Application of pea protein hydrolysate in intervention of gestational diabetes

Abstract

The invention discloses application of pea protein hydrolysate in intervention of gestational diabetes, and belongs to the field of biomedicine. Specifically, the invention relates to application of pea protein hydrolysate in preparation of drugs for intervening gestational diabetes. Compared with the prior art, the invention reveals that PPH can relieve GDM mouse insulin resistance and improve GDM mouse serum and placenta lipid disorder, and verifies that PPH can regulate PI3K / AKT / mTOR / PPAR gamma to improve placenta lipid accumulation and protect the placenta structure; and the placenta lipid metabolism change caused by GDM can be recovered.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of pea protein hydrolysate in intervening in gestational diabetes mellitus. Background Art

[0002] Gestational diabetes mellitus (GDM) is currently the most common pregnancy complication and also increases the risk of other pregnancy complications, including preterm birth and preeclampsia. In young women, the incidence of gestational diabetes is increasing. Gestational diabetes increases the risk of long-term complications such as obesity, impaired glucose metabolism, and cardiovascular diseases in both mothers and infants. Approximately 60% of women with a history of GDM will develop type 2 diabetes in the future. This leads to a vicious cycle of obesity and diabetes, threatening the health of the entire population.

[0003] Currently, the clinical management plan for GDM is mainly a stepped intervention strategy. The basic management focuses on dietary intervention combined with exercise. When lifestyle intervention fails to meet the standard, insulin is still the preferred drug treatment option. However, due to the widespread significant insulin resistance in GDM patients, oral hypoglycemic drugs are often used in combination in clinical practice. It should be noted that although drugs with potential placental permeability such as glibenclamide and metformin can improve blood glucose control, their impact on the fetal intrauterine metabolic environment is still controversial, and there are concerns about whether the use of oral drugs will have adverse effects on the long-term health of mothers and fetuses.

[0004] Bioactive peptides derived from plants are regarded as edible nutraceuticals with health benefits related to the prevention or treatment of diseases. Peas are a valuable health food, and bioactive peptides with health-promoting properties have been identified in pea protein, such as pea protein hydrolysates (PPH), whose functional properties include anti-diabetic, anti-hypertensive, antioxidant, and antibacterial effects. However, there are few studies or certain gaps in the research on pea hydrolysates in gestational diabetes and lipid metabolism. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention proposes the application of pea protein hydrolysate in intervening in gestational diabetes mellitus.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect of the present invention, it relates to the application of pea protein hydrolysate in the preparation of a drug for intervening in gestational diabetes mellitus.

[0008] Optionally, the intervention in gestational diabetes mellitus includes controlling fasting blood glucose and hepatic glycogen synthesis, or alleviating insulin resistance.

[0009] The second aspect of the present invention relates to the use of pea protein hydrolysate in the preparation of a drug for regulating lipid metabolism.

[0010] Optionally, the regulation of lipid metabolism includes reducing the levels of TC, TG, LDL-C, and HDL-C, or reducing liver lipid accumulation.

[0011] The third aspect of the present invention relates to the use of pea protein hydrolysate in the preparation of a drug for improving placental quality.

[0012] Optionally, the improvement of placental quality includes reducing placental weight, or increasing the proportion of the labyrinth zone in the placenta, especially in the case of excessive placental weight caused by a high-fat diet.

[0013] Optionally, the improvement of placental quality includes the expression levels of placental PI3K, AKT, p-mTOR, and PPARγ

[0014] The fourth aspect of the present invention relates to a drug for treating gestational diabetes, comprising pea protein hydrolysate.

[0015] The fifth aspect of the present invention relates to a meal replacement food, comprising pea protein hydrolysate.

[0016] The sixth aspect of the present invention relates to a method for constructing a mouse model, comprising the following steps:

[0017] Pregnant mice are randomly divided into:

[0018] A normal control group, intragastric administration with distilled water;

[0019] A GDM model group, intragastric administration with distilled water;

[0020] A low-dose intervention group, intragastric administration with pea protein hydrolysate, at a dose of 500 mg / kg body weight of the mouse;

[0021] And, a high-dose intervention group, intragastric administration with pea protein hydrolysate, at a dose of 1000 mg / kg body weight of the mouse;

[0022] During the process of culturing the mice, fasting blood glucose is measured and a glucose tolerance test is performed;

[0023] The mice are sacrificed, and serum, liver, and placenta are taken;

[0024] The insulin content and blood lipid levels in the serum are detected; the liver and placenta are sectioned and stained to observe pathological changes;

[0025] Based on non-target lipidomics, the effect of pea protein hydrolysate intervention on placental lipid metabolism in gestational diabetes mice is analyzed.

