CN117531002A - Application of antibacterial peptide KR-121 in preparation of medicines for regulating intestinal microecological balance - Google Patents
Application of antibacterial peptide KR-121 in preparation of medicines for regulating intestinal microecological balance Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1729—Cationic antimicrobial peptides, e.g. defensins
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses an application of antibacterial peptide KR-121 in preparation of a medicine for regulating intestinal microecological balance, and belongs to the technical field of medicines. The amino acid sequence of the antibacterial peptide KR-121 is shown as SEQ ID NO.1, wherein the amino group of the 1 st amino acid is connected with myristoyl. The invention discloses an antibacterial peptide KR-121 for the first time, which can regulate and control intestinal barrier and intestinal flora steady state, and changes intestinal flora beta-diversity on the premise of not influencing intestinal flora alpha-diversity. The antibacterial peptide KR-121 can promote the growth of Ackermansiaceae, further improve the intestinal barrier function and the bacterial infection prognosis, and provides a new thought and means for treating bacterial infection.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of antibacterial peptide KR-121 in preparation of medicines for regulating intestinal microecological balance.
Background
The intestinal barrier is a barrier structure composed of intestinal epithelial cells, the main function of which is to prevent harmful substances from entering the body while allowing nutrient absorption. Tight-junction proteins are important components of intestinal epithelial cell-to-cell junctions, with ZO-1 and occludin being two common tight-junction proteins. ZO-1 and occludin play an important role in intestinal barrier function. Their expression and dysfunction may lead to disruption of the intestinal barrier, enabling harmful substances and microorganisms to pass through the intestinal wall into the body, triggering systemic inflammation. Studies have shown that down-regulation of ZO-1 and occludin expression levels in sepsis patients correlates with impaired intestinal barrier function. Maintaining normal ZO-1 and occludin expression and function is critical to protecting the integrity and function of the intestinal barrier.
Intestinal microecology is a complex ecological system comprising 100 trillion bacteria and thousands of microorganisms, mainly comprising actinomycota, bacteroidetes, thick-wall mycota and Proteus, and accounting for more than 90% of total intestinal bacteria. Symbiotic bacteria have individual differences, are influenced by various factors such as genes and environment, and play an important role in regulating host health and disease occurrence and development. For example: as a first messenger, regulate gene expression, thereby regulating immune response, maintaining the balance of pro-inflammatory and anti-inflammatory cells in the gut. Symbiotic bacteria can stimulate intestinal epithelial cell synthesis to release antibacterial peptide and promote intestinal mucosa barrier maturation. However, bacterial infection can cause structural and functional disturbance of intestinal symbiotic bacteria, influence intestinal barrier function, and if the intestinal barrier function cannot be recovered and improved in time, intestinal bacteria can be caused to shift, and intestinal bacteremia is caused, sepsis is further aggravated and induced, and malignant circulation is formed.
In summary, there is an interdependent relationship between the intestinal barrier and the intestinal flora. The intestinal flora is critical for the function and immunomodulation of the intestinal barrier, which stability and function can also influence the composition of the intestinal flora. When pathogenic microorganism infection occurs, the organism is in a stress state, and the intestinal epithelium is in ischemia and hypoxia, the cell dysfunction and the capability of secreting the antibacterial peptide are weakened, so that the intestinal microecology is destroyed; and intestinal flora is deregulated, so that intestinal mucosa barrier is destroyed, epithelial cells are apoptotic, and the synthesis of antibacterial peptide is affected. Maintaining the health of the intestinal barrier and promoting intestinal microecological balance are critical for disease prognosis. The research based on regulating intestinal flora provides a new idea for preventing and treating diseases.
