Use of lactobacillus plantarum p101 in the preparation of a product for improving polycystic ovary syndrome related conditions

Abstract

This invention discloses the application of *Lactobacillus plantarum* P101 in the preparation of products for improving symptoms related to polycystic ovary syndrome (PCOS), belonging to the field of microbial medical technology. The *Lactobacillus plantarum* P101 strain of this invention was isolated from homemade pickled vegetables in Ji'an City, Jiangxi Province, with the accession number CCTCC M 2021108, and deposited at the China Center for Type Culture Collection, Wuhan City. *Lactobacillus plantarum* P101 of this invention is a Gram-positive bacterium with the ability to remodel the structure and function of the intestinal flora, and can improve PCOS to a certain extent. The *Lactobacillus plantarum* P101 strain of this invention has high safety and good probiotic properties, and this strain plays a beneficial role in the treatment of ovarian dysfunction and metabolic and sex hormone disorders caused by PCOS.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of Lactobacillus plantarum P101 in the preparation of products for improving symptoms associated with polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic disorders in women of reproductive age. Its main complaints are infertility and metabolic syndrome, exhibiting high clinical heterogeneity. Its core pathophysiological feature is ovarian dysfunction. Typical polycystic ovarian changes accompanied by ovulation disorders or anovulation are a significant cause of infertility. Hyperandrogenemia and insulin resistance are key factors driving the progression of PCOS, acting synergistically on the ovaries to stimulate excessive androgen synthesis in theca cells, interfering with normal antral follicle development and dominant follicle selection, ultimately leading to follicular arrest and ovulation disorders.

[0003] The gut microbiota plays a crucial role in the pathogenesis of PCOS. Patients with PCOS and animal models commonly exhibit gut microbiota dysbiosis, typically characterized by a decrease in the abundance of beneficial bacteria such as Lactobacillus and Akkermansia. Studies have shown that probiotics can regulate gut microbial homeostasis and restore the balance and function of the microbiota. Therefore, it is possible to use probiotics to treat PCOS.

[0004] Lactobacillus plantarum is a type of Gram-positive bacterium, mostly facultative anaerobic, and is recognized as one of the probiotic species. Lactobacillus plantarum is closely related to human life and is widely found in nature, especially in various fermented foods, such as fermented vegetables, cured meats, and fermented dairy products. Furthermore, as a probiotic, Lactobacillus plantarum is also abundant in the human gastrointestinal tract, playing a role in regulating intestinal microbial balance, controlling toxin levels in the body, enhancing immunity, and eliminating harmful substances, thus having significant benefits for human health.

[0005] Lactobacillus plantarum P101, screened from various naturally fermented sauerkraut samples, possesses excellent in vitro antioxidant capacity and good gastrointestinal tolerance, laying the foundation for its colonization and function in the gut. This strain also exhibits strong antibacterial activity, effectively inhibiting the growth of pathogenic bacteria such as Staphylococcus aureus. Previous studies have shown that P101 can regulate the host's gut microbiota structure, promote the proliferation of beneficial bacteria, enhance intestinal barrier function, and inhibit the release of inflammatory factors, thereby exerting a protective effect in various metabolic and inflammatory models. Summary of the Invention

[0006] To address the problems of poor adherence and significant side effects associated with long-term drug use in existing technologies, this invention provides a highly safe and long-term usable probiotic preparation that improves symptoms of polycystic ovary syndrome (PCOS) by regulating the intestinal flora. This invention provides the application of *Lactobacillus plantarum* P101 in the preparation of products for improving PCOS-related symptoms. The strain was isolated from fermented sauerkraut. The *Lactobacillus plantarum* P101 provided by this invention (… Lactiplantibacillus plantarum P101 has good probiotic properties and can maintain the balance and function of intestinal flora.

[0007] To achieve the above objectives, the present invention provides the application of Lactobacillus plantarum P101 in the preparation of products for improving symptoms related to polycystic ovary syndrome. The Lactobacillus plantarum P101 was deposited at the China Center for Type Culture Collection in Wuhan on January 19, 2021, with accession number CCTCC NO: M 2021108.

[0008] Furthermore, the polycystic ovary syndrome-related conditions include one or more of the following: ovarian dysfunction, sex hormone secretion disorders, and glucose and lipid metabolism disorders.

[0009] Furthermore, the ovarian dysfunction includes at least one of the following: polycystic ovarian changes, ovulation disorders, abnormal follicular development, increased follicular atresia, ovarian granulosa cell apoptosis, and oxidative stress damage to ovarian tissue.

