CN113181197A - Application of salidroside in preparation of bacteria inhibiting medicine - Google Patents
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- CN113181197A CN113181197A CN202110431272.7A CN202110431272A CN113181197A CN 113181197 A CN113181197 A CN 113181197A CN 202110431272 A CN202110431272 A CN 202110431272A CN 113181197 A CN113181197 A CN 113181197A
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- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 title claims abstract description 104
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 title claims abstract description 81
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
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Abstract
The invention relates to the technical field of biological pharmacy, in particular to a method for inhibiting escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus by salidroside and application thereof; salidroside reduces the number of neutrophils in inflammatory cells, inhibits the secretion of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta, and reduces the number of colonies of Escherichia coli and/or Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus; experiments prove that salidroside can indeed relieve escherichia coli infection, staphylococcus aureus infection and methicillin-resistant staphylococcus aureus infection; provides a basic pharmacological basis for the follow-up research of the salidroside for preparing medicines for treating large intestine escherichia coli infection, staphylococcus aureus infection and methicillin-resistant staphylococcus aureus infection.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a method for inhibiting salidroside such as escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus and application.
Background
Bacterial infections are infectious diseases caused by bacteria, and bacterial infections may occur in children and adults of any age. Bacteria can infect any area of the body, including the skin, bladder, lungs, intestines, brain, etc. Bacterial infections can also spread in the blood, leading to sepsis. In immunocompromised people, such as granulocytopenia patients, organ transplant patients, and acquired immune dysfunction patients, bacterial infections can be immediately life threatening. Escherichia coli is a gram-negative bacterium, and can cause gastrointestinal tract infection or urinary tract infection of various local tissues and organs of human and various animals under certain conditions. After the escherichia coli is infected, diarrhea, vomiting, gastrospasm and low fever can be caused, and hemorrhagic colitis and hemolytic uremic syndrome can also be caused. Hemolytic uremic syndrome is a serious disease, sometimes even fatal, that can lead to gastric failure, anemia, and internal bleeding. Especially, it is very harmful to infants, adults, those with low immune system, and pregnant women. Staphylococcus aureus is a gram-positive bacterium, widely exists in external environments such as air, water, soil and the like, and has high carrying rate of staphylococcus aureus in nasal cavities, throats, skins and intestinal tracts of people and animals. Under certain conditions, staphylococcus aureus can cause local and systemic infection of human bodies and cause toxic shock. Staphylococcus aureus which is resistant to methicillin, oxacillin, cefradine or Mec gene positive is defined as methicillin-resistant Staphylococcus aureus. With the widespread use and abuse of antibiotics, the rate of methicillin-resistant staphylococcus aureus infections has also increased year by year. The detection rate of methicillin-resistant staphylococcus aureus is increasing year by year in recent years, and the methicillin-resistant staphylococcus aureus reaches 53.88-84.6% reported by domestic hospitals, and becomes one of important pathogenic bacteria of nosocomial infection. Therefore, methicillin-resistant staphylococcus aureus infection, hepatitis B and acquired immunodeficiency syndrome are listed as three persistent infectious diseases which are the most difficult to solve in the world at the same time.
The variety and number of resistant bacteria is increasing due to the widespread use and abuse of antibiotics. In order to reduce the use of antibiotics and to cope with the increasing threat of microbial resistance, it is of great importance to find an alternative to antibiotic therapy.
Rhodiola rosea is called as plateau ginseng and is recorded in the four medical classics for the first time, and the pharmacopoeia records that the rhodiola rosea has the effects of tonifying qi, activating blood circulation and promoting blood circulation and relieving asthma and can be used for treating qi deficiency, blood stasis, chest stuffiness, heartache, stroke, hemiplegia, lassitude and asthma. Salidroside is the main active ingredient of rhodiola, and no report of salidroside for treating escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus infection is found so far.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a method for inhibiting bacteria.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for inhibiting Escherichia coli and/or Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus comprises contacting salidroside with the Escherichia coli and/or Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus. Methods of inhibiting bacteria are divided into in vitro methods and in vivo methods.
