Application of fermented lactobacillus mucilaginosus in preparation of functional food or medicine for inhibiting skin cell ferroptosis

Abstract

The invention discloses application of fermented lactobacillus mucilaginosus in preparation of functional food or medicine for inhibiting skin cell ferroptosis. It is found for the first time that the fermented lactobacillus mucus grx938 can be used for inhibiting skin cell ferroptosis, by orally taking a product or a fermented product containing the grx938 strain, the activity of SLC7A11 / GSH / GPX4 can be improved, the availability of ACSL4-mediated polyunsaturated fat acylation substrates can be reduced, iron-dependent lipid peroxidation and cell death can be inhibited, and the application prospect is wide. Therefore, the gap that ferroptosis nodes are not covered by an existing anti-oxidation scheme is made up. The oral fermented milk also constructs an intestine-skin axis edible daily carrier, and the fermented milk is taken as the carrier, so that the survival and delivery efficiency of lactic acid bacteria is improved, the anti-oxidation and iron metabolism regulation ability of the body is enhanced, and the defects that a pure external product depends on smearing compliance and systematic protection is difficult to realize are overcome.

Description

TECHNICAL FIELD

[0001] The application relates to application of a fermented lactobacillus mucus in preparation of a functional food or medicine for inhibiting skin cell ferroptosis, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Skin cell ferroptosis can induce photoaging, inflammation amplification and barrier damage, and further accelerate wrinkle formation and pigment abnormalities, endangering skin homeostasis and health. The main causes of skin cell ferroptosis include iron ion homeostasis imbalance and lipid peroxidation cascade induced by ultraviolet rays, especially UVB: UVB up-regulates ACSL4 and down-regulates GPX4 and SLC7A11, promotes polyunsaturated lipid acylation and glutathione depletion, enhances ROS generation driven by Fenton reaction, triggers C11-BODIPY oxidation, MDA increase and mitochondrial cristae damage; meanwhile, abnormal activation of P53 / SLC7A11 axis and ERK1 / 2 and other MAPK signals further amplifies iron-dependent membrane lipid peroxidation, eventually leading to the occurrence of ferroptosis and participating in the process of photoaging. In addition, intracellular Fe 2+ accumulation and weakened antioxidant system (decreased GSH and inhibited GPX4 activity) jointly reduce the membrane antioxidant threshold, making lipid peroxidation spread irreversibly, which is the key pathological driving force of skin cell ferroptosis. Existing interventions focus on antioxidant, anti-inflammatory and DNA repair, such as vitamins C / E, plant polyphenols, peptides and nano delivery, which can relieve ROS, inhibit MMP and repair collagen to some extent, but it is difficult to touch the root cause of iron homeostasis and lipid peroxidation, and the transdermal delivery, intracellular accessibility and long-term safety are limited, which are only suitable for prevention or need to be combined with treatment; although some drugs or photoelectric means improve dermal remodeling, they have problems such as irritation, color deposition and large individual differences, and it is difficult to systematically inhibit the ferroptosis pathway. In the background of limited transdermal delivery and long-term safety controversy of drug intervention, the development of non-drug strategies to systematically relieve oxidative stress, restore iron homeostasis and lipid peroxidation threshold, and strengthen the barrier and immune surveillance has the significance of reducing the risk of photoaging and skin cancer, improving compliance and population accessibility, and meets the needs of integrated protection and repair of multi-band light damage.

[0003] Lactic acid bacteria play a key role in skin antioxidant and homeostasis maintenance, which can scavenge reactive oxygen species, alleviate lipid peroxidation, improve barrier and inflammatory microenvironment, and reduce photoaging and oxidative stress-related damage. Lactic acid bacteria antioxidant mainly works through multiple pathways: producing glutathione, exopolysaccharides and intracellular antioxidant enzyme-like activity, improving SOD, CAT, GSH-Px and other defense systems; synthesizing organic acids and polyphenol metabolites to reduce the availability of Fenton reaction substrates, reduce lipid peroxidation and MDA generation; regulating Nrf2-related antioxidant gene expression, improving ROS homeostasis; and improving the bioavailability and delivery efficiency of antioxidants through fermentation, thereby systematically relieving photo-induced oxidative damage. However, so far there is no report on lactic acid bacteria that can functionally alleviate the key link of skin cell ferroptosis (iron homeostasis imbalance, GPX4 inactivation, lipid peroxidation chain reaction), and existing literature focuses on general antioxidant and anti-inflammatory, without verifying the inhibition of ACSL4-driven lipid acylation or the improvement of SLC7A11-GSH-GPX4 axis to block iron-dependent lipid peroxidation. The development of fermented milk and other carrier products using lactic acid bacteria that can inhibit ferroptosis can achieve systematic protection of skin cells and reduce the risk of photoaging in the daily diet, with the advantages of safety, compliance and accessibility. SUMMARY

