| The purpose of this study was to investigate the effects of dietary Paeoniae radix alba extract on the production performance of raccoon dog and the anti-enteritis mechanism of Albiflorin(ALB).Through feeding experiment and slaughter experiment,the effects of different supplemental levels of Paeoniae radix alba extract on growth performance,nutrient digestibility,nitrogen metabolism,serum biochemistry,small intestinal inflammation and morphology of raccoon dog were studied,so as to screen out the appropriate supplemental level of Paeoniae radix alba extract for raccoon dog during growing period.Bacterial genomic DNA was extracted from the samples which collected from the rectal contents of raccoon dog,then the 16 S V3～V4 genes were sequenced to investigate the effects of dietary Paeoniae radix alba extract on intestinal microbial composition and diversity of raccoon dog.The effects of ALB,the main monoterpene glycoside from Paeoniae radix alba extract,on Methotrexate(MTX)-induced enteritis in rats were evaluated,and its anti-enteritis mechanism was discussed.This study is mainly composed of three parts:Part 1: Effects of dietary Paeoniae radix alba extract on growth performance,nutrient digestibility and small intestine morphology of raccoon dog.This experiment was conducted to investigate the effects of Paeoniae radix alba extract on growth performance,nutrient digestibility,nitrogen metabolism,serum biochemistry,intestinal inflammation and morphology of raccoon dog.Fifty healthy male raccoon dogs(120 ± 5 day-old)were randomly divided into 5 groups with 10 replicates in each group and 1 in each replicate.A single factor randomized trial was designed in the experiment.Five levels of Paeoniae radix alba extract were added to the basic diet,and the addition levels were 0 g/kg,1 g/kg,2 g/kg,4 g/kg,8 g/kg respectively.The prefeeding period lasted for 7 days and the trial period lasted for 40 days.The results showed that: The average daily feed intake in group PRA1 and PRA2 was significantly higher than that in other groups(P<0.01),there were no significant differences in final weight,average daily gain and feed/gain among all groups(P>0.05);The dry matter excretion in group PRA8 was significantly higher than that in other groups(P<0.01)while the dry matter digestibility and protein digestibility in group PRA8 were significantly lower than those in other groups(P<0.01).There was no significant difference in fat digestibility among all groups(P>0.05);Nitrogen deposition in group PRA1 and PRA2 was significantly higher than that in group PRA8(P<0.05);With the increasing of dietary Paeoniae radix alba extract content,alkaline phosphatase activity was firstly increased and then decreased,and the activity of PRA2 was significantly higher than that of PRA0(P<0.05),but there were no significant differences in lactate dehydrogenase,aspartate aminotransferase and alanine aminotransferase among all groups(P>0.05);The activity of serum SOD in PRA4 was significantly higher than that in other groups(P<0.01),and there was no significant difference in GSH-Px and MDA in each group(P>0.05);The content of serum Ig A in group PRA2 was significantly higher than that in other groups(P<0.05),and there was no significant difference in Ig G and Ig M in each group(P>0.05);There was no significant difference in serum total protein,albumin,urea and glucose in each group(P>0.05);There was no significant difference in serum triglyceride,cholesterol,low density lipoprotein and high density lipoprotein(P>0.05);The content of TNF-α in intestinal mucosa in group PRA1 and group PRA2 was significantly lower than that in group PRA0(P<0.05);There was no significant difference in villus height and crypt depth among the groups(P>0.05);The ratio of villus height to crypt depth was significantly increased in group PRA2 compared with group PRA0(P<0.05).In conclusion,under the conditions of this experiment,when the dietary Paeoniae radix alba extract supplemental level is 1～2g/kg,it can improve the feed intake and nitrogen deposition,increase the serum Ig A content,decrease the content of TNF-α in small intestinal mucosa,and increase the ratio of villus height to crypt depth of raccoon dog.Part 2: Effects of dietary Paeoniae radix alba extract on the composition and diversity of intestinal microorganisms in raccoon dog.The purpose of this experiment was to investigate the effects of dietary Paeoniae radix alba extract on the composition and diversity of intestinal microorganisms in raccoon dog.The feeding experiment was as sample as experiment 1.On the 41 st day in the slaughtering experiment,4 raccoon dogs in each group were randomly selected from group PRA0,PRA2,PRA4 and PRA8,and the DNA of bacterial group in their rectal contents was extracted for 16 S V3～V4 sequence analysis.The