Further, the concentration of salidroside is 60 uM.
Specifically, the salidroside is contacted with the escherichia coli and/or staphylococcus aureus and/or methicillin-resistant staphylococcus aureus for 24 hours, and the colony number of the escherichia coli is reduced by 37 +/-19% compared with that of a blank group control group; the colony number of the staphylococcus aureus is reduced by 68 +/-20% compared with that of a blank group control group; the colony number of the methicillin-resistant staphylococcus aureus is reduced by 64 +/-10% compared with that of a blank control group, wherein the blank control group is formed by adding PBS (phosphate buffer solution) with the same amount as salidroside into escherichia coli and/or staphylococcus aureus and/or methicillin-resistant staphylococcus aureus.
The invention also aims to find the application of salidroside in preparing medicines for treating bacterial infection.
In order to solve the technical problems, the invention adopts the following technical scheme:
application of salidroside in preparing medicine for treating bacterial infection is provided. The bacteria are Escherichia coli and/or Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus
Further, the salidroside is contacted with the escherichia coli and/or staphylococcus aureus and/or methicillin-resistant staphylococcus aureus for 24 hours, and the colony number of the escherichia coli is reduced by 37 +/-19%; the colony number of the staphylococcus aureus is reduced by 68 +/-20%; the colony number of the methicillin-resistant staphylococcus aureus is reduced by 64 +/-10%.
Further, the salidroside is used for treating the Escherichia coli for 24 hours, and the number of the neutrophils is lower than 15 x 106; the content of the MCP-1 is lower than 40 pg/ml; said IL-6 content is less than 30 pg/ml; said TNF-alpha content is less than 25 pg/ml; the IL-1 beta content is less than 30 pg/ml.
Further, the salidroside is used for treating staphylococcus aureus for 24 hours, and the number of the neutrophils is less than 10 multiplied by 105(ii) a The content of the MCP-1 is less than 50 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; the IL-1 beta content is less than 15 pg/ml.
Further, the salidroside treats the methicillin-resistant staphylococcus aureus for 24 hours, and the number of neutrophils is lower than 10 multiplied by 105; the content of the MCP-1 is less than 100 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; the IL-1 beta content is less than 5 pg/ml.
The third purpose of the invention is to find the application of salidroside in preparing drugs for inhibiting the proliferation of neutrophils and the secretion of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta.
In order to solve the technical problems, the invention adopts the following technical scheme:
application of salidroside in preparing medicines for inhibiting neutrophil proliferation and secretion of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta is provided.
Further, the salidroside is used for treating the Escherichia coli for 24 hours, and the number of the neutrophils is lower than 15 x106(ii) a The content of the MCP-1 is lower than 40 pg/ml; said IL-6 content is less than 30 pg/ml; said TNF-alpha content is less than 25 pg/ml; said IL-1 β content is less than 30 pg/ml;
further, the salidroside is used for treating staphylococcus aureus for 24 hours, and the number of the neutrophils is less than 10 multiplied by 105(ii) a The content of the MCP-1 is less than 50 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; said IL-1 β content is less than 15 pg/ml;
further, the salidroside is used for treating methicillin-resistant staphylococcus aureus for 24 hours, whereinThe number of sex granulocytes is less than 10X 105(ii) a The content of the MCP-1 is less than 100 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; the IL-1 beta content is less than 5 pg/ml.
Further, the use amount of the salidroside in a mouse body is 40 mg/kg.
The invention has the beneficial effects that:
finds that the salidroside can be applied to the preparation of the medicines for treating escherichia coli infection, staphylococcus aureus infection and methicillin-resistant staphylococcus aureus infection. Animal experiments prove that salidroside can indeed relieve escherichia coli infection, staphylococcus aureus infection and methicillin-resistant staphylococcus aureus infection. The invention provides a basic pharmacological basis for the subsequent research of the salidroside for preparing the medicine for treating escherichia coli infection of large intestine, staphylococcus aureus infection and methicillin-resistant staphylococcus aureus infection.