[0010] Furthermore, the sex hormone secretion disorder includes at least one of hyperandrogenemia, elevated luteinizing hormone (LH) / follicle-stimulating hormone (FSH) ratio, abnormal estradiol secretion, and elevated androstenedione levels.

[0011] Furthermore, the glucose and lipid metabolism disorder includes at least one of insulin resistance, elevated fasting blood glucose, elevated fasting insulin, impaired glucose tolerance, elevated triglycerides, elevated total cholesterol, elevated low-density lipoprotein cholesterol, and abnormal weight gain.

[0012] Furthermore, the product is a medicine, health food, or dietary supplement.

[0013] Furthermore, the product is an oral preparation, which includes one of the following: powder, capsule, tablet, granule, oral liquid, and suspension.

[0014] Furthermore, the product contains 1×10⁻⁶ live bacteria of *Lactobacillus plantarum* P101. 9 ~ 1×10 11 CFU / g or 1×10 9 ~ 1×10 11 CFU / mL.

[0015] Furthermore, the product also contains pharmaceutically or food-acceptable excipients, including one or more of lyophilization protectants, prebiotics, dietary fiber, fillers, disintegrants, and lubricants.

[0016] Furthermore, the freeze-drying protectant is one or more of skim milk powder, maltodextrin, trehalose, and vitamin C.

[0017] Furthermore, the prebiotic is one or more of fructooligosaccharides, galactooligosaccharides, inulin, and resistant dextrin.

[0018] Furthermore, the product also contains active ingredients for improving polycystic ovary syndrome, including one or more of metformin, short-acting oral contraceptives, inositol, vitamin D, and coenzyme Q10.

[0019] Furthermore, the daily dosage of the product in mouse experiments was: 1 × 10⁻⁶ live Lactobacillus plantarum P101 cells. 8 ~ 1×10 9 CFU / animal / day, with a continuous dosing cycle of no less than 4 weeks.

[0020] Furthermore, after administration, the number of corpora lutea in mice increased by 65-70%, while the number of cystic follicles in mice decreased by 50-60%.

[0021] Furthermore, the product improves polycystic ovary syndrome-related symptoms by regulating the gut microbiota-bile acid metabolism-IL-22 / JAK1 / STAT3 ovarian axis.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention, Lactobacillus plantarum P101, regulates the intestinal flora. The bile acid metabolism-IL-22 / JAK1 / STAT3 ovarian axis has a significant therapeutic effect on ovarian function impairment and metabolic and sex hormone disorders induced by hyperandrogenism in PCOS. Attached Figure Description

[0023] Figure 1 The figure shows how Lactobacillus plantarum P101 improves ovarian function impairment in PCOS mice induced by hyperandrogenemia according to a specific embodiment of this application (where * indicates comparison with "PCOS group", * indicates p<0.05, and ** indicates p<0.01). Figure 2 The figure shows how Lactobacillus plantarum P101 improves glucose metabolism disorder in PCOS mice induced by hyperandrogenemia according to a specific embodiment of this application (where * indicates compared with "PCOS", * indicates p<0.05, and ** indicates p<0.01). Figure 3The figure shows how Lactobacillus plantarum P101 improves sex hormone disorders in PCOS mice induced by hyperandrogenemia in a specific embodiment of this application (where * indicates comparison with "PCOS group", * indicates p<0.05, and ** indicates p<0.01). Figure 4-6 The figure shows how Lactobacillus plantarum P101 improves ovarian function damage, metabolic disorders, and sex hormone disorders in hyperandrogen-induced PCOS mice by regulating the gut microbiota-bile acid metabolism-IL-22 / JAK1 / STAT3 ovarian axis in a specific embodiment of this application (where * indicates compared with the "PCOS group", * indicates p<0.05, and ** indicates p<0.01). Detailed Implementation

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Activation and cell preparation of existing Lactobacillus plantarum P101 1. Experimental strain: The Lactobacillus plantarum P101 used in this example is the commercially available standard strain Lactobacillus plantarum P101 (purchased from the China Center for Type Culture Collection, which is a publicly available and legally obtainable strain with no intellectual property disputes).

[0027] 2. Culture medium preparation: MRS liquid culture medium (per liter): 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 1.0 mL Tween 80, with the remainder being distilled water. Adjust the pH to 6.5±0.2, autoclave at 121℃ for 15 min, and cool for later use. MRS solid culture medium is prepared by adding 15.0 g / L agar to the above formula.

[0028] 3. Strain activation: The glycerol-containing bacteria preserved at low temperature were streaked onto MRS solid medium plates and cultured in a facultative anaerobic environment at 37°C for 48 h. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C and 100 r / min for 24 h to obtain the primary seed culture. The primary seed culture was then transferred to fresh MRS liquid medium at an inoculation rate of 3% (v / v) and cultured under the same conditions for 24 h to obtain the secondary seed culture.

