Application of inulin in prevention of filial generation obesity and / or bone loss caused by mother generation obesity

Abstract

The invention relates to the technical field of new application of inulin, in particular to application of inulin in preparation of a composition for preventing filial generation obesity and / or bone loss caused by mother generation obesity. According to the present invention, the inulin is applied to the mother generation, such that the expression of GLP-1 is promoted, the osteogenic differentiation is activated, the Bacteroides, the Muraculum and the Akkermansia bacteria are improved, and the Alistipes, the Blautia and the unclassified-Oscilospiraceae bacteria are reduced so as to prevent the obesity and / or the bone loss of the offspring. According to the invention, the inulin is fed to the maternal generation, so that the metabolic disorder and bone health of the male offspring can be improved, and the inulin intervention effect of the maternal generation before pregnancy is more obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of new applications of inulin, and particularly relates to the application of inulin in preventing obesity and / or bone loss in offspring caused by obesity in the maternal generation. Background Art

[0002] Human dietary patterns have been changing with social development and technological progress, and high-fat diets (HFDs) are prevalent around the world. Long-term high-fat diets cause overnutrition in the body, and imbalance between energy intake and expenditure leads to obesity. Studies have found that exposure of offspring to maternal obesity or gestational obesity in early life can cause permanent changes in offspring appetite regulation, neurodevelopment, and metabolism, thereby increasing the prevalence of obesity, cognitive and emotional disorders, hypertension, and diabetes throughout the life cycle. At present, the number of obese pregnant women is increasing worldwide. Obesity in pregnant women not only harms themselves, but also has adverse effects on their offspring. Some studies have also found that maternal high-fat diets may affect the metabolic state of offspring in a sex-dependent manner. High-fat diets in maternal mice can worsen glucose tolerance and impair insulin sensitivity in male offspring, and change metabolic circadian rhythms, but no significant effects are found in female offspring. More and more epidemiological surveys and animal experimental results show that maternal high-fat diets can affect the bone health of offspring in early life. A large cohort study of 50,000 Danish mother-infant pairs found that mothers who consumed a high-fat Western diet increased the risk of forearm fractures in their offspring between birth and 16 years of age. Chen et al.'s study showed that high-fat diet-induced maternal obesity in mice affects fetal osteoblast differentiation, resulting in significant impairment of skull and vertebral bone formation and skeletal ossification in the offspring's embryonic period. In addition to affecting intrauterine fetal development, a high-fat diet in the mother also affects postnatal bone development and peak bone mass in adulthood. When the mother ate a high-fat diet during pregnancy and lactation, osteogenic genes decreased, adipogenic genes increased, and bone metabolism was impaired in adolescent offspring. Studies have shown that maternal obesity can affect the bone health of offspring, but the mechanism and preventive measures of the effects of a high-fat diet on the development of the offspring's skeletal system have not yet been clarified and need further exploration.

[0003] Enteroendocrine L cells located on the surface of the lumen have receptors and substrate transporters that can sense nutrients affecting the secretion of the intestinal hormone GLP-1. Studies have found that GLP-1 interacts with bones through GLP1-R to promote osteogenic differentiation, and PI3K / AKT may be a classic signaling pathway related to GLP-1R function. With the increasing attention to early life nutrition and health and the increasing incidence of maternal obesity, the study of the health effects of obese mothers on their offspring has become a research hotspot in recent years. We need to explore potential nutritional factors and develop rigorous and comprehensive scientific nutrition strategies for obese mothers and their offspring. Summary of the invention

[0004] In view of the above technical problems, the present invention provides the use of inulin in preventing obesity and / or bone loss in offspring caused by obesity in the mother.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention provides an application of inulin in preparing a composition for preventing obesity and / or bone loss in offspring caused by obesity in the maternal generation.

[0007] As an embodiment, the inulin is administered to the mother before pregnancy, during pregnancy and / or during lactation.

[0008] As an embodiment, the mother is obese before pregnancy, and the inulin is administered to the mother before pregnancy, during pregnancy and during lactation.

[0009] As an embodiment, the mother is obese during pregnancy and lactation, and the inulin is administered to the mother during pregnancy and lactation.

[0010] As an embodiment, the inulin is used in the composition in an amount of 5% to 15% by mass.

[0011] In the present invention, the inulin reduces the body fat content of male and female offspring.

[0012] In the present invention, the inulin increases the bone tissue of male offspring 43 Ca, 44 Ca, 31 P content, promoting the metabolic balance of mineral elements in the bones of male offspring.

[0013] In the present invention, the inulin increases the expression levels of colon genes GCG, GLUT2, SAGLT1, and GPR43 in male offspring and the secretion of functionally related GLP1-R.

[0014] In the present invention, the inulin promotes the secretion of GLP-1 in male offspring and activates osteogenic differentiation.

