| Background:Functional gastrointestinal diseases(FGIDs)refer to a group of diseases which have gastrointestinal symptoms without obvious organic changes.FGIDs are always combined with mental diseases,such as anxiety and depression,and some patients with psychological disorders also represent gastrointestinal symptoms.Both FGIDs and psychological disorders have sexual divergence.The levels of ovarian hormone and the status of menopause influence the incidence and severity of both diseases.While visceral hypersensitivity is alleviated with the decrease of ovarian hormones,anxiety and depression are aggravated.Gut microbiota is also significantly influenced by ovarian hormones.Compared with premenopausal women,the structure,abundance and diversity of intestinal flora are significantly altered in postmenopausal women.Berberine(BBR)is able to ameliorate colorectal cancer,arthritis and osteoporosis through regulating gut microbiota.However,whether BBR could improve the anxiety-like behaviors of female ovariectomized rats via regulating intestinal microbes has not been demonstrated.BBR is also capable to alleviate type 2 diabetes,obesity and energy metabolism by modulating intestinal flora.Ovariectomy induces abnormality in glucose and lipid metabolism,the regulatory effects and possible mechanisms of BBR on such kind of metabolic abnormality deserve further study.As a kind of soy isoflavone,daidzein can be metabolized into equol by gut microbes.Equol is capble to activate estrogen receptor ? and protect central nervous system.However,whether BBR could influence the levels of equol via regulating gut microbiota is unclear.Objective:1.To observe the influence of berberine on the levels of equol in female ovariectomized rats;2.To observe the influence of berberine on anxiety-like behaviors and metabolic abnormalities in female ovariectomized rats.Methods:1.Part 1:A week after ovariectomy,11 week female specific pathogen free(SPF)rats were treated with BBR or vehicle through oral gavage for 28 days,and then fecal samples were collected.Both open field test and elevated plus maze test were conducted to detect the anxiety-like behaviors.After the behavior test,all rats were sacrificed and serum samples were collected.A week after ovariectomy,11 week female germ free(GF)rats were treated with BBR or vehicle through oral gavage for 28 days,and then fecal samples were collected.Both open field test and elevated plus maze test were conducted to detect the anxiety-like behaviors.After the behavior tests,all rats were sacrificed and serum samples were collected.A week after ovariectomy,11 week female GF rats were transplanted with feces from SPF ovariectomized rats and BBR treated rats,and the fecal suspensions were gavaged for 3 consecutive days.After 28 days of colonization,fecal samples were collected.Both open field test and elevated plus maze test were conducted to detect the anxiety-like bahaviors.Then all rats were sacrificed and serum samples were collected.For fecal samples,the 16S rRNA sequencing was utilized to analyze intestinal microbiota,and the liquid chromatography-mass spectrometry(LC-MS)was used to analyze the levels of equol.For serum samples,the enzyme linked immunosorbent assay(ELISA)was applied to detect the levels of estradiol,and the LC-MS was utilized to measure the levels of progesterone and equol.2.Part2:A week after ovariectomy,11 week female SPF rats were treated with BBR or vehicle through oral gavage for 28 days.Then all rats were sacrificed and samples were collected.For fecal samples,the 16S rRNA sequencing was utilized to analyze gut microbiota,and the non-targeted metabolomics was used to detect differential metabolites and metabolic pathways.For serum samples,the auto biochemical analyzer was applied to measure the blood glucose,total cholesterol,triglyceride and high-density lipoprotein,and the non targeted-metabolomics was used to determine differential metabolites and metabolic pathways.Results:1.Part 1:(1)Compared with sham operated SPF female rats,ovariectomized SPF rats exhibited anxiety-like behaviors,and BBR treatment significantly alleviated the anxiety.The levels of equol and the ratio of equol to daidzein in both feces and serum were significantly decreased after ovariectomy,while elevated after BBR administration.Microbiota sequencing showed that,after ovariectomy,the abundance of Lactobacillus,Akkermansia,Bacteroides and Bifidobacterium were significantly dercreased,while BBR treatment elevated their abundance significantly.The abundance of Akkermansia was positively correlated with that of Bacteroides,Parabacteroides and Lactobacillus.The abundance of Lactobacillus,Akkermansia and Bacteroides were positively correlated with the improvement of anxiety-like behaviors and the levels of equol in both feces and serum.