Drawings
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
FIG. 1: the number of neutrophils after the escherichia coli infection treatment;
FIG. 2: bacterial colony number after escherichia coli infection treatment;
FIG. 3: the content of inflammatory factor MCP-1 after the escherichia coli infection treatment;
FIG. 4: the content of an inflammatory factor IL-6 after the escherichia coli infection treatment;
FIG. 5: the content of inflammatory factor TNF-alpha after the escherichia coli infection treatment;
FIG. 6: the content of an inflammatory factor IL-1 beta after the escherichia coli infection treatment;
FIG. 7: average fluorescence intensity of BacLightgreen in vitro phagocytosis escherichia coli experiments of salidroside group and PBS group;
FIG. 8: the number of neutrophils after the staphylococcus aureus infection treatment;
FIG. 9: bacterial colony number after staphylococcus aureus infection treatment;
FIG. 10: the content of inflammatory factor MCP-1 after the staphylococcus aureus infection treatment;
FIG. 11: the content of an inflammatory factor IL-6 after the staphylococcus aureus infection treatment;
FIG. 12: inflammatory factor TNF-alpha content after staphylococcus aureus infection treatment;
FIG. 13: the content of an inflammatory factor IL-1 beta after the staphylococcus aureus infection treatment;
FIG. 14: degree of infiltration after treatment of staphylococcus aureus infection;
FIG. 15: average fluorescence intensity of BacLightgreen in vitro phagocytosis staphylococcus aureus experiments of salidroside group and PBS group;
FIG. 16: the number of neutrophils after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 17: bacterial colony number after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 18: the content of inflammatory factor MCP-1 after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 19: the content of an inflammatory factor IL-6 after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 20: the content of inflammatory factor TNF-alpha after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 21: the content of an inflammatory factor IL-1 beta after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 22: the infiltration degree after methicillin-resistant staphylococcus aureus infection treatment;
FIG. 23: average fluorescence intensity of BacLightgreen in vitro phagocytosis methicillin-resistant Staphylococcus aureus experiments of salidroside group and PBS group.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
EXAMPLE 1 Effect of Salidroside on treatment of E.coli infection
The method comprises the steps of injecting escherichia coli to the abdominal cavity to cause infection and inflammation of mice, performing different treatments and grouping, and then detecting the number of neutrophils in the abdominal cavity of the mice, the number of escherichia coli colonies and the level of inflammatory factors to verify that salidroside can inhibit escherichia coli infection and has the effect equivalent to that of antibiotics.
1 animals
C57/BL6 mice, male, 10-12 weeks old, 20-25 g in weight, purchased from the center of third-force medical laboratory animals, and normally kept in clean animal rooms.
2 experimental grouping
PBS group: administration of PBS therapy
Antibiotic group: treatment with 12.5mg/kg ciprofloxacin
Salidroside group: administration of Salidroside 40mg/kg treatment
3 Experimental methods
The experimental animals were anesthetized with ether and injected intraperitoneally with E.coli (5X 10)6c.f.u), after 2 hours, 0.5ml PBS was injected intraperitoneally as PBS group; administering salidroside (40mg/kg) as salidroside group; ciprofloxacin treatment given at 12.5mg/kg was the antibiotic group.
In order to examine whether salidroside has a therapeutic effect on escherichia coli infection, mice are killed 24 hours after treatment, lavage fluid is obtained from the abdominal cavity of the mice by using 1ml of PBS to calculate the total cell number and centrifuged, the neutral granulocytes are labeled by using Ly6G antibody, F4/80+ and Ly6G + cell populations are detected by flow cytometry to calculate the neutral granulocytes, the supernatant after centrifugation is used for detecting the levels of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta in the peritoneal lavage fluid by flow cytometry, and the escherichia coli colony number is calculated by using a coating plate method to detect the therapeutic effect of different methods on escherichia coli infection.
4. Statistical method
Data are presented as mean ± standard deviation, and one-way variance analysis (indexing one-way ANOVA) was performed using GraphPad prism8.0,. p.ltoreq.0.05,. p.ltoreq.0.01,. p.ltoreq.0.001,. p.ltoreq.0.0001.