[0015] In the present invention, the inulin increases the abundance of Bacteroides, Muribaculum and Akkermansia bacteria in the intestinal tract of male offspring, and reduces the abundance of Alistipes, Blautia and unclassified-Oscillospiraceae bacteria.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. Obesity before pregnancy and high fat intake during pregnancy cause obesity in male and female offspring, and male offspring suffer more serious damage to blood lipid metabolism and bone health than female offspring. The present invention can improve the metabolic disorder and bone health of male offspring by feeding inulin to the mother.

[0018] 2. The present invention explores the changes related to the leptin pathway in male and female offspring. The study found that the leptin and leptin receptor genes in the bone tissue of male offspring with maternal obesity before pregnancy and high fat during pregnancy decreased. Intervention with maternal inulin increased the expression of leptin receptor genes in the bone tissue of offspring, promoted osteogenic differentiation, and improved bone health.

[0019] 3. The present invention explores the mechanism of damage and repair in male offspring caused by a high-fat diet of the maternal generation. The study found that maternal inulin intervention can improve the intestinal morphological structure damage of high-fat male offspring, and improve to varying degrees the reduction in GLP-1 secretion and the expression of colon-related genes GCG, GLUT2, SAGLT1, GPR43, and GLP-1R in male offspring caused by a high-fat diet of the maternal generation, thereby promoting the expression of GLP1-R gene in bone tissue, activating the downstream PI3K / AKT pathway and promoting osteogenic differentiation. Among them, the effect of maternal inulin intervention before pregnancy is more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Timeline for animal experimental treatment of mother and offspring mice;

[0021] Figure 2 The effects of high-fat and inulin diets at different times on serum LDC-C and HDL-C in offspring mice, where A is the male offspring and B is the female offspring;

[0022] Figure 3 This is the result of HE staining to analyze the liver pathological morphology (100×);

[0023] Figure 4 HE staining images of tibia and femur tissues of offspring, where A is male offspring and B is female offspring;

[0024] Figure 5 The effects of high-fat and inulin diets at different times on bone serum markers in offspring mice, where A is the male offspring and B is the female offspring;

[0025] Figure 6 The effects of high-fat and inulin diets at different times on the expression of bone-related genes in offspring mice, where A is the male offspring and B is the female offspring;

[0026] Figure 7 The effects of high-fat and inulin diets at different times on leptin levels in bone tissue of offspring mice, where A is the male offspring and B is the female offspring;

[0027] Figure 8 HE staining of colon tissue;

[0028] Fig. 9This is an analysis of the diversity and abundance of intestinal flora, where A is the dilution curve, B is the principal coordinate analysis method, C is the Chao1 index, D is the Ace index, E is the Simpson index, and F is the Shannon index;

[0029] Fig.10 Correlation analysis of intestinal microbiota at the phylum level, where A is the species distribution histogram at the phylum level, B is the species distribution histogram at the genus level, and C is the LDA value distribution histogram at the genus level. 10 ) is greater than 4;

[0030] Fig.11 The relative abundance analysis of differential bacterial communities at the genus level;

[0031] Fig.12 The effects of high-fat and inulin diets at different times on fecal GLP-1 levels in offspring mice;

[0032] Fig.13 The effects of high-fat and inulin diets at different times on the expression of GLP-1 related genes in offspring mice;

[0033] Fig.14 To investigate the effects of high-fat and inulin diets at different stages on the expression of GLP1-R pathway-related genes in the bone tissue of offspring mice. DETAILED DESCRIPTION

[0034] The present invention provides an application of inulin in the preparation of a composition for preventing maternal obesity from causing offspring obesity and / or bone loss. As an embodiment, the maternal obesity includes but is not limited to maternal obesity caused by a high-fat diet. The present invention finds that high-fat mothers can improve lipid metabolism disorders and bone health of their offspring by feeding inulin. As an embodiment, maternal inulin intervention is used to enhance the expression of leptin receptor genes in bone tissue of male offspring, improve the destruction of intestinal morphology and structure of high-fat male offspring, improve the reduction of GLP-1 secretion in male offspring caused by maternal high-fat diet and the reduction of colon-related genes GCG, GLUT2, SAGLT1, GPR43, and GLP-1R expression, thereby promoting bone tissue GLP1-R gene expression, activating the downstream PI3K / AKT pathway to promote osteogenic differentiation, and achieving the technical effect of improving male offspring metabolic disorders and bone health.

[0035] The present invention has no special limitation on the source of inulin, which can be selected from Jerusalem artichoke, chicory tubers, dahlia tubers, thistle roots as raw materials for conventional preparation, or can be selected from commercially available products.

[0036] In one embodiment, the inulin is administered to the mother before pregnancy, during pregnancy and / or during lactation, which is a method of maternal inulin intervention. In one embodiment, the inulin is administered before pregnancy, during pregnancy and / or during lactation for at least four consecutive weeks, and the inulin is administered at least once a day. In one embodiment, the mother is obese before pregnancy, and the inulin is administered before pregnancy, during pregnancy and during lactation (i.e., maternal pre-pregnancy inulin intervention). In one embodiment, the mother is obese during pregnancy and lactation, and the inulin is administered during pregnancy and lactation (i.e., maternal pregnancy inulin intervention).