[0004] The first object of the present application is to provide the application of fermented L.mucosus in the preparation of functional food or medicine for inhibiting skin cell ferroptosis. The second object of the present application is to provide a fermented milk for inhibiting skin cell ferroptosis.

[0005] Technical solution: The application of fermented L.mucosus in the preparation of functional food or medicine for inhibiting skin cell ferroptosis, wherein the strain is fermented L.mucosus grx938.

[0006] Further, the fermented L.mucosus grx938 is used to improve P21, P53, SLC7A11 and GPX4 gene expression.

[0007] Further, the fermented L.mucosus grx938 is used to reduce skin cell apoptosis.

[0008] Further, the functional food is fermented milk.

[0009] The fermented milk for inhibiting skin cell ferroptosis is fermented by fermented L.mucosus grx938.

[0010] Further, the preparation steps of the fermented milk include: high-temperature sterilization of whole milk, inoculation of fermented L.mucosus grx938 after cooling, post-ripening after fermentation, and obtaining.

[0011] Further, the inoculation amount of the fermented L. muciadis grx938 is 1-5%.

[0012] Further, the fermentation condition is 37-42℃ for 16-24 h.

[0013] Further, the post-ripening condition is 0-6℃ for 16-24 h.

[0014] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application first discovers that the fermented L. muciadis grx938 can be used to inhibit skin cell ferroptosis, which can inhibit iron-dependent lipid peroxidation and cell death by increasing SLC7A11 / GSH / GPX4 activity and reducing ACSL4-mediated polyunsaturated fatty acyl substrate availability through oral products or fermented products containing grx938 strains, thereby filling the gap of the iron death node not covered by the existing antioxidant scheme. Oral fermented milk also constructs a "gut-skin axis" edible daily carrier, which uses fermented milk as a carrier to improve the survival and delivery efficiency of lactic acid bacteria, enhances the body's antioxidant and iron metabolism regulation capacity, and overcomes the shortcomings of simple external products that rely on application compliance and are difficult to achieve systematic protection. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The results of macroscopically scoring the skin of mice.

[0016] Figure 2 The appearance of the skin of the irradiated area of the mice.

[0017] Figure 3 The apoptosis of the skin cells of the mice.

[0018] Figure 4 The expression of iron death-related genes. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further described below in combination with the drawings.

[0020] Example 1 Preparation of fermented milk The fermentation bacteria used in the present application are fermented L. muciadis grx938 (patent application CN202410777612.5 Antioxidant functional fermented L. muciadis and its application in mead-flavored fermented milk).

[0021] The whole milk was sterilized at 95℃ for 5 min, cooled to 37℃, and 1×10 9 cfu / mL-9×10 9 cfu / mL of fermented L. muciadis grx938 was inoculated at an inoculation amount of 3%, and fermented at 37℃ for 24 h. After fermentation, it was placed at 4℃ for post-ripening for 24 h. After post-ripening, it was stored at 4℃ for standby.

[0022] Example 2

[0023] 1. Experimental grouping and modeling 24 SPF athymic nude mice, weighing 18-22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. After a week of adaptive feeding, the experimental mice were randomly divided into 4 groups, 6 mice in each group, namely the blank group, the model group, the positive drug group, and the grx938 fermented milk group. The specific grouping and treatment methods of the animal experiment are as follows: every other day, the mice were irradiated with ultraviolet light according to the determined dose (the blank group was not irradiated); the mice in the blank group were given physiological saline 15 mL / kg / d by gavage every day, the mice in the model group were given physiological saline 15 mL / kg / d by gavage every day, the mice in the positive drug group were given vitamin E solution by gavage every day, and the mice in the grx938 fermented milk group were given grx938 fermented milk by gavage every day.