Intestinal flora plays an important role in the occurrence and development of bacterial infections, and akkermansia (akkermansia) is considered a beneficial bacterium. Ackermand can positively affect bacterial infection in several ways: (1) enhancing the integrity of intestinal barrier and reducing the permeability of intestinal tract. It promotes the expression of tight junction proteins of intestinal epithelial cells, maintains the integrity of the intestinal barrier, reduces the transfer of harmful substances and bacteria into the blood, and avoids exacerbation of infection. (2) Has antiinflammatory effect, and can reduce inflammatory reaction. It can inhibit the production of inflammatory mediators, regulate the activity of immune cells, and reduce inflammatory response caused by infection. Thus, ackerman bacteria play a positive role in the prognosis of bacterial infections and inflammatory responses.
There is a complex association between antibacterial peptides and intestinal flora: the expression of the intestinal antibacterial peptide requires the participation of intestinal flora, and the antibacterial peptide induced by the intestinal flora can kill pathogenic bacteria and maintain intestinal microecology, such as: HD5 and CRAMP can shape structural components of the intestinal flora and maintain intestinal mucosal barrier function.
Patent document CN 114181293A discloses a modified human antibacterial peptide LL-37 KR-121, which retains the active structure of human antibacterial peptide LL-37, extends the N-terminal of the modified human antibacterial peptide LL-37, and connects a fatty acid chain to form amphiphilic self-assembled nano particles, specifically, the amino acid sequence is GGGKRIVQRIKDFLR, and the N-terminal of the peptide chain is connected with myristic acid. The bactericidal activity is obviously improved through the modification, but the effect of the bactericidal composition in the intestinal flora and the intestinal barrier still belongs to the blank field.
Disclosure of Invention
The invention aims to provide an antibacterial peptide capable of improving the structure of intestinal flora and enhancing the barrier function of the intestinal tract, and provides a new idea for clinical treatment and prognosis of intestinal damage caused by pathogenic microorganism infection such as bacteria.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides application of an antibacterial peptide KR-121 in preparation of medicines or functional foods for regulating intestinal microecological balance, wherein the amino acid sequence of the antibacterial peptide KR-121 is shown as SEQ ID NO.1, and myristoyl is connected to the amino group of amino acid at position 1.
Specifically, the antibacterial peptide KR-121 does not affect the alpha-diversity of the intestinal flora, changes the beta-diversity of the intestinal flora, increases the ratio of Akkermansidae (Akkermansicae) in the intestinal flora, and reduces the ratio of Staphylococcus opportunistic bacteria (Staphylococcus) and Streptococcus (Streptococcaceae) in the intestinal flora.
The research of the invention shows that aiming at healthy populations, the antimicrobial peptide KR-121 obviously changes the microbiota composition of the intestinal flora on the premise of not changing the richness of the intestinal flora, wherein the relative abundance of Ackermann bacteria is obviously increased, and the relative abundance of staphylococcaceae and streptococcaceae is obviously reduced. Aiming at sepsis, after the antimicrobial peptide KR-121 is pretreated, the resistance to bacterial infection is obviously enhanced, and the survival rate is improved.
The expression level of related proteins such as intestinal epithelial tight junction protein ZO-1, occludin and the like is obviously reduced after the intestinal barrier is damaged, and the intestinal epithelial cells undergo apoptosis. Studies show that the antibacterial peptide KR-121 treatment can effectively increase the expression level of the intestinal barrier related proteins ZO-1 and occludin and improve the intestinal barrier injury.
Further, the medicament is a medicament for improving intestinal barrier damage and/or intestinal flora modification caused by microbial infection. Pathogenic microorganism infection can cause impaired intestinal barrier function and the intestinal flora structure changes significantly.
The manifestations of intestinal barrier damage include: increased apoptosis of intestinal epithelial cells and decreased expression levels of the intestinal barrier associated protein ZO-1, occludin; the intestinal villi is obviously shed, a large amount of inflammatory cells infiltrate, and the intestinal wall becomes thin and other tissue pathological injuries. Studies have shown that treatment with antimicrobial peptide KR-121 can significantly improve the above symptoms.