[0037] The inulin is used in the composition in an amount of 5% to 15% by mass. In one embodiment, the inulin is used in the composition in an amount of 10% by mass. Unless otherwise specified, % in the present invention refers to mass percentage.

[0038] The present invention has no particular limitation on the preparation form of the composition, which may be any one of liquid, powder, stick, pill and tablet.

[0039] The maternal inulin intervention of the present invention can significantly reduce the body fat content of male and female offspring mice with maternal obesity before pregnancy or high fat during pregnancy, especially the body weight of pre-pregnancy inulin intervention is significantly reduced.

[0040] The maternal inulin intervention of the present invention can significantly reduce the liver index, spleen index and thymus index of male offspring caused by maternal obesity before pregnancy or high fat during pregnancy.

[0041] The maternal inulin intervention of the present invention improves blood lipid metabolism of male offspring with maternal obesity before pregnancy or high fat during pregnancy.

[0042] In one embodiment, the offspring bone loss is a change in the tibia and femur of the male offspring. In one embodiment, the offspring bone loss is a change in the trabeculae. The inulin intervention of the present invention before or during pregnancy can increase the bone tissue of the male offspring with obese mothers before pregnancy or high fat during pregnancy. 43 Ca, 44 Ca, 31 The increased P content increases the activity of osteoblasts in male offspring, reduces the activity of osteoclasts, promotes bone absorption, promotes the metabolic balance of mineral elements in the bone, improves the bone damage of male offspring caused by the high-fat diet of the maternal generation, increases the ability to resist external forces, and reduces the risk of fractures.

[0043] The inulin intervention before or during pregnancy of the present invention significantly increases the expression level of leptin receptor (LEPR) in the bone tissue of male offspring with obesity before or high fat during pregnancy.

[0044] The inulin intervention before or during pregnancy of the present invention significantly increases the expression of colon genes GCG, GLUT2, SAGLT1, and GPR43 in male offspring with obesity before pregnancy or high fat during pregnancy, promotes GLP-1 secretion, and significantly increases the expression of GLP1-R to promote metabolism by binding with GLP-1. Among them, the GCG gene is the precursor gene of the intestinal hormone GLP-1, and knocking out the distal intestinal GCG gene will reduce the level of circulating GLP-1 in mice. GLUT2 and SAGLT1 are carbohydrate receptors, and GPR43 is a lipid receptor. Changes in the expression of these three genes can cause changes in the content of GLP-1. GLP1-R does not affect the secretion of GLP-1, but it will affect the functional effect of GLP-1.

[0045] The inulin intervention before pregnancy of the present invention promotes the expression of the intestinal hormone GLP-1 in the bone tissue of male offspring whose mothers are obese before pregnancy or have high fat during pregnancy, activates the intracellular PI3K / AKT pathway, promotes osteogenic differentiation and prevents bone loss in offspring. The composition containing inulin of the present invention can also be added with an active ingredient that promotes the expression of GLP-1.

[0046] The inulin of the present invention prevents bone loss in male offspring with maternal obesity before pregnancy or high fat during pregnancy by increasing the abundance of Bacteroides, Muribaculum and Akkermansia bacteria and reducing the abundance of Alistipes, Blautia and unclassified-Oscillospiraceae bacteria. Bacteroides, Muribaculum and Akkermansia bacteria can also be added to the composition containing inulin of the present invention.

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] In the specific embodiment of the present invention, all data were analyzed and graphed using Graphpad Prism, IBM SPSS Statistics 21, and Origin 2018 software. One-way ANOVA was performed using Duncan's test, and data were presented as mean ± standard error (mean ± SD). Significant differences were expressed as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0051] Example 1

[0052] 1. Experimental Animals and Grouping

[0053] Male mice of the father generation were randomly divided into cages and fed with normal diet, and female mice of the mother generation were randomly divided into five groups, named as follows: Figure 1 As shown: 16 mice in the common diet group (MC-C), 16 mice in the high-fat diet group during pregnancy (MC-H), 16 mice in the high-fat inulin diet group during pregnancy (MC-I), 16 mice in the high-fat obesity group before pregnancy (MH-H), and 16 mice in the high-fat inulin diet group before pregnancy (MI-I). Among them, the mice in the high-fat diet group were fed with 60% high-fat diet, the mice in the high-fat inulin diet group were fed with high-fat inulin diet, and the mice in the other groups were fed with common diet, and the inulin added in the high-fat inulin diet was 10% by mass (inulin was purchased from Qinghai Ruihu Biological Resources Development Co., Ltd., inulin ≥90%, average degree of polymerization 20-60).