[0024] Table 1 Information of animal experiment grouping

[0025] Modeling method: the ultraviolet radiation source was about 30 cm away from the back skin of the mice. The experiment lasted for 8 weeks, and the ultraviolet radiation was performed every other day. The irradiation was performed at 9 am on the irradiation day, 1 hour before administration. The minimum erythema dose incremental method, which is currently recognized and scientific internationally, was used. The irradiation dose was 1 MED (100 mJ / cm 2 ) in the first week, and then increased by 1 MED every week until the irradiation dose stopped increasing at 4 MED in the fourth week. The irradiation dose was continued at 4 MED until the end of the experiment in the eighth week, and the total irradiation dose reached 9 J / cm 2 .

[0026] Before the end of the experiment, the mice were sacrificed by decapitation after blood sampling, and the back skin tissue of the mice was taken out, 1 cm 2 placed in 10% paraformaldehyde for fixation, and used for skin histopathology research. The remaining skin tissue was stored in a-80°C refrigerator for later use.

[0027] 2. Macroscopic evaluation of skin The irradiated area of the back of the mice was photographed every week, and the appearance of the irradiated area of the mice was recorded. The irradiated area of the mice was observed, mainly including the roughness of the skin, the generation of erythema, the degree of wrinkle, the change of thickness, and the elasticity retention of the skin, etc. The macroscopic score of the back skin of the mice was evaluated, and the grade range was 0-6 (0 grade represented normal skin, and 6 grade represented severe photoaging skin). The examination lasted for 8 weeks.

[0028] Table 2 Macroscopic evaluation criteria for skin damage

[0029] Eight weeks after UVB modeling, the model group mice developed deep wrinkles and mild skin laxity, accompanied by erythema. Mice treated with fermented mucin lactis (grx938 fermented milk) or vitamin E via gavage showed only a few shallow wrinkles and no obvious erythema. The scoring results are shown below. Figure 1 The appearance of the skin in the irradiated area of ​​the mouse is shown in the figure. Figure 2 This indicates that fermented mucin lactis (grx938) fermented milk or vitamin E intervention can significantly improve skin damage caused by UVB.

[0030] 3. Evaluation of skin cell apoptosis Fixed tissue samples were dehydrated for 60 min each in 75%, 85%, 95% I, 95% II, 100% I, and 100% II ethanol solutions, then cleared in xylene I and II for 45 min each, followed by permeabilization in paraffin I, paraffin II, and paraffin III for 60 min each. The samples were then embedded in paraffin and sectioned. The paraffin sections were baked, then immersed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 3 min, anhydrous ethanol II for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, and pure water for 2 min. The sections were then transferred to a humidified chamber, and 20 μg / ml Proteinase K working solution was added to each sample. The mixture was incubated at 37°C for 30 min. The samples were washed thoroughly with PBS three times, 5 min each time. The PBS around the tissue was blotted off with absorbent paper. Sufficient TUNEL assay solution was added to each slide, and the slides were incubated at 42°C in the dark for 1 h. Wash away excess detection solution with PBS, add DAPI and incubate in the dark for 3 min to stain the nuclei, then rinse with PBS to remove excess DAPI. Blot the slide dry with absorbent paper, mount with mounting solution containing an anti-fluorescence quencher, and then observe the acquired image under a fluorescence microscope. (DAPI UV excitation wavelength 330-380 nm, emission wavelength 420 nm, emits blue light; TUNEL excitation wavelength 510-560 nm, emission wavelength 590 nm, emits red light) Eight weeks after UVB modeling, the skin cell apoptosis status was as follows: Figure 3 As shown, red represents dead cells and blue represents live cells. The number of apoptotic skin cells in the model group was significantly higher than that in the other groups (p < 0.05). After intervention with fermented milk of *Lactobacillus mucinus* GRX938 or vitamin E by gavage, there was no significant difference in the number of apoptotic cells in the skin tissue of mice compared with the control group. This indicates that fermented milk of *Lactobacillus mucinus* GRX938 can significantly improve skin cell apoptosis induced by UVB.

