis of mouse small intestinal epitheliumsMechanisms underlying the decline of mouse growth and the damage of small intestinal mucous membrane resulted from potato glycoalkaloidswere investigated using the in vitro cell culture technology and flow cytometry to understand effects of glycoalkaloids on mouse intestinal health in the levels of cytology and molecules.?-chaconine,which is the most abundant glycoalkaloid in potato,was used in the in vitro experiment to treat mouse small intestinal epithelium.In vitro Cell culture experiments were carried out,three concentrations of ?-chaconine were treated with 0 g/mL,0.4 g/mL and 0.8 g/mL,each treatment had 3 replicates,the MTT,cell cycle and apoptosis of cells were measured in 24 h,28h and 72 h during cell culture,and the apoptosis was detected by TUNEL.The results showed that 1)the G0/G1 increased(P<0.05)and S stage decreased(P<0.05)with dose and time of incubation.G2/M tended to decrease with dose and incubation time and the difference between the 0.8 ?g/ml and the control groups was significant at 72 h(P<0.05).The rate of apoptosis significantly increased at 24,48 and 72 h in the ?-chaconine treatments compared with the control group(P<0.05).Experiment 4: Effects of cellular mechanical barrier in mouse small intestinal epitheliumTo study effects of ?-chaconine on intestinal membrane permeability and consequent influence on intestinal health and to understand the mechanisms of ?-chaconine affectingthe cellular mechanical barrier of mouse intestinal epitheliums,a study was conducted using thein vitrocell culture,Western blotting,immunofluorescence and fluorescence quantitative PCR techniques.Mouse small intestinal epitheliums were incubated with a series of ?-chaconine concentrations at 0(control),0.4,0.8 ?g/ml of and TEER value,alkaline phosphatase(AKP),lactate dehydrogenase(LDH),and the distribution,relative expression and mRNA expression of ZO-1 and Occludin proteins were measured at 24,48 and 72 h of incubation.The results showed that TEER value tended to decrease with the dose of ?-chaconine and incubation time.Addition of 0.4 ?g/ml of ?-chaconine did not significantly change TEER value at 24 and 48 h of incubation(P>0.05),but 0.8 ?g/ml did(P<0.05).However,after 72 h of incubation,the concentrations at 0.4 and 0.8 ?g/ml both significantly differed from the control(P<0.05).Cellular AKP and LDH activities increased with ?-chaconine dose and incubation time.With an exception of 0.4 ?g/ml treatment at 24 h,both 0.4 and 0.8 ?g/ml treatments increased AKP and LDH activities at 24,48 and 72 h compared with the control(P<0.05).?-chaconinesignificantly changed the distribution of tight junction proteins(ZO-1 and Occludin)in the surface of cell membrane(P<0.05)and significantly decreased the relative expression of ZO-1 and Occludin proteins(P<0.05).The mRNA expression of these proteins in intestinal epitheliums tended to decrease with the dose of supplementation and incubation time and ?-chaconine concentrations at 0.4 and 0.8 ?g/ml resulted in significantly lower expression than the control at 24,48 and 72 h of incubation with an exception of 0.4 ?g/ml at 24 h.Experiment 5: Effects ?-chaconine on the antioxidant ability of mouse small intestinal epitheliumsTo study effects of ?-chaconine on the antioxidant ability of mouse small intestinal epitheliums for the understanding of molecular mechanisms underlying oxidation damage of these cells with ?-chaconine,an experiment was conducted using an in vitrocell culture incubation and fluorescence quantitative PCR techniques.Mouse small intestinal epitheliumswere incubated with 0,0.4 and 0.8 ?g/ml of ?-chaconine and sampled at 24,48 and 72 h for the determination of the contents of SOD and T-GSH,the activities of MDA,CAT,GSH-Px and ?-GCS,and the expression of antioxidant enzyme genes SOD,CAT,GSH-Px and ?-GCS.The results showed that ?-chaconine enhanced the activity of antioxidant enzyme MDA in mouse small intestinal epithelium at 24,48 and 72 h(P<0.05),whereas it decreased T-GSH content and the activities of SOD,CAT,GSH-Px and ?-GCS(P<0.05)for both 0.4 and 0.8 ?g/ml treatments at 24,48 and 72 h except 0.4 ?g/ml at 24 h.The mRNA expression of SOD gene decreased with the dose of ?-chaconine and incubation time.The expression for ?-chaconine treatments was not significantly different from that for the control at 24 h(P>0.05),but differences between the experimental and control groups were significant after 48 and 72 h of incubation(P<0.05).The mRNA expression of CAT and ?-GCS genes decreased also with ?-chaconine dose and incubation time.Addition of ?-chaconine at 0.4 and 0.8 ?g/ml significantly changed the expression at 24,48 and 72 h(P<0.05).A decrease in the mRNA expression of GSH-Px gene was also found with the increase of ?-chaconine dose and incubation time.The expression of this gene was lower for the experimental groups than for the control group at all sampling time(P<0.05)except for 0.4 ?g/ml at 24 h.In conclusion,?-chaconine retarded the development of cells at the stage of G0/G,impacted the synthesis of DNA,and inhibited cell proliferation,resulting in apoptosis which depended on dose and incubation time.?-chaconine also decreased the activity of antioxidant enzymes and the mRNA expression of relevant genes and consequently decreased cellular antioxidant ability.Different concentrations of ?-chaconine differentially damaged cellular membrane permeability,inhibited cytoactivity,and further decreased the relative expression of cellular tight junction proteins ZO-1 and Occludin and mRNA expression of relevant genes,resulting in a decrease in the mechanical barrier function of intestine.?-chaconine accelerated theapoptosis progress of small intestinal epitheliums and decreased mechanical barrier function due to decreased antioxidant ability.As a result,?-chaconine among glycoalkaloids caused the damage of small intestinal mucosal morphology and structure,the shortening of the fine hair heights of duodenum,jejunum and ileum,the increase in the depth of crypt,and the decrease in the ratio of fine hair height and crypt depth,which resulted in a decrease in nutrient uptake by small intestinal membrane,a decrease in Alpha and Beta diversity index of caecum microbes and a decrease in absorption and utilisation of nutrients in intestine,and consequently resulted in adecrease in average liveweight gain and average daily feed intake,and anincrease in the ratio of feed to liveweight gain,which finally decreased in animal performance. |
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