[0054] After the experiment, the five groups of maternal mice were placed in the same cage with the paternal mice (female: male = 2:1), and the maternal mice were fed the same diet during the same cage. The mice were judged to be pregnant by checking the vaginal plug. After the mice were successfully conceived, they were raised in a single cage. After the pregnant mice in the high-fat diet group during pregnancy were separated into single cages, they were fed with a 60% high-fat diet instead of a normal diet, and the high-fat inulin diet group during pregnancy was changed to a high-fat inulin diet. The feeding methods of the other groups remained unchanged. The female mice gave birth naturally after three weeks of pregnancy, and the offspring were breastfed for four weeks after birth. The feeding method of the maternal mice during lactation remained unchanged. The offspring mice were used as the research objects, and there were 21 male offspring mice and 27 female offspring mice in the common diet group; 18 male offspring mice and 23 female offspring mice in the high-fat diet group during pregnancy; 19 male offspring mice and 24 female offspring mice in the high-fat inulin diet group during pregnancy; 16 male offspring mice and 9 female offspring mice in the high-fat obesity group before pregnancy; 22 male offspring mice and 22 female offspring mice in the high-fat inulin diet group before pregnancy. The offspring mice were divided into five groups accordingly: common diet feeding group (CC-C), high-fat diet feeding group during pregnancy (CC-H), high-fat inulin diet feeding group during pregnancy (CC-I), high-fat obesity group before pregnancy (CH-H), and high-fat inulin diet feeding group before pregnancy (CI-I), and each group was divided into male and female.

[0055] 2. Determination of basic indicators

[0056] The offspring mice of each group were weighed, and the distance from the nose tip to the anus was measured after the mice were killed to obtain the body length. The body fat (abdominal groove fat, gonadal fat, periintestinal fat), spleen, thymus, and liver were weighed and the Lee's index, body fat percentage, and organ index were calculated according to the following formula:

[0057]

[0058] Effects of high-fat diet and inulin intervention at different periods on the basic indicators of offspring mice. The basic indicators of male offspring mice are shown in Table 1:

[0059] Table 1 Basic indicators of male offspring mice

[0060]

[0061] Note: N=8-10. Different letters in the same row of data represent significant differences (p<0.05).

[0062] As can be seen from Table 1, compared with the normal diet group, the weight of male offspring mice in the two high-fat groups showed an increasing trend, among which the offspring of the pre-pregnancy obesity group was heavier, with an average weight of 11.72g; the weight of pre-pregnancy inulin intervention decreased significantly, and there was no difference in the weight of offspring from the normal diet group (p<0.05); the weight of male offspring after inulin intervention during pregnancy showed a decreasing trend, but the difference was not significant. Compared with the normal diet group, the body fat rate of male offspring in the pre-pregnancy obesity model group and the high-fat diet group during pregnancy increased significantly (p<0.05), and the body fat content was significantly reduced after inulin intervention (p<0.05). The liver index, spleen index and thymus index of male offspring in high-fat diet of the mother at different periods increased, and decreased to varying degrees after inulin intervention. The above results show that inulin intervention at different periods can improve the fat increase and organ abnormalities of male offspring caused by high-fat diet of the mother.

[0063] The basic index results of female offspring mice are shown in Table 2:

[0064] Table 2 Basic indicators of female offspring mice

[0065]

[0066] Note: N=8-10. Different letters in the same row of data represent significant differences (p<0.05).

[0067] As can be seen from Table 2, compared with the normal feed group, the weight and body fat percentage of female offspring mice in the two high-fat groups increased significantly, and the Lee's index showed an increasing trend. Inulin intervention at different times can reduce their weight and body fat percentage. There is no significant difference in the organ index of female offspring between different groups. The spleen index increased with inulin before pregnancy, which may be due to the fact that inulin greatly stimulates the immune system, causing the increase.

[0068] The above research results show that both pre-pregnancy obesity and high-fat diet during pregnancy will lead to weight gain, increased fat percentage, and increased body fat accumulation in male and female offspring. The organ indexes of male offspring are significantly different, while the differences in female offspring are not obvious. Pre-pregnancy obesity or high-fat diet during pregnancy has a greater impact on the basic physiological functions of male offspring.

[0069] 3. Serum high-density lipoprotein and low-density lipoprotein content determination

[0070] The blood sample was placed in a -4℃ refrigerator and centrifuged at 3500r / min for 10min after a period of time. The upper serum was collected and repeated once. The serum was divided and stored in a -80℃ refrigerator for subsequent experiments. The HDL-L and LDL-L content were determined using the Shanghai ELISA kit. The specific steps were performed according to the corresponding instructions. The effects of high-fat diet and inulin intervention at different times on serum high-density lipoprotein and low-density lipoprotein in offspring mice Figure 2 The results showed that maternal obesity before pregnancy or high fat intake during pregnancy had a significant impact on the blood lipid metabolism of male offspring, and inulin had an improving effect.