[0029] 4. Preparation of live bacterial suspension: Centrifuge the secondary seed culture at 8000 r / min for 5 min, discard the supernatant, wash twice with PBS, and then resuspend in 1 / 50 volume of buffer solution to adjust the bacterial concentration to 1×10⁻⁶. 9 ~1×10 10 CFU / mL, refrigerated at 4℃ for later use in subsequent experiments. Example 1: Establishment of PCOS Mouse Model and Experimental Grouping 1. Experimental animals: Female C57BL / 6 mice, 3 weeks old, weighing 10±2g, were purchased from Henan Skobes Biotechnology Co., Ltd. They were housed in a barrier environment with a constant temperature of 22±2℃ and a 12h light-dark cycle, with free access to food and water. The experiment began after 1 week of acclimatization.

[0031] 2. PCOS model establishment: Mice were injected subcutaneously with dehydroepiandrosterone (DHEA) (60 mg / kg body weight) dissolved in 0.2 mL of sesame oil (MCE, USA) once a day for 21 consecutive days; mice in the blank control group were injected subcutaneously with 0.2 mL of sesame oil daily.

[0032] Criteria for successful model establishment: ① Microscopic observation of vaginal smears showed that the mice were in a persistent estrus cycle with disordered estrous cycles; ② Serum testosterone (T) levels were significantly elevated; ③ HE staining of ovarian tissue showed polycystic changes, which meet the core characteristics of the Rotterdam PCOS diagnostic criteria.

[0033] 3. Experimental grouping and drug administration: Mice were randomly divided into 3 groups of 6 mice each. The specific grouping and drug administration regimens are as follows: Blank control group (Ctrl): 0.2 mL of sesame oil was injected subcutaneously daily for 21 consecutive days, followed by normal feeding for 2 weeks; PCOS model group (PCOS): 60 mg / kg DHEA (dissolved in 0.2 mL sesame oil) was injected subcutaneously daily for 21 consecutive days, followed by normal feeding for 2 weeks; P101 intervention group (PCOS+P101): daily subcutaneous injection of 60 mg / kg DHEA (dissolved in 0.2 mL sesame oil) + gavage administration of 100 μL 1×10 9CFU / mL Lactobacillus plantarum P101 suspension was administered continuously for 21 days, followed by continuous gavage administration of 100 μL (1×10) 9 CFU / mL Lactobacillus plantarum P101 suspension for 2 weeks.

[0034] During the drug administration period, the mice were observed daily. On day 21 of the experiment, vaginal smears were prepared to observe changes in the estrous cycle. The smears were stained with hematoxylin and eosin (HE) and then observed under a microscope to determine the main cell types and stages of the estrous cycle. Proestrus (P) was dominated by irregularly shaped nuclear epithelial cells. Estrus (E) was dominated by layered, anucleate, keratinized epithelial cells. Metestrus (M) was dominated by keratinized epithelial cells and leukocytes. Interestrus (D) was dominated by a large number of leukocytes.

[0035] Example 2: The effect of Lactobacillus on improving glucose and lipid metabolism disorders in PCOS mice OGTT Experiment: Mice underwent an oral glucose tolerance test (OGTT) on day 32 of drug administration. Mice were placed in clean cages between 8:00 PM and 9:00 PM, allowed free access to water, and fasted for 12 hours before the OGTT. Mice were restrained, their tails were disinfected with 75% medical alcohol, and 0.5 mm of the tail was cut off. The first drop of blood was discarded, and the blood was allowed to drip naturally onto blood glucose test strips. Fasting blood glucose levels were measured and recorded as the 0-minute blood glucose level of the OGTT test. Immediately after measurement, body weight was measured, and mice were administered 20% glucose solution at 10 μL / g via gavage. Blood glucose levels were measured at 30, 60, 90, and 120 minutes after gavage. Blood glucose levels at each time point were compared between different groups, and a blood glucose curve was plotted. The area under the curve (AUC) was calculated.

[0036] The results are as follows Figure 2 Blood glucose levels in mice in groups A and B Figure 2 As shown by the area under the blood glucose level curve in the middle B, the glucose tolerance of PCOS model mice induced by hyperandrogenism is impaired, and Lactobacillus plantarum P101 can improve their impaired glucose tolerance.

[0037] The results showed that the area under the OGTT curve in the PCOS group was significantly higher than that in the Ctrl group (P<0.01); after intervention with Lactobacillus plantarum P101, the fasting blood glucose of mice was significantly reduced, proving that Lactobacillus can effectively correct PCOS-related glucose metabolism disorders.

