| Objective: The study aims to investigate the anti-hypoxia effects of citrus tangerine pith extract and on the function of intestines in mice under a hypoxia environment,and to analyze the components of flavonoids in order to intribute to the development and utilization of tangerine,and to provide reference for the research of new anti-hypoxia supplements.Methods: 1.Sixty healthy male Balb/c mice were randomly divided into the blank control group,rhodiola control(rhodiola,400 mg/kg),experimental-L group(low dose citrus tangerine pith extract,15 mg/kg),experimental-M(medium dose citrus tangerine pith extract,25 mg/kg)and experimental-H(high dose citrus tangerine pith extract,35mg/kg)groups.The effects of low,medium and high doses of citrus tangerine pith extract in mice exposed to normal pressure airtight hypoxia condition were explored.2.Forty healthy male Balb/c mice were randomly divided into the BN(blank normal oxygen group),BH(blank hypoxia group),TH(citrus tangerine pith extract hypoxia group,15 mg/kg)and RH(rhodiola hypoxia group,400 mg/kg)groups.The mice were gavaged with different foods for 15 consecutive days.From the 6th day,they were exposed to a plateau capsule that simulated the 6000 m altitude.Intestinal movement test was used to explore the effect of citrus tangerine pith extract on intestinal peristalsis function of mice exposed to the hypoxia environment.The morphology of ileum was analyzed by HE staining.The contents of inflammatory factors IL-6,TNF-α and IFN-γ in ileum were determined by ELISA,while the contents of D-LA,DAO,SOD and GSH in serum were determined through ELISA and manual colorimetry,respectively.Occludin and Claudin-1 were localized and semi-quantitatively analyzed with immunohistochemical assay,and their expressions were determined by RT-q PCR method.3.Forty healthy male Balb/c mice were randomly divided into the BN(blank normal oxygen group),BH(blank hypoxia group),TH(citrus tangerine pith extract hypoxia group,15 mg/kg)and RH(rhodiola hypoxia group,400 mg/kg)groups.The mice were gavaged with different foods for 15 consecutive days.From the 6th day,they were exposed to a plateau capsule that simulated the 6000 m altitude.Fecal samples were collected at the end of the experiment,and the changes of gut microbiota were analyzed by 16 S r RNA high-throughput sequencing technology.4.The constituents of flavonoids(quercetin,myricetin,luteolin,kaempferol,apigenin,narirutin,naringin,hesperidin,neohesperidin,nobiletin,naringenin,tangeretin,hesperetin)were analyzed by HPLC.5.Sixth healthy male Balb/c mice were randomly divided into the blank control group,citrus tangerine pith extract group,3-flavonoids group and 9-flavonoids group.The effects of citrus tangerine pith extract flavonoids compounds on mice exposed to normal pressure airtight hypoxia condition were observed.Results: 1.Observations of the survival time of mice under normal pressure airtight hypoxia conditions showed that in comparison to the blank control group,the survival time of mice was extended by 9.79% by rhodiola and 17.15% by citrus tangerine pith extract.The difference was statistically significant(P<0.05).2.(1)The results of small intestine movement experiments indicated that the ink propulsion rate of the BH group was significantly higher than that of the BN group(P<0.05),and that citrus tangerine pith extract significantly reduced the ink propelling rate of the TH group compared with the BH group(P<0.05).(2)The pathological section of ileum showed that compared with the the BN group,the ileum villi in the BH group were collapsed,shortened,broken,and arranged disorderly.Its mucosal layer became thinner and the ileum tissue was infiltrated with inflammatory cells.In the TH group,ileum villi were elongated,complete and orderly,accompanied by thickened mucosa.Morphological measurements of pathological sections demonstrated that the length of ileum villus and the thickness of ileum mucosa in the BH group were significantly lower than of the BN group,while the crypt depth was significantly increased(P<0.05).However,citrus tangerine pith extract induced a significant increase of the length of ileum villus and the thickness of the ileum mucosal layer,but this intervention led to a significant decrease of crypt depth in the BH group(P<0.05).(3)The results of ileum ELISA showed that levels of IL-6,TNF-α and IFN-γ were elevated in the BH group compared with that of the BN group,and that there was significant difference between the former two results(P<0.05).In contrast,the contents of IL-6,TNF-α and IFN-γ were significantly decreased after the intervention of citrus tangerine pith extract(P<0.05).(4)The results of ileum immunohistochemical staining showed that,unlike the BN group,the hypoxia exposure could significantly decrease the expression of tight junction protein in ileum in the BH group(P<0.05).The intervention of citrus tangerine pith extract caused a significant increase in the expression of tight junction protein in the ileum of the TH group(P<0.05).