[0071] 4. Hematoxylin-eosin staining of liver tissue:

[0072] HE staining was used to perform HE staining on the liver tissues of male and female offspring in different groups to observe their pathological phenotypes. Figure 3 As shown, the staining results showed that compared with the group fed with ordinary feed, the high-fat group during pregnancy had more small fat droplets, and the high-fat obesity model group before pregnancy had more large fat droplets, and the inflammatory infiltration in the two groups was not obvious; after inulin intervention, the fat droplet vacuoles decreased and the fat content of liver tissue decreased, indicating that inulin intervention can reduce the fat content in the liver of male and female offspring mice, and can also improve the increased inflammation in the liver tissue of male offspring caused by the high-fat diet of the mother.

[0073] 5. Determination of bone performance indicators:

[0074] (1) Bone tissue morphology staining

[0075] HE staining was used to observe the changes in the morphology of the tibia and femur of the offspring mice, especially the changes in trabeculae. Figure 4 Among A and B, Figure 4 Figure A shows the male offspring. The trabeculae of the femur / tibia tissue in the normal diet group were denser and connected in a network arrangement, and the bone microstructure was intact. The number of femur / tibia trabeculae in the different high-fat diet groups decreased, the trabeculae became thinner, the gaps became larger, and more fat vacuoles could be seen above the proximal epiphysis of the tibia. Compared with the high-fat group, the trabeculae in the inulin group increased, thickened, and more tightly arranged, with fewer fat vacuoles, and the bone microstructure morphology was significantly improved. Figure 4 Figure B is the offspring female rats. Compared with the normal diet group, the number of trabeculae in the pre-pregnancy obesity group decreased and the bone microstructure was slightly damaged; the microscopic changes of the femur / tibia in the other groups were not obvious. The above results show that this diet pattern of the mother has a significant effect on the bone microstructure of the offspring male rats.

[0076] (2) Mechanical properties of bone tissue

[0077] The three-point mechanical experiment was used to detect the ability of male and female offspring to resist external forces in different groups. The experimental results are shown in Table 3. The three mechanical parameters of male offspring in the CC-I and CI-I groups increased to varying degrees. This indicates that pre-pregnancy obesity and pre-pregnancy high fat diets lead to reduced mechanical properties of male offspring bones, increased bone brittleness, and increased risk of fractures. After inulin intervention, the ability to resist external forces increased and the risk of fractures decreased. There were no significant differences in the maximum bending stress, maximum bending load, and elastic modulus among the female offspring groups. This indicates that the high-fat and high-fat inulin diets of the mother at different stages have a greater effect on the ability of male offspring to resist external forces than female mice.

[0078] Table 3 Bone mechanical parameters of offspring mice

[0079]

[0080] Note: Different letters in the same row of data indicate significant differences (p<0.05, N=4~5).

[0081] (3) Determination of mineral element content in bones

[0082] ICP-MS was used to determine the content of calcium and phosphorus, and the linear equation correlation coefficient was greater than 0.9900. 43 Ca, 44 Ca, 31 The results of P content are shown in Table 4, which show that compared with the male and female offspring of the normal diet group, the bone tissue of the two high-fat groups 43 Ca, 44 Ca, 31 P content decreased, and inulin intervention could improve the bone tissue of male offspring 43 Ca, 44 Ca, 31 P content, there was no difference in female mice, but there was a significant difference in male mice (p < 0.05). This indicates that inulin intervention at different stages of the maternal generation will cause 43 Ca, 44 Ca, 31 The increased P content promotes the metabolic balance of mineral elements in bones and improves bone damage in offspring caused by the high-fat diet of the mother.

[0083] Table 4 Bone mineral content of offspring mice

[0084]

[0085] (4) Determination of bone biometabolism markers in mouse serum

[0086] The results of bone serum markers in the offspring mice are shown in Figure 5, indicating that high-fat diet at different stages of the maternal generation led to decreased osteoblast activity in male offspring, inhibited bone formation, increased osteoclast activity, promoted bone resorption, and caused bone metabolism disorders, which were improved after inulin intervention. High-fat diet and inulin intervention at different stages of the maternal generation had no significant effect on female mice.

[0087] (5) Expression of related genes in bone tissue

[0088] The results of bone-related gene expression in the offspring mice are shown in Figure 6 , indicating that maternal obesity before pregnancy or high fat intake during pregnancy has a great impact on the expression of related genes in the bone tissue of male offspring. Inulin intervention can inhibit the increased differentiation of male osteoclasts caused by high-fat diet of the mother at different stages, promote osteogenic differentiation, regulate the balance of bone metabolism in male offspring, and is beneficial to bone health.