Pathogenic microbial infections result in an increase in the number of pro-inflammatory related bacteria such as Clostridia UCG-014 and a decrease in the number of Akkermannia (Akkermansiaceae). Studies show that the antibacterial peptide KR-121 can be used for treating the intestinal flora, so that the structure of the intestinal flora can be promoted to be normal, and the proportion of beneficial bacteria can be increased. Specifically, ackermaniaceae (Akkermansiaceae) is significantly up-regulated in the intestinal flora.
Further, the microbial infection is a celiac-derived bacterial infection. The bacterial infection may be, but is not limited to, caused by acinetobacter baumannii.
The disease caused by infection with pathogenic microorganisms such as bacteria may be, but is not limited to, enterogenic bacteremia and sepsis.
Further, the medicine is a medicine for preventing and treating secondary infection. Research shows that when the antibacterial peptide KR-121 is used for treating infection, the antibacterial peptide KR-121 not only effectively disinfects, but also regulates the intestinal barrier function and the intestinal flora steady state, improves long-term prognosis and improves the resistance to secondary infection. In particular, the secondary infection may be, but is not limited to, a sepsis secondary infection.
The invention also provides a pharmaceutical composition for treating intestinal barrier damage and/or intestinal flora structural change caused by microbial infection, which comprises an effective dose of the antibacterial peptide KR-121 and a pharmaceutically acceptable carrier.
When the antibacterial peptide KR-121 is used as a medicament, preparation can be performed by referring to the prior mode.
The invention has the beneficial effects that:
the invention discloses an antibacterial peptide KR-121 for the first time, which can regulate and control intestinal barrier and intestinal flora steady state, and changes intestinal flora beta-diversity on the premise of not influencing intestinal flora alpha-diversity. Further mechanism researches show that the antibacterial peptide KR-121 can promote the growth of Akkermanni (Akkermansiaceae), further improve the intestinal barrier function and the disease prognosis, and provide a new thought and means for treating infectious diseases.
Drawings
FIG. 1 shows analysis of intestinal flora alpha-diversity, beta-diversity and significant difference flora of healthy mice 14 days after intraperitoneal injection of KR-121, wherein A is alpha-diversity; b is beta-diversity; c is the relative abundance of the intestinal Acidovorax (Akkermansiaceae), the Staphylococcus (Staphylococcus) and the Streptococcus (Streptococcaceae). In the figure, p values less than 0.05 indicate statistical differences; pre-KR-121-0d: healthy mice prior to KR-121 treatment; pre-KR-121-14d: healthy mice after 14d intraperitoneal injection of KR-121.
FIG. 2 shows the effect of KR-121 pretreatment on resistance to bacterial infection, wherein A is the survival rate of mice infected with bacteria 14 days after intraperitoneal injection of KR-121; b is the plasma fluorescein isothiocyanate glucan (FITC-dextran) level of the bacteria infected mice after 14 days of intraperitoneal injection of KR-121, reflecting the intestinal permeability; c is KR-121, 14 days after intraperitoneal injection, bacterial infection mice intestinal epithelial tunnel staining detects apoptosis of intestinal epithelial cells; d is the expression level of the protein detected by ZO-1, occludin immunofluorescence staining of intestinal epithelium of the bacteria infected mice after 14 days of intraperitoneal injection of KR-121; e is the Western blot result of ZO-1, occludin in intestinal epithelium of mice infected by bacteria after 14 days of intraperitoneal injection of KR-121. In the figure, p values less than 0.05 indicate statistical differences; the control group is a sublethal dose of Acinetobacter baumannii infected mice; the pre-KR-121-14d group is mice infected with sub-lethal dose of Acinetobacter baumannii after 14 days of intraperitoneal injection of KR-121; ZO-1: a tight junction protein; occludin: a tight junction protein; beta-actin: reference protein.
FIG. 3 shows the improvement of intestinal barrier and intestinal flora in mice with bacterial infection by the long-term application of KR-121, wherein A is the recovery of body weight of mice with bacterial infection; b is the level of fluorescein isothiocyanate glucan (FITC-dextran) of the plasma of a bacterial infected mouse, and reflects the intestinal permeability; c is intestinal flora alpha-diversity; d is intestinal flora β -diversity.