results showed that:at the phylum level,the dominant bacteria in group PRA0 were Firmicutes(42.33% ± 26.15%),Proteobacteria(32.88%±20.07%),Fusobacteria(9.78%±5.72%);The dominant bacteria in group PRA2 were Proteobacteria(44.81%±21.63%),Firmicutes(30.83%±19.76%),Bacteroidetes(18.50%±21.62%);The dominant bacteria in group PRA4 were Firmicutes(73.78%±12.87%),Fusobacteria(8.13% ± 8.42%),Proteobacteria(5.76% ± 2.62%);The dominant bacteria in group PRA8 were Firmicutes(80.66%±20.40%),Proteobacteria(19.22%±20.34%),Bacteroidetes(0.04%±0.03%).The relative abundance of Firmicutes in group PRA8 was significantly higher than that in group PRA0(P<0.05).At the genus level,the dominant bacteria in group PRA0 were Streptococcus(20.98%±13.14%),Desulfovibrio(14.17%±17.96%),Staphylococcaceae＿Staphylococcus(9.82%±18.45%);The dominant bacteria in group PRA2 were Streptococcus(14.70% ± 23.08%),Bergeyella(8.68% ±17.32%),Lactococcus(6.37%±8.26%);The dominant bacteria in group PRA4 were Streptococcus(20.28% ± 23.15%),Staphylococcaceae＿Staphylococcus(12.74% ± 22.15%),Weissella(8.18% ±16.29%);The dominant bacteria in group PRA8 were Staphylococcaceae＿Staphylococcus(29.79%±12.65%),Enterococcus(11.31%±19.50%),Acinetobacter(10.48%±17.57%).Faith＿pd index showed that there were significant difference between group PRA0 and group PRA8(P<0.05).The results of PCo A analysis showed that there were differences between group PRA8 and other groups based on Jaccard distance and Bray-Curtis distance(P<0.05);The results of difference analysis between groups showed that there was significant separation between group PRA8 and group PRA0 based on Jaccard distance and unweighted Uni Frac distance(P<0.05).To sum up,the dominant bacteria in intestinal of raccoon dog were Firmicutes and Proteobacteria.Paeoniae radix alba extract can significantly increase the richness of Firmicutes and Bacteroidetes.Part 3: Effect of Albiflorin on methotrexate-induced small enteritis in rats and its mechanism.The purpose of this study was to explore the effect of Albiflorin,the main monoterpene component in the Paeoniae radix alba extract,on enteritis induced by MTX in rats and to explore its possible mechanism.Male SD rats were randomly divided into four groups: group control,group methotrexate(MTX),group MTX ALB(20 mg/kg)and group MTX ALB(40 mg/kg).The two groups of ALB was given ALB 20 mg/kg or 40 mg/kg daily for 6 days.The rats in the group control and group MTX were perfused with the same amount of normal saline.After 3 days of intragastric administration of ALB,the rats in group of MTX and the two groups of ALB were intraperitoneally injected with MTX 7 mg/kg/d for 3 days.At the same time,the rats in the group control were injected with the same amount of normal saline.The results showed that: ALB significantly increased body weight and intestinal weight of rats with MTX-induced enteritis(P<0.05).The disease activity index(DAI)score was also decreased after ALB administration(P<0.01).In histological examination,ALB significantly attenuated inflammatory cells infiltration and promoted survival of goblet cells(P<0.01).Additionally,ALB reduced malondialdehyde,glutathione and superoxide dismutase levels and suppressed reactive oxygen species activity in the intestinal tissues of MTX-treated rats(P<0.05).In the detection of inflammatoryassociated factors,we observed that ALB significantly decreased CD68+ cells infiltration(P<0.01),inhibited Myeloperoxidase activity(P<0.01),and suppressed intercellular cell adhesion molecule-1 and cyclooxygenase-2 expression in MTX-treated intestinal tissues(P<0.01).Moreover,inflammasome NLR family pyrin domain containing 3(NLRP3)was found to be increased in MTX-exposed intestine,while ALB effectively inhibited NLRP3 expression(P<0.01),as well as the caspase-1 p20 and interleukin(IL)-1β and 18(P<0.01).Finally,NF-κB p65 phosphorylation and nuclear translocation was also demonstrated to be blocked upon ALB treatment(P<0.01).In conclusion,ALB alleviated MTXinduced enteritis via inhibiting the NF-κB/NLRP3 signaling pathway.Conclusion: to sum up,when the dietary Paeoniae radix alba extract is 1～2 g/kg,it is helpful to increase the feed intake and nitrogen deposition of raccoon dog,increase the content of serum Ig A,decrease the content of TNF-α in intestinal mucosa,increase the ratio of villus height to crypt depth,and improve the richness of intestinal Firmicutes and Bacteroidetes.Albiflorin,the active component of Paeoniae radix alba extract,can inhibit oxidative stress and inflammation,inhibit the activation of NLRP3 inflammatory bodies,regulate the expression of NF-κB,and alleviate the process of enteritis in rats.That is,Albiflorin attenuates methotrexate-induced enteritis by inhibiting NF-κB/NLRP3 signal pathway. |
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