[0038] Example 3: The effect of Lactobacillus on improving ovarian function impairment in PCOS mice Ovarian tissue morphology observation: After blood collection and sacrifice of mice, bilateral ovaries were aseptically isolated, and surrounding adipose tissue was removed. One ovary was fixed with 4% paraformaldehyde for 24 h, then dehydrated in a gradient, embedded in paraffin, serially sectioned (5 μm thick), stained with hematoxylin and eosin (HE), and the morphology of ovarian tissue was observed under an optical microscope. The number of corpora lutea and cystic follicles in the ovary was counted, and the improvement of polycystic ovarian changes was assessed.

[0039] The results are as follows Figure 1 The diagrams and quantitative analysis of the estrous cycles in the three groups of AD mice show that the estrous cycle in the hyperandrogen-induced PCOS model mice is disordered, as shown in the figure. Figure 1 The typical HE staining of ovaries, number of corpora lutea, and quantitative analysis of cystic follicles in the three groups of EG mice are shown in the figure. The number of corpora lutea and the number of cystic follicles are reduced in the PCOS model mice induced by hyperandrogenism. The intervention of Lactobacillus plantarum P101 significantly improved the estrous cycle of mice, increased the number of corpora lutea, and reduced the number of cystic follicles.

[0040] The results showed that the ovaries of mice in the Ctrl group had normal structure, with healthy follicles at all levels and abundant corpora lutea; the ovaries of mice in the PCOS group showed polycystic changes and a significant reduction in the number of corpora lutea; after intervention with Lactobacillus plantarum P101, the polycystic changes in the ovaries of mice were alleviated, the number of cystic follicles decreased, and the number of corpora lutea increased.

[0041] Estrogenic cycle recovery: Mice in the Ctrl group maintained a regular estrous cycle; mice in the PCOS group remained in estrus and completely lost their estrous cycle rhythm; the estrous cycle disorder in mice in the PCOS+P101 group was significantly reversed, suggesting that Lactobacillus plantarum P101 can improve ovulation dysfunction in PCOS mice.

[0042] Example 4: The effect of Lactobacillus on improving sex hormone secretion disorders in PCOS mice Sample collection and testing: Mice were sacrificed at week 5 by blood collection from the eyeballs. Serum was separated by centrifugation at 3000 r / min and 4℃ for 15 min. The serum was measured using an enzyme-linked immunosorbent assay (ELISA) kit, strictly following the instructions. The core sex hormone indicators in the serum were measured: testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and the LH / FSH ratio was calculated.

[0043] The results are as follows Figure 3 The serum androgen (T), LH, FSH levels and LH / FSH ratios of the three groups of mice in the AD model showed that the sex hormone disorder in the hyperandrogen-induced PCOS model mice was present, while the levels in the Lactobacillus plantarum P101 treatment group tended to be similar to those in the control group.

[0044] The results showed that the PCOS group mice exhibited typical PCOS sex hormone disorder characteristics, with significantly elevated serum androgen (T), LH levels, and LH / FSH ratio (P<0.01). After intervention with Lactobacillus plantarum P101, the above sex hormone indicators were significantly improved, proving that the Lactobacillus described in this invention can significantly correct PCOS-related sex hormone secretion disorders. Example 5: Exploring the intervention effect of Lactobacillus plantarum P101 on PCOS mice based on intestinal bile acid metabolism and the IL-22 signaling pathway. Mouse feces, intestinal tissue, and serum samples were collected. The composition of the intestinal flora was analyzed by 16S rRNA sequencing. Serum bile acid profiles were detected by liquid chromatography-mass spectrometry (LC-MS). The mRNA expression levels of TGR5, GATA3, and IL-22 in intestinal tissues were detected by qRT-PCR. Ovarian tissues were collected, and the protein expression levels of IL-22, IL-22RA, JAK1, and P-STAT3 were detected by IHC.

[0046] The results are as follows Figure 4 As shown in Figure A, the abundance of *Bacteroides commonis* with bile salt hydrolase activity in the intestines of PCOS model mice induced by hyperandrogenemia was significantly increased, such as... Figure 4 As shown in Figure B, the concentrations of bile acids TUDCA and GDCA in the serum of PCOS model mice induced by hyperandrogenemia were significantly decreased. Figure 4 As shown in Figure C, the mRNA levels of bile acid receptor TGR5, its key downstream transcription factor GATA3, and IL-22 were significantly decreased in the intestines of PCOS model mice induced by hyperandrogenemia. After intervention with *Lactobacillus plantarum* P101, these indicators tended to return to control levels. This indicates that *Lactobacillus plantarum* P101 reduced the abundance of *Bacteroides* in the intestines of PCOS model mice, thereby increasing the reduced IL-22 levels in these mice.