[0089] 6. Effects of high-fat diet and inulin intervention at different stages on the leptin / leptin receptor pathway in offspring mice

[0090] The results of leptin pathway-related gene expression in bone tissue of offspring mice are shown in Figure 7 The results showed that the leptin content / leptin receptor gene expression in bone tissue, in male offspring, compared with CC-C group, the CC-H and CH-H groups had significantly lower leptin (LEP) and leptin receptor gene (LEPR) expression levels, CI-I and CC-I inulin groups significantly upregulated LEPR mRNA expression, and had little effect on LEP mRNA expression. In female offspring, there was no significant difference between the groups, and the maternal diet had little effect on the leptin / leptin receptor signaling pathway in female offspring, so the final phenotypic differences in female offspring were not obvious.

[0091] The above study found that the impact on male mice under this model is greater than that on female mice. Therefore, male offspring mice were used as research subjects to explore the potential mechanism by which the intestine-bone signaling axis mediates the effects of high-fat diet and inulin intervention at different periods on the bone health of male offspring.

[0092] Example 2

[0093] 1. Effects of high-fat diet and inulin intervention at different stages on intestinal tissue morphology of male offspring mice

[0094] HE staining was used to observe the pathological morphology of colon tissue. Figure 8In the male offspring CC-C group, the colon tissue structure was clear and complete, the inflammatory cell infiltration was less, and the number of goblet cells was more. Compared with the CC-C group, the colon tissue arrangement of the CC-H and CH-H groups was disordered, the number of goblet cells decreased, the crypt structure was destroyed, and there was inflammatory infiltration; the tissue gap of CC-H increased. The arrangement of the intestinal mucosa of the two inulin intervention groups CC-I and CI-I became regular, the goblet cells increased, and the tissue gap of the CC-I group became smaller, which improved the colon tissue morphological damage caused by high fat, but there was still a small amount of inflammatory infiltration. Inulin intervention repaired the changes in intestinal tissue morphology of male offspring caused by maternal obesity before pregnancy and high fat during pregnancy to a certain extent.

[0095] 2. Effects of high-fat diet and inulin diet at different times on intestinal flora of male offspring mice

[0096] (1) Analysis of intestinal flora richness and diversity

[0097] The Rarefaction Curve can be used to determine whether the sample sequencing volume is sufficient. If the curve is flat, it means that the sequencing depth has basically covered all species in the sample, and subsequent analysis can be performed, such as Fig. 9 The values ​​of each group shown in A in the middle tend to be stable as the sequencing amount increases, indicating that subsequent analysis can be carried out.

[0098] Beta diversity analysis was used to reflect the differences between different groups, and the results of cylindrical coordinate analysis were as follows Fig. 9 As shown in B. CC-I and CI-I are close to CC-C group, indicating that maternal inulin intervention makes the intestinal flora composition of male offspring mice similar to that of control mice. The closeness between CC-H and CH-H, and between CC-I and CI-I groups is caused by the same high-fat or high-fat inulin diet of their maternal generations.

[0099] The intestinal flora diversity was analyzed using four indicators in the Alpha diversity analysis: Chao1 and Ace index reflecting the number of species and Shannon and Simpson index reflecting species diversity. The results showed that compared with the CC-C group, the Chao1 index ( Fig. 9 C), Ace Index ( Fig. 9 D), Shannon Index ( Fig. 9 There was no significant change in the F group, but the two inulin groups (CC-I and CI-I) decreased to varying degrees, indicating a decrease in the number and diversity of species. Compared with the CC-C group, the Simpson index ( Fig. 9Middle E) had no significant change, and the index decreased after intervention with inulin during pregnancy; the Simpson index of the pre-pregnancy high-fat group (CH-H) increased, indicating that its community diversity decreased, and the corresponding inulin group (CI-I) decreased, indicating that maternal inulin intervention had a slight recovery effect on community diversity. In summary, the number and diversity of intestinal species in male offspring of maternal high-fat diet did not decrease significantly, while the number and diversity of intestinal species in male offspring of maternal inulin intervention decreased slightly.

[0100] (2) Differences in intestinal microbial composition

[0101] Further analysis was conducted on the changes in the intestinal microbiota of male offspring at the phylum and genus levels to explain the effects of high-fat and inulin diets on male offspring at different stages. Fig.10 A in the figure is a bar graph of species distribution at the top ten phyla in terms of abundance. The present invention found that compared with the CC-C group, the relative abundance of Firmicutes in the intestinal flora of male offspring fed a high-fat diet increased, while the relative abundance of Bacteroidetes decreased. After inulin intervention, the relative abundance of Firmicutes in the intestinal flora of male offspring decreased, while the relative abundance of Bacteroidetes increased. In particular, the relative abundance of Verrucomicrobia increased significantly after inulin intervention, indicating that inulin intervention decomposes polysaccharides and produces short-chain fatty acids by increasing the relative abundance of Verrucomicrobia, thereby exerting the beneficial effects of inulin.