(5)Observations of the relative expression of tight junction protein genes in ileum revealed that,compared with the BN group,the expression of tight junction protein in ileum was significantly decreased in the BH group after hypoxia exposure(P<0.05).After the intervention of citrus tangerine pith extract,the expression of tight junction protein in the ileum of the TH group was significantly increased(P<0.05).(6)Serum ELISA results of mice demonstrated that DLA and DAO in mice of the BH group were more significantly increased than in the BN group upon hypoxia exposure(P<0.05).After the intervention of citrus tangerine pith extract,the serum D-LA content in the TH group was significantly decreased(P<0.05),but the alternation of DAO was insignificant(P>0.05).(7)Serum biochemical results of mice showed that compared with the BN group,SOD in serum of the BH group was significantly increased after hypoxia exposure(P<0.05),while GSH was significantly decreased(P<0.05).After the intervention of citrus tangerine pith extract,SOD content in serum of the TH group was significantly decreased(P<0.05),while GSH was significantly increased(P<0.05).3.(1)The results of α diversity of intestinal microflora of mice after 10 days of hypoxia exposure showed that compared with the BN group,there was a decrease in the Chao1 index and ACE index in the BH group and BH group.Moreover,a more obvious decrease of community richness was observed in the TH group.(2)As shown in the results of community diversity,the Shannon index and Simpson index in the BH and TH groups were slightly increased.The intestinal flora diversity of the TH group was decreased.Nevertheless,there was no significant difference between the three groups(P>0.05).(3)The results of phylum level analysis showed that Firmicutes and Bacteroidetes were two main phyla in the gut microbiota of mice in each group.In contrast to the BN group,the relative abundance of Bacteroidetes in the BH group decreased from 73.52% to 64.93%,while that of Firmicutes increased from 24.78% to33.87% after hypoxia exposure.Upon the intervention of citrus tangerine pith extract,the relative abundance of Bacteroidetes in the TH group decreased from 64.93% to57.19%,while that of Firmicutes increased from 33.87% to 41.06% compared with the BH group.(4)The results of genus level analysis showed that most of the relative abundance was increased in the BH group,including Lactobacillus,Bacteroides,Alloprevotella,Unidentified＿Lachnospiraceae,Unidentified＿Ruminococcaceae,Roseburia and Ruminiclostridium.By contrast,the relative abundance of Akkermansia and Alistipes decreased.After treatment with citrus tangerine pith extract,the TH group had the higher relative abundances of Lactobacillus and Akkermansia than the BH group.However,the lower relative abundances of some bacteria was achieved,including Bacteroidia,Alloprevotella,Unidentified＿Lachnospiraceae,Alistipes,Unidentified＿Ruminococcaceae,Roseburia,Ruminiclostridium and Blautia.4.HPLC analysis showed that the contents of hesperidin,quercetin and narirutin in tangerine pith extract were the highest,which were 288.51,159.32 and 147.58 μg/ m L,respectively.followed by the levels of hesperetin and naringenin,which were 27.03 and24.25 μg/ m L,respectively.Besides,the contents of neohesperidin,naringin,kaempferol and myricetin were the lowest,corresponding to 11.9,8.47,6.06,3.97 μg/m L,respectively.However,other compounds were not detected,such as nobiletin,tangeretin,leteolin or apigenin.5.Compared with the blank control group,the survival time of mice in other groups was prolonged by 15.3%,16.7% and 21.4%,respectively,corresponding to Citrus tangerine pith extract,3-Flavonoids group and 9-Flavonoids group.These differences were statistically significant(P<0.05).However,there were no significant differences between groups of Citrus tangerine pith extract,3-Flavonoids groups,and9-Flavonoids groups(P>0.05).Conclusions: 1.Citrus tangerine pith extract can significantly prolong the survival time of mice under normal pressure airtight hypoxia condition(P<0.05),and possesses a strong anti-hypoxia effect.2.Ten days of exposure to hypoxia condition can impair ileal function,ileal structure and intestinal barrier function in mice.The intervention of citrus tangerine pith extract has a certain protective effect on the ileum of mice under hypoxia exposure conditions,which alleviates intestinal inflammation to some extent and restores the antioxidant function as well as barrier function of the intestinal tract.3.Ten days of exposure to hypoxia condition can change the structure and composition of intestinal flora in mice.After the intervention of citrus tangerine pith extract,the intestinal flora of mice can be altered greatly compared with that of hypoxia exposure,and the abundance of beneficial bacteria in the intestine,especially Lactobacillus,is significantly increased.4.The results of HPLC showed that there are abundant flavonoids in the extract of citrus tangerine pith,and the top three types of flavonoids are hesperidin,quercetin and narirutin.5.The anti-hypoxia effect of citrus tangerine pith extract may be related to its abundant flavonoids components such as hesperidin,quercetin and narirutin. |
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