[0047] The results are as follows Figure 5 As shown, serum IL-22 levels in PCOS model mice induced by hyperandrogenemia were significantly decreased, and tended to return to control levels after intervention with Lactobacillus plantarum P101. Serum IL-22 concentration was significantly positively correlated with the levels of specific bile acids (such as GDCA and TUDCA).

[0048] The results are as follows Figure 6 As shown, compared with the PCOS model mice induced by hyperandrogenism, the protein expression levels of IL-22, IL-22RA1, JAK1, and P-STAT3 in the ovaries were all increased under the intervention of P101.

[0049] The results showed that compared with the Ctrl group, the abundance of *Bacteroides* in the intestine of PCOS mice was significantly increased, and the serum levels of bile acids TUDCA and GDCA, as well as the mRNA expression of intestinal TGR5, GATA3, and IL-22, were significantly decreased. After intervention with *Lactobacillus plantarum* P101, the abundance of *Bacteroides* in the intestine was significantly reduced, and the serum bile acid profile and the expression of intestinal-related genes were significantly improved, suggesting that *Lactobacillus plantarum* P101 can regulate intestinal bile acid metabolism. The expression of IL-22 and downstream pathway proteins (IL-22RA1, JAK1, P-STAT3) in the ovarian tissue of PCOS mice was significantly decreased. After intervention with *Lactobacillus plantarum* P101, the expression of IL-22 / STAT3 pathway-related proteins in the ovary was significantly upregulated, suggesting that *Lactobacillus plantarum* P101 can improve ovarian dysfunction in PCOS mice by activating the ovarian IL-22 signaling pathway.

[0050] The above results indicate that *Lactobacillus plantarum* P101 can improve ovarian function damage, metabolic and sex hormone disorders in hyperandrogen-induced PCOS model mice to some extent by regulating the gut microbiota-bile acid metabolism-IL-22 / JAK1 / STAT3 ovarian axis. This includes restoring normal estrous cycles, restoring normal levels of cystic follicles and corpus luteum, improving impaired glucose tolerance, and restoring serum sex hormone levels. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. The application of *Lactobacillus plantarum* P101 in the preparation of products for improving symptoms related to polycystic ovary syndrome, characterized in that... The *Lactobacillus plantarum* P101 was deposited at the China Center for Type Culture Collection in Wuhan on January 19, 2021, with accession number CCTCC NO: M 2021108.

2. The application according to claim 1, characterized in that, The conditions associated with polycystic ovary syndrome include one or more of the following: ovarian dysfunction, sex hormone secretion disorders, and glucose and lipid metabolism disorders.

3. The application according to claim 1, characterized in that, The product is a medicine, health food, or dietary supplement.

4. The application according to claim 1, characterized in that, The product is an oral preparation, which includes one of the following: powder, capsule, tablet, granule, oral liquid, and suspension.

5. The application according to claim 1, characterized in that, The product contains 1×10⁻⁶ live bacteria of *Lactobacillus plantarum* P101. 9 ~ 1×10 11 CFU / g or 1×10 9 ~ 1×10 11 CFU / mL.

6. The application according to claim 1, characterized in that, The product also contains pharmaceutically or food-acceptable excipients, including one or more of freeze-drying protectants, prebiotics, dietary fiber, fillers, disintegrants, and lubricants; preferably, the freeze-drying protectant is one or more of skim milk powder, maltodextrin, trehalose, and vitamin C; and the prebiotic is one or more of fructooligosaccharides, galactooligosaccharides, inulin, and resistant dextrin.

7. The application according to claim 1, characterized in that, The product also contains active ingredients for improving polycystic ovary syndrome, including one or more of metformin, short-acting oral contraceptives, inositol, vitamin D, and coenzyme Q10.

8. The application according to claim 1, characterized in that, The daily dosage of the product in mouse experiments was: 1 × 10⁻⁶ live Lactobacillus plantarum P101 bacteria. 8 ~ 1×10 9 CFU / animal / day, with a continuous dosing cycle of no less than 4 weeks.

9. The application according to claim 8, characterized in that, After administration, the number of corpora lutea in mice increased by 65-70%; the number of cystic follicles in mice decreased by 50-60%.

10. The application according to claim 1, characterized in that, The product improves symptoms associated with polycystic ovary syndrome by regulating the gut microbiota-bile acid metabolism-IL-22 / JAK1 / STAT3 ovarian axis.

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