[0102] The LDA value is calculated based on the characteristics of the rich differences in intestinal flora, and the biomarkers (biomakers) with statistical differences are found based on the LDA value distribution bar graph. Fig.10 Figure B is a stacked column chart of the top 15 species in terms of genus abundance. Fig.10C in the figure is the LDA value distribution bar graph at the genus level. Combine the LDA value distribution bar graph and the species stacking chart to find the intestinal flora with high abundance that causes differences between different groups. The analysis revealed the dominant bacteria at the genus level in the top fifteen different groups of abundance: unclassified-Lachnospiraceae, Alloprevotella, and unclassified-Muribaculaceae in the CC-C group; Blautia, unclassified-Oscillospiraceae, and Erysipelatoclostridium in the CC-H group; Parabacteroides, Lachnoclostridum, and Parasutterell in the CC-I group; Ligilactrobacillus, Alistipes, Escherichia-shigella, and unclassified-Enterobacteriaceae in the CH-H group; and Akkermansia and Bacteroides in the CI-I group. The genera whose abundances were consistent with the phenotypic trends were selected, namely Blautia, Alistipes, Bacteroides, unclassified-Oscillospiraceae, and Muribaculum (see Fig.11 ), Bacteroides and Muribaculum bacteria showed the same trend of change. Compared with the CC-C group, the relative abundance in the intestine of male offspring in the high-fat group (CC-H and CH-H) decreased, and the relative abundance in male offspring in CI-I and CC-I groups increased after inulin intervention. The abundance trends of Alistipes, Blautia and unclassified-Oscillospiraceae were consistent among different groups, with higher relative abundance in male offspring in the high-fat group (CC-H and CH-H), and lower relative abundance in male offspring in the inulin group (CI-I and CC-I) and the control group (CC-C). Akkermansia is considered to be the "next generation of beneficial microorganisms". The relative abundance of Akkermansia in different groups is as follows: Fig.11 As shown, the relative abundance of Akkermansia in the intestine of male offspring in the high-fat group was reduced, and Akkermansia was significantly enriched in the male offspring in the maternal inulin intervention group, and the relative abundance of Akkermansia in male offspring in the high-fat inulin group before pregnancy was high. Chiplot indicates significant differences, the pinker the color, the greater the significant difference, and the bluer the color, the smaller the difference.

[0103] In summary, inulin intervention in the maternal generation can increase the abundance of beneficial bacteria related to bone metabolism in the intestines of male offspring, reduce the abundance of harmful bacteria related to bone metabolism, regulate intestinal flora, promote osteoblast differentiation, inhibit osteoclast differentiation, and improve the bone health of male offspring.

[0104] 3. Effects of high-fat diet and inulin intervention at different times on intestinal hormone GLP-1 and related pathways in male offspring mice

[0105] (1) Determination of intestinal hormone GLP-1 in feces of male offspring mice

[0106] Feces of four-week-old male offspring mice were collected and stored at -80℃. Biomarker Corporation was commissioned to sequence the V3-V4 region of the 16SrRNA gene of the intestinal flora. The experimental steps were to extract DNA from the samples using a DNA extraction kit, and then perform DNA amplification, library structure and sequencing. The enzyme-linked immunosorbent assay (ELISA) kit of Shanghai ELISA Biotechnology Co., Ltd. was used to detect the content of intestinal hormone GLP-1. The specific operation steps were carried out according to the instructions, and the results were analyzed on the Biomarker cloud platform.

[0107] Test results such as Fig.12 As shown: Compared with the CC-C group, the GLP-1 content in the feces of CC-H and CH-H mice was significantly decreased; the GLP-1 content in the feces of CC-I and CI-I mice was significantly increased, indicating that inulin intervention improved the decrease in intestinal GLP-1 content in male offspring caused by the high-fat diet of the maternal generation. The intestinal hormone GLP-1 can mediate the series connection between the intestine-bone axis, and the change in its content is one of the reasons for the changes in bone metabolism in male offspring mice under the maternal dietary pattern.

[0108] (2) Colon-related gene expression

[0109] Five genes related to the production, secretion, and function of GLP-1 in the colon were selected for analysis. The proglucagon gene GCG is the precursor gene for GLP-1 synthesis, GLUT2 and SAGLT1 are carbohydrate receptors, GPR43 is a lipid receptor, and GLP1-R affects the function of GLP-1.