(2)Compared with sham operated GF female rats,ovariectomized GF rats exhibited anxiety-like behaviors,while BBR treatment could not ameliorate the anxiety.The levels of daidzein could be measured in both feces and serum of GF rats,while the levels of equol were not able to be detected.(3)Compared with ovariectomized GF rats transplanted with feces from SPF ovariectomized rats,ovariectomzied GF rats transplanted with feces from SPF BBR-treated rats exhibited less anxiety-like behaviors and higher levels of both equol and equol to daidzein in feces and serum Microbiota sequencing showed that,the abundance of Lactobacillus,Akkermansia,and Bacteroides were significantly higher in GF rats transplated with feces from BBR-treated rats compared with GF rats transplanted with feces from SPF ovariectomized rats.The abundance of Lactobacillus and Akkermansia were positively correlated with the improvement of anxiety-like behaviors and the levels of equol in serum.2.Part 2:(1)Compared with sham operated SPF female rats,the body weight,levels of blood glucose,total cholesterol and triglyceride were significantly elevated after ovariectomy,and BBR treatment significantly decreased the body weight,levels of blood glucose and lipid.Compared with sham operated GF female rats,the body weight,levels of blood glucose and lipid were also significantly increased after ovariectomy,however,BBR treatment showed no effects.(2)Microbiota sequencing showed that,after ovariectomy,the abundance of Ruminococcus and Prevotella were significantly increased.BBR administration significantly elevated the abundance of Lactobacillus and Bifidobacterium,and decreased the abundance of Ruminococcus and Prevotella.The abundance of Bifidobacterium was negatively correlated with the levels of blood glucose and triglyceride,the abundance of Prevotalla and Ruminococcus were positively correlated with the levels of blood triglyceride.KEGG pathway annotation of intestinal flora showed that compared with sham operated rats,carbon metabolism and carbon fixation pathway were significantly enriched after ovariectomy;compared with ovariectomized rats,starch and sucrose metabolism,glycolysis and gluconeogenesis,fatty acid degradation and glucagon signaling pathway were significantly enriched after BBR intervention.(3)Fecal metabolism showed that the levels of pyruvic acid were higher in ovariectomized rats compared with sham operated rats,and the levels of both N-acetyl-L-glutamine and 12(13)-DiHOME were lower in ovariectomized group.BBR treatment significantly decrease the levels of linoleic acid,leucine and arachidonic acid methyl ester compared with ovariectomized rats.KEGG pathway annotation of fecal metabolites showed that both steroid hormone biosynthesis and phenylalanine,tyrosine and tryptophan biosynthesis pathway were different between sham operated rats and ovariectomized rats.Valine,leucine and iso leucine biosynthesis,linoleic acid metabolism and unsaturated fatty acids biosynthesis pathway were altered after BBR intervention.Serum metabolism showed that the levels of lactic acid,pyruvic acid,methionine and linoleic acid were higher in ovariectomized rats compared with sham operated rats,and the levels of maltose,citric acid,tryptophan,linoleic acid and arachidonic acid were significantly lower after BBR reatment.KEGG pathway annotation of serum metabolites showed that pyruvate metabolism,gluconeogenesis or glycolysis,linoleic acid metabolism and phenylalanine,tyrosine and tryptophan biosynthesis pathway were significantly different between sham operated rats and ovariectomized rats.Starch and sucrose metabolism,glyoxylate and dicarboxylic acid metabolism,linoleic acid metabolism,phenylalanine metabolism and arachidonic acid metabolism pathway were altered after BBR treatment.Conclusions:BBR treatment enriched Lactobacillus,Bacteroides,Bifidobacterium and Akkermansia,elevated the levels of both equol and the ratio of equol to daidzein,and alleviated anxiety-like behaviors of ovarietomized rats.The results indicated that BBR was able to improve ovarian hormone deficiency-induced anxiety-like behaviors via enriching equol-producing gut microbes.BBR treatment significantly increased the abundance of Bifidobacterium,decreased the abundance of Ruminococcus and Prevotella.The dicarboxylic acid metabolism,linoleic acid metabolism,phenylalanine metabolism and valine,leucine and iso leucine biosynthesis pathway were significantly different after BBR administration.The levels of citric acid,linoleic acid,12(13)-DiHOME,leucine and tryptophan were regulated with BBR treatment.After BBR intervention,the body weight and the levels of blood glucose and lipids were lower than ovariectomized rats.The results indicated that BBR was able to ameliorate ovarian hormone deficiency-induced obesity and metabolic disorders through influencing both gut microbiota and metabolomics. |
PDF Full Text Request