[0031] 4. Expression of genes related to ferroptosis The skin tissues of the mice in each group were ground, and the total RNA of the Caco-2 cells in each group was extracted using the UNIQ-10 column Trizol total RNA extraction kit. The concentration and purity of the sample RNA were detected by the absorbance values of the sample at 260 nm and 280 nm. Reverse transcription was performed using the Novozyme HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit. q-PCR analysis was performed using the ABI Prism 7300 detection system, and all primers were designed according to the whole genome data using Oilgo 7.0 software, as shown in Table 3. All q-PCR reactions were performed using the Novozyme AceQ qPCR SYBR Green Master Mix kit. The qPCR reaction was performed according to the following conditions: pre-denaturation: 95℃ for 30 s, cycle reaction: 95℃ for 10 s, 60℃ for 30 s, 40 cycles.

[0032] Table 3 Primer sequences

[0033] After UVB modeling, the expression of P21, P53, SLC7A11 The transcription level of the gene was significantly down-regulated compared with the blank group, GPX4 The transcription level of the gene was significantly up-regulated by 1 times compared with the blank group, indicating that the damage induced by UVB inhibited the typical P53→P21 stress / cell cycle arrest axis, and the cysteine transporter SLC7A11 The inhibition of will limit the synthesis of GSH, and the anti-ferroptosis threshold will decrease, GPX4 The mild compensatory up-regulation is not enough to offset the promotion of iron-dependent lipid peroxidation, and the overall state is "increased ferroptosis susceptibility and insufficient compensation". After the mice were intervened by gavage with fermented mucus lactobacillus grx938 fermented milk, the expression of P21, P53, SLC7A11 The transcription level of the gene was significantly up-regulated compared with the blank group, GPX4 The transcription level of the gene was significantly up-regulated by 6 times compared with the blank group, indicating that the P53→P21 axis was activated, the DNA damage response and cell cycle checkpoint were enhanced, which helped to inhibit the erroneous proliferation of damaged cells and promote repair; the up-regulation SLC7A11 Restores cysteine input and GSH generation, and superimposes GPX4 Strong up-regulation significantly strengthens the SLC7A11-GSH-GPX4 anti-ferroptosis axis, inhibits iron-dependent lipid peroxidation, and significantly inhibits the lipid peroxidation chain reaction induced by UVB. After the mice were intervened by gavage with vitamin E, the expression of P21 and GPX4 The transcription level of the gene had no significant change compared with the blank group, P53 and SLC7A11Gene transcription levels were significantly downregulated compared to the blank group, indicating that vitamin E mainly provides free radical scavenging rather than upstream transcription remodeling: it failed to activate the P53→P21 checkpoint, nor did it restore SLC7A11 mediated cysteine import, resulting in an incomplete SLC7A11-GSH-GPX4 axis, making it difficult to form a mechanistic inhibition of ferroptosis.

Claims

1. Use of a fermented Lactobacillus muci- age in the manufacture of a functional food or drug for inhibiting ferroptosis of skin cells, characterized in that, The strain is fermented Lactobacillus muciage grx938.

2. Use according to claim 1, characterized in that, The fermented lactobacillus muci gen grx938 is used for improving P21、 P53, SLC7A11 and GPX4 gene expression amount.

3. Use according to claim 1, characterized in that, The fermented Lactobacillus muciage grx938 is used for reducing skin cell apoptosis.

4. Use according to claim 1, characterized in that, The functional food is fermented milk.

5. A fermented milk for inhibiting ferroptosis of skin cells, characterized by, The fermented milk is fermented by the fermented Lactobacillus muciage grx938.

6. The fermented milk according to claim 5, characterized in that, The preparation steps of the fermented milk include: high-temperature sterilization of whole milk, inoculation of the fermented Lactobacillus muciage grx938 after cooling, post-ripening after fermentation is completed, and obtaining.

7. The fermented milk according to claim 6, characterized in that, The inoculation amount of the fermented Lactobacillus muciage grx938 is 1-5%.

8. The fermented milk according to claim 6, characterized in that, The fermentation condition is 37-42℃ for 16-24 h.

9. The fermented milk according to claim 6, characterized in that, The post-ripening condition is 2-6℃ for 16-24 h.

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