[0110] RT-qPCR was used to determine the relative expression of each gene. Fig.13As shown in the results, compared with the CC-C group, the relative expression of GCG in the CC-H and CH-H groups decreased significantly, GLP-1 synthesis was impaired, and the GCG gene expression in the CC-I and CI-I groups was upregulated, among which the expression in CI-I was not significantly different from that in the CC-C group. The maternal inulin intervention had the effect of promoting the synthesis of GLP-1 in male offspring. Compared with the CC-C group, the GLUT2 gene expression in the CH-H group decreased significantly, and the GLUT2 gene expression in the CI-I group was upregulated after inulin intervention. There was no significant difference in the GLUT2 gene expression between the CC-H and CI-I groups. The SAGLT1 gene expression in the CC-H and CH-H groups was significantly lower than that in the CC-C group. After inulin intervention, the SAGLT1 gene expression in the CI-I and CC-I groups was upregulated, indicating that inulin intervention affected the expression of the sugar receptors GLUT2 and SAGLT1 genes, causing changes in the secretion and content of GLP-1 in male offspring. Compared with the CC-C group, the high-fat group reduced the expression of the short-chain fatty acid receptor GPR43 gene, and the inulin group upregulated the expression of the GPR43 gene, indicating that after inulin intervention, more short-chain fatty acids were produced in the intestine of male offspring to activate the L cell GPR43 receptor and promote the secretion of GLP-1. Compared with the CC-C group, the expression of the GLP1-R gene in the CC-H and CC-H groups was downregulated, and the expression level of the GLP1-R gene was significantly restored in the CI-I and CC-I groups after inulin intervention, indicating that the secreted GLP-1 combines with GLP1-R to play a metabolic role in reducing intestinal inflammation.

[0111] In summary, the relative expression levels of colon GLP-R genes in male offspring were analyzed. Inulin intervention during maternal pregnancy promoted the expression of colon SAGLT1 and GPR43 genes, and inulin intervention before maternal pregnancy promoted the expression of GLUT2, SAGLT1 and GPR43 genes, thereby promoting GLP-1 secretion.

[0112] (3) Relative mRNA expression levels of GLP1-R, PI3K, and AKT in bone tissue

[0113] RT-qPCR was used to measure the expression of femur-related genes in male offspring of different groups. Fig.14 As shown in the figure, it can be seen that the relative expression of GLP1-R, PI3K, and AKT mRNA in the femur of male offspring decreased after maternal high-fat diet at different periods, and the three genes of offspring were significantly upregulated after pre-pregnancy inulin intervention, while the upregulation of GLP1-R and AKT genes was not obvious after intervention in the pregnancy inulin group. This indicates that maternal inulin intervention can promote the expression of GLP1-R in osteoblast precursor cells of male offspring, activate the intracellular PI3K / AKT pathway, and promote osteogenic differentiation.

[0114] In summary, the feces of male offspring with maternal obesity or high fat diet during pregnancy showed a significant decrease in intestinal hormone GLP-1, down-regulated the expression of colonic GLP-1 precursor gene GCG, secretion-related genes SAGLT1 and GPR43, and function-related GLP-1 receptor gene. The secretion-related gene GLUT2 gene expression of male offspring in the maternal obesity group was down-regulated. After inulin intervention, GLP-1 content increased and gene expression increased. The gene expression of GLP-1R, PI3K, and AKT in bone tissue of male offspring with maternal obesity or high fat diet during pregnancy decreased. After pre-pregnancy inulin intervention, the gene expression levels of GLP-1R, PI3K, and AKT increased significantly. There was no significant difference in GLP-1R, PI3K, and AKT genes after inulin intervention during pregnancy. Maternal inulin intervention promoted the expression of GLP1-R in osteoblast precursor cells of male offspring, activated the intracellular PI3K / AKT pathway, and promoted osteogenic differentiation.

[0115] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of inulin in preparing a composition for preventing obesity and / or bone loss in offspring caused by obesity in the mother.

2. The use according to claim 1, characterized in that: The inulin is administered to the mother before conception, during pregnancy and / or during lactation.

3. The use according to claim 2, characterized in that: The mother was obese before pregnancy, and the inulin was administered to the mother before pregnancy, during pregnancy and during lactation.

4. The use according to claim 2, characterized in that: The mother is obese during pregnancy and lactation, and the inulin is administered to the mother during pregnancy and lactation.

5. The use according to any one of claims 1 to 4, characterized in that: The inulin is used in the composition in an amount of 5% to 15% by mass.

6. The use according to any one of claims 1 to 4, characterized in that: The inulin reduces the body fat content of male and female offspring.

7. The use according to any one of claims 1 to 4, characterized in that: The inulin increases the bone tissue of male offspring 43 Ca, 44 Ca, 31 P content, promoting the metabolic balance of mineral elements in the bones of male offspring.

8. The use according to any one of claims 1 to 4, characterized in that: The inulin increases the expression levels of colon genes GCG, GLUT2, SAGLT1, and GPR43 in male offspring and the secretion of functionally related GLP1-R.

9. The use according to claim 8, characterized in that: The inulin promotes the secretion of GLP-1 in male offspring and activates osteogenic differentiation.

10. The use according to any one of claims 1 to 4, characterized in that: The inulin increases the abundance of Bacteroides, Muribaculum and Akkermansia bacteria in the intestinal tract of male offspring, and decreases the abundance of Alistipes, Blautia and unclassified-Oscillospiraceae bacteria.

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