FIG. 4 is a graph showing the effect of KR-121 on the intestinal flora of mice infected with bacteria for a long period of time, analyzed at the mycota level.
FIG. 5 is a graph showing the effect of the prolonged application of KR-121 on the intestinal flora of mice infected with bacteria at the mycological level.
FIG. 6 shows the effect of KR-121 long-term treatment on secondary infection survival.
FIG. 7 shows the effect of KR-121 long-term treatment on decongestive edema in the intestine following infection.
FIG. 8 is a graph showing the effect of KR-121 long-term treatment on bacterial load in whole body tissue organs after secondary infection, PLF: peritoneal lavage fluid, liver: liver, spleen: spleen, lung: lung, kidney: and (3) kidneys.
FIG. 9 shows detection of inflammatory factors following KR-121 long-term treatment.
FIG. 10 shows plasma levels of fluorescein isothiocyanate dextran (FITC-dextran) in mice following infection with KR-121 for long-term treatment.
FIG. 11 is an illustration of the effect of KR-121 long term treatment on the intestinal barrier of secondary infections, wherein A is intestinal epithelial tunnel staining for detection of intestinal epithelial apoptosis; b is the expression level of the protein detected by immunofluorescence staining of intestinal epithelium ZO-1 and occludin; c is the Western blot result of intestinal epithelium ZO-1, occludin.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The amino acid sequence of KR-121 used in the following examples is shown in SEQ ID NO.1, specifically: gly (Gly) 1 -Gly 2 -Gly 3 -Lys 4 -Arg 5 -Ile 6 -Val 7 -Gln 8 -Arg 9 -Ile 10 -Lys 11 -Asp 12 -Phe 13 -Leu 14 -Arg 15 A myristoyl group is attached to the amino group of Gly at position 1. Synthesized by Shanghai Tao Pu polypeptide Co., ltd.
Example 1: effect of KR-121 on alpha-diversity and beta-diversity of intestinal flora
1. Experimental animal
SPF class 6-8 week healthy male C57BL/6J mice.
2. Experimental method
All animals were fed ad libitum, fed with water, and after 1 week of adaptive feeding, the mice were collected faeces, designated pre-KR-121-0d, frozen in liquid nitrogen, and subjected to high throughput sequencing of the 16S rDNA amplicon for subsequent intestinal flora. Mice were then given daily i.p. injections of KR-121 (3 mg/kg), and after 14 days of continuous i.p. injections, the mouse faeces were collected, designated pre-KR-121-14d, frozen in liquid nitrogen, and the difference in flora composition was compared in faecal DNA samples from mice of pre-KR-121-0d and pre-KR-121-14d groups, after subsequent intestinal flora 16S rDNA amplicon high throughput sequencing.
The high throughput sequencing procedure for 16S rDNA amplicon is briefly described: (1) DNA extraction: total DNA is extracted from the sample using a microbial DNA extraction kit. (2) 16S rDNA amplification: a universal primer pair for the 16S rRNA gene was selected to PCR amplify 16SrDNA from the bacterial 16S rDNA variable region (V3-V4). (3) And (3) purifying a PCR product: the size and purity of the PCR products were checked using gel electrophoresis. (4) Library construction and quantification: library construction was performed using Illumina platform. The purified library was quantified using a real-time PCR method. (5) High throughput sequencing: multiple libraries with different barcode were pooled and high throughput sequenced. (6) Data analysis: and performing quality control on the original sequencing data.
The samples were analyzed for microbial alpha diversity, beta diversity, and relative abundance based on OUT (Operational Taxonomic Units) for microbial classification. Ace index, chao index and OTUs reflect the alpha-diversity of intestinal flora of healthy mice before and after KR-121 treatment; restriction primary coordinate analysis (CPCoA) and primary coordinate analysis (PCoA) reflect healthy mouse intestinal flora β -diversity before and after KR-121 treatment.