[0026] The beneficial effects of the present invention:

[0027] Compared with the prior art, the present invention reveals that PPH can alleviate insulin resistance in GDM mice, improve serum and placental lipid disorders in GDM mice, and verifies that PPH can protect the placental structure by regulating PI3K / AKT / mTOR / PPARγ to improve placental lipid accumulation; and can restore the changes in placental lipid metabolism caused by GDM. The present invention provides a new idea for the intervention and treatment of GDM and its drug development, and expands the new potential value of PPH in the treatment of GDM and in food therapy or meal replacement products.

[0028] The mouse model constructed by the present invention can characterize the mechanism of action of PPH in pregnant diabetic mice, specifically including:

[0029] (1) Prove that PPH can alleviate insulin resistance in GDM mice.

[0030] (2) Prove that PPH can improve serum and placental lipid disorders in GDM mice.

[0031] (3) Prove that PPH can protect the placental structure by regulating PI3K / AKT / mTOR / PPARγ to improve placental lipid accumulation.

[0032] (4) Prove that PPH can restore the changes in placental lipid metabolism caused by GDM. Description of the Drawings

[0033] The present invention will be further described below with reference to the drawings.

[0034] Figure 1 It is a schematic diagram of the research process of the pea protein hydrolyzate of the present application on diabetes in pregnant mice;

[0035] Figure 2 It is a schematic diagram of the experimental results of blood glucose, insulin resistance, and hepatic glycogen synthesis attenuation in mice in the examples of the present application;

[0036] Figure 3 It is a schematic diagram of the detection results of serum TC, TG, LDL-C, and HDL-C levels in mice in the examples of the present application;

[0037] Figure 4 It is a schematic diagram of the detection results of the histopathological characteristics of the liver tissue of mice in the examples of the present application;

[0038] Figure 5 It is a schematic diagram of the detection of fetal, placental weight, and placental pathological characteristics of mice in the examples of the present application;

[0039] Figure 6 It is the protein expression of the PI3K / AKT / mTOR / PPARγ signaling pathway in mice in the examples of the present application;

[0040] Figure 7 The effect of PPH intervention on the placental lipid profile of GDM mice in the embodiments of this application;

[0041] Figure 8 The volcano plot of differential metabolites screened based on multivariate statistical analysis in the embodiments of this application;

[0042] Figure 9 The lipid change results of the CON group and the GDM group in the embodiments of this application;

[0043] Figure 10 The lipid metabolism detection results of GDM mice in the embodiments of this application. Detailed implementation manners

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0045] In some embodiments of the present invention, as Figure 1 shown, the exploration process of the effect of pea protein hydrolysate on diabetes in pregnant mice is disclosed, including the following steps:

[0046] Establishment of GDM mouse model and PPH intervention: 60 four-week-old SPF C57BL / 6J female mice and 30 four-week-old SPF C57BL / 6J male mice were housed in an SPF sterile barrier environment at the Dingjiaqiao Campus of Southeast University. The animal room experimental facility certificate number is SYXK (Su) 2021-0022. The feeding environment conditions are: 12h light and dark alternation, temperature 20.0℃~26.0℃, relative humidity range 40%~70%. During the experiment, the mice were free to eat and drink, and the bedding was changed every two days to keep the environment clean. The mice were adaptively fed for one week, and then randomly divided into four groups according to body weight: normal control group (distilled water gavage, n=15), model group (distilled water gavage, n=15), low-dose intervention group (PPH-L gavage, 500mg / kg, n=15) and high-dose intervention group (PPH-H gavage, 1000mg / kg, n=15). Among them, except for the normal control group, which was fed with XTCON50 basic maintenance feed, the other three groups were fed with XTHF45 high-fat and high-sugar feed. The normal control group and the model group were intervened by oral gavage with distilled water every day, and the high-dose and low-dose intervention groups were intervened by oral gavage with pea protein hydrolyzate dissolved in distilled water to make PPH suspension every day. After four weeks of continuous feeding, the male and female mice were caged overnight at a ratio of 2:1. No intervention was performed during the cage cohabitation. The presence of vaginal plugs was checked at 8:00 the next morning. If there was a vaginal plug, it indicated that mating was successful and was recorded as gestational age of 0day (GD0). After that, gavage intervention before cage cohabitation continued until the experiment was terminated on GD18. Before cage cohabitation, the weight of mice was recorded every week. After cage cohabitation, the weight of pregnant mice was recorded on GD0, GD10, GD14 and GD18. Fasting blood glucose (FBG) was measured after fasting for 8h three days before cage cohabitation, GD10, GD14 and GD18. Oral glucose tolerance test (OGTT) was performed after fasting for 8 h on GD14. Random blood glucose of mice was recorded one day before the end of the intervention. This experiment was approved by the Animal Experiment Ethics Committee of Southeast University, approval number: 20230708008.