The results are shown in FIGS. 1-6, and the numbers of neutrophils, colonies and inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta were significantly reduced in the salidroside group and antibiotic group compared with the PBS group, and the salidroside groupBacterial colony count 2.32x1053.70x10 compared to PBS blank5The reduction is about 37 percent, which shows that salidroside and antibiotics have treatment effect on escherichia coli infection; compared with the antibiotic group, the salidroside group has no obvious difference in the number of neutrophils, the number of colonies and the inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta, and shows that the salidroside has basically equivalent effect on ciprofloxacin.
Example 2 Salidroside promotes macrophage phagocytosis of Escherichia coli in vitro
PBS or salidroside is added into macrophages cultured in vitro for pretreatment, then escherichia coli marked by a molecular probe BacLightgreen is added, and the phagocytosis capacity of the macrophages on the escherichia coli is detected by detecting the average fluorescence intensity of the BacLightgreen in the macrophages through flow cytometry.
1 animals
C57/BL6 mice, male, 10-12 weeks old, 20-25 g in weight, purchased from the center of third-force medical laboratory animals, and normally kept in clean animal rooms.
2 experimental grouping
PBS group: administration of PBS therapy
Salidroside group: administration of salidroside treatment
3 Experimental methods
The experimental animals were anesthetized with ether and then intraperitoneally injected with 1.5 ml of 3% thioglycollic acid medium. After 72 hours, lavage the mouse abdominal cavity with 5ml PBS, centrifugate and get peritonitis cell, put the above-mentioned cell into low adhesion culture dish of DMEM cell culture solution already containing 10% fetal calf serum to culture for 12 hours, remove the suspension cell and leave and is the primary mouse abdominal cavity macrophage. Macrophages given PBS 24 hours of incubation were the PBS group; the macrophages incubated with salidroside (60uM) were given as the salidroside group.
In order to examine the influence of salidroside on the in vitro escherichia coli phagocytosis capacity of macrophages, the macrophages are taken, a molecular probe BacLightgreen marked escherichia coli is added for in vitro incubation for 30 minutes (the ratio of cells to bacteria is 1:10), the macrophages are marked by a fluorescence marked F4/80 antibody after being collected, and the average fluorescence intensity of BacLightgreen in the macrophages is detected by flow cytometry so as to reflect the escherichia coli phagocytosis capacity of the macrophages. The results are expressed as relative values and are shown in FIG. 7.
EXAMPLE 3 Effect of Salidroside on treatment of Staphylococcus aureus infection
The staphylococcus aureus infection of the skin of a mouse is induced by injecting staphylococcus aureus into a skin sac cavity, different treatments are given and grouped, and then the number of neutrophils in the abdominal cavity of the mouse, the number of staphylococcus aureus colonies and the level of inflammatory factors are detected to verify that salidroside can inhibit the staphylococcus aureus infection and the effect is equivalent to that of antibiotics.
1 animals
C57/BL6 mice, male, 10-12 weeks old, 20-25 g in weight, purchased from the center of third-force medical laboratory animals, and normally kept in clean animal rooms.
2 experimental grouping
PBS group: administration of PBS therapy
Antibiotic group: administration of 50mg/kg vancomycin treatment
Salidroside group: administration of Salidroside 40mg/kg treatment
3 Experimental methods
Mice were given a dorsal subcutaneous injection of 2ml of air to create a cavity, 3 days later at which a further injection of 2ml of air was made. After 3 days, the required amount of Staphylococcus aureus, salidroside or vancomycin was mixed in 0.5ml PBS, the experimental animals were anesthetized with ether, and the skin capsule cavity was injected with Staphylococcus aureus resistant (2X 10)5F.u), after 2 hours, the skin was injected intracapsular with 0.5ml PBS for PBS group; administering salidroside (40mg/kg) as salidroside group; the antibiotic group was treated with 50mg/kg vancomycin.