Next, the significantly different populations of intestinal flora of mice in the pre-KR-121-0d group and the pre-KR-121-14d group were analyzed.
3. Experimental results
3.1 as shown in figure 1 a, the two groups of mice were compared for intestinal flora differences by inter-sample alpha diversity analysis (Ace index, chao index and OTUs), with no statistical differences in alpha diversity.
As shown in fig. 1B, the pre-KR-121-0d group (red dots) was found to be centrally distributed on the left side of the graph by inter-sample beta diversity analysis (restricted primary coordinate analysis, CPCoA, and primary coordinate analysis, PCoA), while the pre-KR-121-14d group (pink dots) was centrally distributed on the right side of the graph, with no cross-overlap with the pre-KR-121-0d group, indicating significant differences in intestinal flora structure between the pre-KR-121-0d group and the pre-KR-121-14d group mice.
The results of the alpha-diversity and beta-diversity analysis show that the intraperitoneal injection of KR-121 does not change the richness of the intestinal flora, but significantly changes the microbiota composition of the intestinal flora, indicating that the intraperitoneal injection of KR-121 changes the microbiota composition of the intestinal flora of the healthy mice.
3.2 analysis of the significantly different populations of intestinal flora in mice from groups pre-KR121-0d and pre-KR121-14d, as shown in FIG. 1C, the pre-KR121-14d groups were significantly up-regulated in the intestinal flora by Akkermansidae (Akkermansiaceae) and significantly down-regulated in the intestinal flora by Staphylococcus (Staphylococcus) and Streptococcus (Streptococcaceae).
Example 2: effect of KR-121 pretreatment on resistance to bacterial infection
1. Experimental animal
SPF grade 6-8 week healthy male C57BL/6J mice, mice were intraperitoneally injected with a sublethal dose (2X 10) 7 CFU) acinetobacter baumannii (ATCC 19606), a sepsis model was constructed.
2. Experimental method
All animals were fed ad libitum, and after 1 week of adaptive feeding, mice were given daily KR-121 intraperitoneal injections (3 mg/kg), and after 14 days of continuous intraperitoneal injections, mice were given a sublethal dose of Acinetobacter baumannii (ATCC 19606) (2X 10) 7 CFU), designated pre-KR-121-14d.
Abdominal injection of sublethal dose of Acinetobacter baumannii (ATCC 19606) (2X 10) into healthy mice not subjected to KR-121 intraperitoneal injection 7 CFU) as a control group.
Two groups of mice after infection are respectively infused with 600mg/kg of fluorescein isothiocyanate dextran (FITC-dextran) orally, blood is collected through an orbital venous plexus after 4 hours, and the levels of FITC-dextran in the blood are detected to judge the intestinal permeability.
Mice survival was observed every 12 hours. 8-12 hours after infection, the mice were anesthetized and bled to an anticoagulant tube. The mice were fixed, the abdominal cavity was opened, part of the ileum was cut 4-5cm away from the ileocecal valve, one part was fixed with 4% paraformaldehyde, and the other part was frozen with liquid nitrogen.
4% paraformaldehyde-fixed ileum was paraffin-embedded, sectioned, tunnel stained and observed for intestinal epithelial apoptosis.
4% paraformaldehyde-fixed ileum was paraffin-embedded, sectioned, immunofluorescent stained for ZO-1, occludin, and intestinal epithelial ZO1, occludin protein levels were observed.
And cutting part of the ileum tissues frozen by liquid nitrogen, extracting proteins, and carrying out Western blot detection of ZO-1 and occludin.
3. Experimental results
Survival statistics as shown in figure 2 a, the survival rate of mice in pre-KR-121-14d group was significantly higher than that in control group.
Intestinal permeability results as shown in figure 2B, plasma FITC-dextran levels were significantly lower in pre-KR-121-14d mice than in control, indicating that KR-121 pretreatment reduced the increase in intestinal permeability caused by infection.