[0047] (2) Regulatory effect of PPH on glucose metabolism in GDM mice:

[0048] like Figure 2 As shown in the results, PPH intervention reduced fasting insulin levels (FINS), alleviated insulin resistance, and restored liver glycogen synthesis in GDM mice, but had no effect on fasting blood glucose (FBG), random blood glucose (RBG), and oral glucose tolerance levels (OGTT).

[0049] Fasting insulin levels in the GDM group ( Figure 2 A) and insulin resistance index ( Figure 2B) in it increased, and the fasting blood glucose increased significantly ( Figure 2 C) in it, and the oral glucose tolerance decreased ( Figure 2 D-E) in it, and the hepatic glycogen synthesis attenuated ( Figure 2 G) in it. High-dose PPH could significantly reduce the insulin level and insulin resistance index of GDM mice, and restore hepatic glycogen synthesis, while low-dose PPH intervention had no significant effect ( Figure 2 C) in it. In addition, the effects of PPH intervention on FBG, OGTT and body weight were not significant ( Figure 2 C-E) in it.

[0050] (3) Study on the mechanism of action of PPH:

[0051] The insulin content in the serum of experimental mice was measured by a commercial enzyme-linked immunosorbent assay (ELISA) kit. After the serum was diluted according to the instructions of the test kit, the four lipid items were measured and calculated, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

[0052] The results are shown in Table 1 and Figure 3 as follows. The serum TC level in the GDM group was significantly higher than that in the CON group. Low-dose and high-dose PPH interventions decreased the TC level by 37.22% and 33.82% respectively. Although there was no significant difference in the TG level between the CON group and the GDM group, low-dose PPH intervention decreased the TG level of GDM mice from 1.73 mmol / L to 1.4 mmol / L. Although the LDL-C level in the GDM group showed an increasing trend compared with the CON group, the difference between the groups was not statistically significant (p>0.05). However, PPH intervention showed a significant regulatory effect: LDL-C in both the low-dose and high-dose groups decreased significantly. There was no significant difference in the change of HDL-C level among the groups.

[0053] Table 1 Serum lipid metabolism results

[0054]

[0055] Note: #: Compared with the GDM group, the difference was statistically significant (p<0.05); n = 8

[0056] After the liver tissue was ground and homogenized, the four lipid items were measured and calculated according to the instructions. Mouse liver paraffin sections were made and observed under a microscope for pathological changes of liver tissue after hematoxylin / eosin (H&E) staining, glycogen staining (PAS) and oil red staining.

[0057] As Figure 4 shown, the H&E staining results showed that PPH could improve the large number of lipid vacuoles in the liver of GDM mice ( Figure 4A) in. The Oil Red staining results showed that PPH intervention could reduce lipid accumulation in the livers of GDM mice ( Figure 4 B) in. In addition, in line with the histopathological changes, quantitative analysis of liver lipids ( Figure 4 C-F) in revealed the regulatory effect of PPH. Both low and high doses of PPH significantly reversed the increase in total liver cholesterol induced by GDM. Notably, the high dose of PPH showed a more comprehensive regulatory efficacy: not only did it decrease the TG level, but it also reduced the LDL-C level and increased the HDL-C level.

[0058] Paraffin sections of mouse placentas were made, sectioned perpendicular to the long axis of the placenta, and stained with hematoxylin / eosin (H&E) to observe the structural changes of the mouse placenta.

[0059] Compared with the CON group, the placental weight in the GDM group was significantly increased, and both low and high doses of PPH intervention could effectively reverse this trend. Compared with the CON group, the fetal weight in the GDM group increased, but the difference was not significant (Table 2, Figure 5 A and B) in. At the same time, the proportion of the labyrinth area, the main site of nutrient exchange, in the placenta was significantly reduced in the GDM group compared with the CON group, and the decrease in the proportion of the labyrinth layer was improved after PPH intervention ( Figure 5 C and D) in.

[0060] Table 2. Placenta and fetal weights (g)

[0061]

[0062] Note: #: Compared with the GDM group, the difference was statistically significant (p < 0.05);

[0063] n = 8

[0064] The expression levels of key molecules in the PI3K / AKT / mTOR / PPARγ pathway in the placenta were detected by Western blot; the effect of PPH intervention on lipid metabolism in GDM placentas was analyzed based on non-target lipidomics.

[0065] Compared with the CON group, the expression levels of p-mTOR and PPARγ in the GDM group were significantly increased, and there was no significant difference in the expression levels of PI3K and AKT between the two groups. After PPH intervention, the levels of PI3K, AKT, p-mTOR, and PPARγ were all significantly decreased ( Figure 6 ).