The results of small intestine tunnel staining are shown in fig. 2C, blue fluorescence indicates nuclei, green fluorescence indicates apoptotic intestinal epithelial cells, and pre-KR-121-14d group intestinal epithelial apoptosis is significantly less than control group, indicating that KR-121 pretreatment reduced intestinal epithelial apoptosis caused by infection.
The results of small intestine ZO-1, occludin immunofluorescence staining are shown in FIG. 2D, blue fluorescence indicates nuclei, green fluorescence indicates ZO-1, red fluorescence indicates occludin, and higher fluorescence intensity indicates higher protein levels. The expression level of pre-KR-121-14d group intestinal epithelium ZO-1, occludin is significantly higher than that of control group, indicating that KR-121 pretreatment increases the expression of intestinal barrier-related protein, and reduces the intestinal barrier damage caused by infection.
As shown in the Western blot results of ZO-1 and occludins in the small intestine, the expression level of ZO-1 and occludins in the pre-KR-121-14d group of intestinal epithelium is obviously higher than that of the control group, which indicates that the KR-121 pretreatment increases the expression of the protein related to the intestinal barrier and reduces the damage of the intestinal barrier caused by infection.
The results show that the resistance of the mice to bacterial infection is remarkably improved after 14 days of intraperitoneal injection of KR-121.
Example 3: long term application of KR-121 to improve intestinal barrier and intestinal flora of bacterial infection
1. Experimental method
The experimental mice were randomized into 4 groups after 1 week of adaptive feeding, namely, a lethal dose bacterial infection model group (cantretated group) (n=7), a lethal dose bacterial infection KR-121 treatment group (KR-121-14 d group) (n=8), a lethal dose bacterial infection meropenem (MEM) treatment group (MEM-14 d group) (n=7), a healthy group (H group) (n=7), and specifically:
untreated group: 0h intraperitoneal injection of minimum lethal dose of Acinetobacter baumannii (2.8X10) 7 CFU), without medication;
KR-121-14d group: 0h intraperitoneal injection of minimum lethal dose of Acinetobacter baumannii (2.8X10) 7 CFU) +KR-121 (3 mg/kg) was injected intraperitoneally daily for 14 consecutive days;
MEM-14d group: 0h intraperitoneal injection of minimum lethal dose of Acinetobacter baumannii (2.8X10) 7 CFU) + continuous 14 days of intraperitoneal injection of MEM (13 mg/kg);
group H: 7: 0h of intraperitoneal injection of physiological saline and continuous 14 days of intraperitoneal injection of physiological saline;
all animals were free to eat, drink, and the Untreated group died entirely within 48 hours. KR-121-14d, MEM-14d, H were weighed daily, fecal samples were collected on day 14, frozen in liquid nitrogen, and the intestinal flora 16S rDNA amplicon was subjected to high throughput sequencing.
On day 14, KR-121-14d, MEM-14d, and H mice were orally infused with 600mg/kg of fluorescein isothiocyanate dextran (FITC-dextran), and 4 hours later were bled through the orbital venous plexus to detect FITC-dextran levels in the blood.
2. Experimental results
As shown in FIG. 3A, the KR-121-14d group recovered significantly faster than the MEM-14d group.
As shown in FIG. 3B, the levels of FITC-dextran in the blood of the KR-121-14d group were significantly lower than those of the MEM-14d group, comparable to those of the H group, indicating that KR-121-14d treatment significantly restored the intestinal barrier function of the bacterially infected mice as compared to the MEM-14d treatment.
As shown in fig. 3C, the results of the intestinal flora analysis showed that the alpha diversity (Ace index, chao index, OTUs) was significantly reduced among the bacterial-infected mice intestinal flora samples in MEM-14d treated group compared to group H, while the alpha diversity (Ace index, chao index, OTUs) was recovered in KR-121-14d treated group, with no statistical difference from group H.
As shown in FIG. 3D, the differences in intestinal flora of KR-121-14D group, MEM-14D group and H group mice were compared by an inter-sample diversity (β -diversity) analysis. The results of CPCoA and PCoA analysis of the three groups of mice intestinal flora based on non-weighted Unifrac show that there is no cross overlap between groups H, MEM-14d, KR-121-14d (purple, blue, red dots), indicating significant differences in intestinal flora structure between groups KR-121-14d, MEM-14d, and H mice.
As shown in FIG. 4, three groups of mice were analyzed at the phylum level for the presence of significantly different groups of bacteria in KR-121-14d, MEM-14d, and H groups of mice. The group KR-121-14d, verrucomicrobiota (Verrucom) is significantly up-regulated in the intestinal flora and the group MEM-14d, proteus (Proteus) is significantly down-regulated in the intestinal flora, since MEM kills a large number of gram-negative Proteus in the intestinal tract while treating bacterial infections.
As shown in FIG. 5, further analysis at the bacterial family level, it was found that the Akkermaceae (Akkermanaseae) of the KR-121-14d treatment group was significantly up-regulated, whereas the Enterobacteriaceae (Enterobacteriaceae) of the MEM-14d treatment group was significantly down-regulated, since MEM was indiscriminately challenged to gram-negative bacteria, and not only Acinetobacter baumannii, but also Enterobacteriaceae was killed, causing structural disturbance of intestinal flora, enterococcus became dominant bacteria, and aggravating the possibility of secondary infection. In the KR-121-14 treatment group, intestinal flora is balanced and intestinal barrier related probiotics Acremonium (Akkermansiaceae) are increased while Acinetobacter baumannii infection is treated.
Example 4: KR-121 long-term treatment has protective effect on secondary infection
1. Experimental method
A lethal dose bacterial infection and treatment model was established as in example 3, after treatment was completedE.coli E.coil (1X 10) was intraperitoneally injected into group H, KR-121-14d, MEM-14d, respectively, for 3 days 7 CFU), a mouse secondary infection model was constructed.
All animals were fed ad libitum and the survival rate of three groups of mice was observed for 12 h.
After infection, three groups of mice were each orally infused with 600mg/kg of fluorescein isothiocyanate dextran (FITC-dextran), and 4 hours later were bled through the orbital venous plexus to detect FITC-dextran levels in the blood. In addition, 8 hours after infection, mice were anesthetized, 3mL of pre-chilled PBS was injected intraperitoneally, and after sufficient gentle massaging of the abdomen, peritoneal lavage fluid was withdrawn for subsequent bacterial counts and inflammatory factor detection. The abdominal cavity was then opened and three groups of mice were observed for enteric congestion and edema. And shearing part of livers, spleens, lungs and kidneys, weighing, grinding, gradient diluting with PBS, inoculating on LB solid medium, culturing for 16-18h, and counting bacteria. Three groups of mouse ileum were excised and fixed with 4% paraformaldehyde, the fixed ileum was paraffin-embedded, sectioned, tunnel stained and observed for intestinal epithelial apoptosis.
And centrifuging the peritoneal lavage fluid, taking the supernatant, and respectively detecting the levels of inflammatory factors TNF-alpha, IL-1 beta and IL-10 in the peritoneal lavage fluid according to the detection specification of an ELISA kit.
2. Experimental results
As shown in FIG. 6, the survival rate of mice infected with secondary to KR-121-14d+E.coil group was significantly increased compared to H+E.coil group and MEM-14d+E.coil group.
As shown in FIG. 7, KR-121-14d+E.coil group showed significantly reduced intestinal congestion edema in mice subsequently infected with KR-121-14d+E.coil group compared to H+E.coil group and MEM-14d+E.coil group.
As shown in FIG. 8, KR-121-14d+E.coil group peritoneal lavage fluid, liver, spleen, lung, kidney whole body tissue organ bacterial load was significantly lower than H+E.coil group and MEM-14d+E.coil group.