[0066] In addition, in this example, the effect of PPH intervention on the lipid profile of GDM mouse placentas was analyzed based on non-target lipidomics. As Figure 7As shown, the PCA score scatter plots of all samples are within the 95% confidence interval. There is no overlap in metabolites between the CON group and the GDM group, suggesting significant differences in mouse placental metabolism. Notably, there is only partial overlap between the metabolites in the GDM group and the PPH treatment group, thus suggesting the effect of the intervention. Differentially metabolized substances were screened based on multivariate statistical analysis, and the results were determined according to the P value of the Student’s t test and the VIP (Variable Importance In the Projection) value of the variables in the first principal component of the OPLS-DA model, with P < 0.05 and VIP > 1 as the basic criteria. The volcano plot shows that compared with the CON group, the GDM group has 193 upregulated metabolites and 1732 downregulated metabolites ( Figure 8 in A of Figure 8 ). There are 247 upregulated metabolites and 78 downregulated metabolites ( Figure 8 in B of

[0067] ) and 4295 upregulated metabolites and 189 downregulated metabolites ( Figure 9 in C of Figure 9 between the GDM group and the low-dose and high-dose PPH groups, respectively. Figure 9 in C of

[0068] In the results of lipid changes between the CON group and the GDM group ( Figure 10 in A of Figure 10 ), the relative change percentages of the contents of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are negative, indicating higher contents in the CON group, while in the GDM group and PPH-L ( Figure 10 in B of

[0069] ), between the PPH-H groups ( Figure 9 in C of Figure 9 ), the relative change percentages of PC and PE are positive, indicating higher contents in the PPH intervention groups.

[0068] Between the CON group and the GDM group, many PCs and PEs involved in glycerophospholipid metabolism are downregulated in GDM mice ( Figure 10 in A of Figure 10 ), indicating that the decreased activity of the GP metabolic pathway leads to GDM. Other affected pathways include retrograde endocannabinoid signaling, linoleic acid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism. Compared with the GDM group, the metabolically different pathways in the PPH-H group ( Figure 10 in C of Figure 10 in B of

[0069] ) mainly include autophagy, retrograde endocannabinoid signaling, glycerophospholipid metabolism, fatty acid synthesis, and unsaturated fatty acid biosynthesis. The differentially metabolized pathways between the GDM group and the PPH-L group ( Figure 10 in B of

[0069] ) are almost the same as those between the CON group and the GDM group, including glycerophospholipid metabolism, retrograde endocannabinoid signaling, linoleic acid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism.

[0069] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0070] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and the descriptions in the specification are only used to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. Use of pea protein hydrolysate in the preparation of a drug for intervening in gestational diabetes mellitus.

2. Use of the pea protein hydrolyzate according to claim 1 in the preparation of a medicament for intervening in gestational diabetes, characterized in that, The intervention in gestational diabetes mellitus includes controlling fasting blood glucose and hepatic glycogen synthesis, or alleviating insulin resistance.

3. Use of pea protein hydrolysate in the preparation of a drug for regulating lipid metabolism.

4. Use of the pea protein hydrolysate according to claim 3 in the preparation of a drug for regulating lipid metabolism, characterized in that, The regulation of lipid metabolism includes reducing total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, or reducing hepatic lipid accumulation.

5. Use of pea protein hydrolysate in the preparation of a drug for improving placental quality.

6. Use of the pea protein hydrolysate according to claim 5 in the preparation of a drug for improving placental quality, characterized in that, The improvement of placental quality includes reducing placental weight, or increasing the proportion of the labyrinthine zone in the placenta.

7. Use of the pea protein hydrolysate according to claim 5 in the preparation of a drug for improving placental quality, characterized in that, The improvement of placental quality includes the expression levels of placental PI3K, AKT, p-mTOR and PPARγ.

8. A drug for treating gestational diabetes, characterized in that, It includes pea protein hydrolysate.

9. A meal replacement food, characterized in that, It includes pea protein hydrolysate.

10. A method for constructing a mouse model, characterized in that It includes the following steps: Pregnant mice are randomly divided into: Normal control group, gavaged with distilled water; GDM model group, gavaged with distilled water; Low-dose intervention group, gavaged with pea protein hydrolysate at a dose of 500 mg / kg body weight of mice; And, high-dose intervention group, gavaged with pea protein hydrolysate at a dose of 1000 mg / kg body weight of mice; During the cultivation of mice, fasting blood glucose determination and glucose tolerance test are carried out; The mice are sacrificed, and serum, liver and placenta are taken; The insulin content and blood lipid level in the serum are detected; the liver and placenta are sectioned and stained to observe pathological changes; Based on non-target lipidomics, the effect of pea protein hydrolysate intervention on placental lipid metabolism in gestational diabetes mellitus mice is analyzed.

Images