As shown in FIG. 9, KR-121-14d+E.coil group peritoneal lavage fluid TNF- α, IL-1β and IL-10 levels were significantly reduced.
As shown in FIG. 10, the levels of FITC-dextran in the blood of KR-121-14d+E.coil were significantly lower than those of the H+E.coil and MEM-14d+E.coil, indicating that KR-121-14d+E.coil intestinal permeability was significantly lower than those of the H+E.coil and MEM-14d+E.coil.
As shown in FIG. 11A, the small intestine tunnel staining results, blue fluorescence indicated nuclei, green fluorescence indicated apoptotic intestinal epithelial cells, and the KR-121-14d+E.coil group had significantly less intestinal epithelial apoptosis than the H+E.coil group and MEM-14d+E.coil group, indicating that KR-121-14d significantly reduced intestinal epithelial apoptosis caused by secondary infection after bacterial infection in mice.
As shown in FIG. 11B, the results of immunofluorescence staining of small intestine ZO-1, occludin, blue fluorescence indicated nuclei, green fluorescence indicated ZO-1, red fluorescence indicated occludin, and higher fluorescence intensity indicated higher protein levels. The expression level of ZO-1 and occludin in intestinal epithelium of KR-121-14d+E.coil group is obviously higher than that of H+E.coil group and MEM-14d+E.coil group, which shows that after the KR-121-14d is used for treating sepsis mice, the expression of protein related to intestinal barrier is increased, and the damage of the intestinal barrier caused by secondary infection is reduced.
As shown in FIG. 11C, the Western blot results of ZO-1 and occludin in the small intestine show that the expression level of ZO-1 and occludin in the intestinal epithelium of KR-121-14d+E.coil group is obviously higher than that of the ZO-1 and occludin in the H+E.coil group and MEM-14d+E.coil group, and the expression of protein related to intestinal barrier is increased after the KR-121-14d is used for treating mice with secondary infection, so that the damage of the intestinal barrier caused by secondary infection is reduced.
The results show that the KR-121 can improve the intestinal flora structure of mice infected by bacteria, promote recovery, increase the proportion of beneficial bacteria, improve the resistance to secondary infection and strengthen the intestinal barrier function compared with MEM.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (10)
1. The application of the antibacterial peptide KR-121 in preparing medicines or functional foods for regulating intestinal microecological balance is characterized in that the amino acid sequence of the antibacterial peptide KR-121 is shown as SEQ ID NO.1, wherein the amino group of amino acid 1 is connected with myristoyl.
2. Use according to claim 1, wherein the antimicrobial peptide KR-121 does not affect the α -diversity of the intestinal flora, alters the β -diversity of the intestinal flora and increases the ratio of ackerman bacteria in the intestinal flora.
3. The use according to claim 1, wherein the antimicrobial peptide KR-121 increases the expression level of the intestinal barrier associated protein ZO-1, occludin.
4. The use according to claim 1, wherein the medicament is a medicament for ameliorating intestinal barrier damage and/or intestinal flora modification caused by a microbial infection.
5. The use of claim 4, wherein the manifestation of intestinal barrier damage comprises: increased apoptosis of intestinal epithelial cells and decreased expression levels of the intestinal barrier associated protein ZO-1, occludin.
6. The use according to claim 4, wherein the antimicrobial peptide KR-121 promotes the return of the intestinal flora structure to normal and increases the proportion of beneficial bacteria.
7. The use according to claim 4, wherein the microbial infection is a bacterial infection of abdominal origin.
8. The use according to claim 7, wherein the bacterial infection is caused by acinetobacter baumannii.
9. The use according to claim 1, wherein the medicament is a medicament for the prophylaxis and treatment of secondary infections.
10. A pharmaceutical composition for treating intestinal barrier damage and/or intestinal flora structural change caused by microbial infection, which is characterized by comprising an effective dose of antibacterial peptide KR-121 and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the antibacterial peptide KR-121 is shown as SEQ ID NO.1, and myristoyl is connected to the amino group of amino acid 1.
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