In order to examine whether salidroside has a treatment effect on staphylococcus aureus infection or not, mice are sacrificed 24 hours after treatment, 1ml of PBS is used for irrigating the skin capsule cavity of the mice to obtain lavage fluid, the total cell number is calculated and centrifuged, Ly6G antibody which is fluorescently labeled is used for labeling neutrophilic granulocytes, F4/80+ and Ly6G + cell populations are detected through flow cytometry, the neutrophilic granulocytes number is calculated, the supernatant after centrifugation is used for detecting the levels of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta in the peritoneal lavage fluid through flow cytometry, the staphylococcus aureus colony number is calculated through a coating plate method, and the inflammatory infiltration degree of the skin is observed through HE staining. To test the therapeutic efficacy of different methods for treating staphylococcus aureus infections.
4. Statistical method
Data are presented as mean ± standard deviation, and one-way ANOVA (order one-way ANOVA) was performed using GraphPad prism 8.0.
The results are shown in fig. 7-13, compared with the PBS group, the numbers of neutrophils, colonies, and inflammatory factors MCP-1, IL-6, TNF- α, and IL-1 β in the salidroside group and the antibiotic group were all significantly reduced, the number of bacterial colonies 228c.f.u. in the salidroside group was reduced by about 68% compared with the PBS blank group 708, HE staining showed that the degree of inflammatory cell infiltration was low, indicating that salidroside has a therapeutic effect on staphylococcus aureus infection; compared with the antibiotic group, the salidroside group has no obvious difference in the number of neutrophils, colony number, inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta, and HE skin staining shows that the infiltration degree of inflammatory cells has no obvious difference, which indicates that the effect of salidroside and vancomycin is basically equivalent.
Example 4 Salidroside promotes macrophage phagocytosis of Staphylococcus aureus in vitro
PBS or salidroside is added into macrophages cultured in vitro for pretreatment, staphylococcus aureus marked by a molecular probe BacLightgreen is added, and the phagocytic capacity of the macrophages on the staphylococcus aureus is detected by detecting the average fluorescence intensity of the BacLightgreen in the macrophages through flow cytometry.
1 animals
C57/BL6 mice, male, 10-12 weeks old, 20-25 g in weight, purchased from the center of third-force medical laboratory animals, and normally kept in clean animal rooms.
2 experimental grouping
PBS group: administration of PBS therapy
Salidroside group: administration of salidroside treatment
3 Experimental methods
The experimental animals were anesthetized with ether and then intraperitoneally injected with 1.5 ml of 3% thioglycollic acid medium. After 72 hours, lavage the mouse abdominal cavity with 5ml PBS, centrifugate and get peritonitis cell, put the above-mentioned cell into low adhesion culture dish of DMEM cell culture solution already containing 10% fetal calf serum to culture for 12 hours, remove the suspension cell and leave and is the primary mouse abdominal cavity macrophage. Macrophages given PBS 24 hours of incubation were the PBS group; the macrophages incubated with salidroside (60uM) were given as the salidroside group.
In order to examine the influence of salidroside on the capability of phagocytizing staphylococcus aureus by macrophages in vitro, the macrophages are taken, a molecular probe BacLightgreen marked staphylococcus aureus is added for incubation for 30 minutes in vitro (the ratio of cells to bacteria is 1:10), the macrophages are marked by a fluorescence marked F4/80 antibody after being collected, and the average fluorescence intensity of BacLightgreen in the macrophages is detected by flow cytometry so as to reflect the capability of the macrophages for phagocytizing the staphylococcus aureus. The results are expressed as relative values and are shown in FIG. 15.
EXAMPLE 5 Effect of Salidroside on treatment of methicillin-resistant Staphylococcus aureus infection
The method comprises the steps of inducing the skin of a mouse to resist the methicillin-resistant staphylococcus aureus infection by injecting staphylococcus aureus into a skin sac cavity, carrying out different treatments and grouping, and then detecting the number of neutrophils in the abdominal cavity of the mouse, the number of colonies of the methicillin-resistant staphylococcus aureus and the level of an inflammatory factor so as to verify that salidroside can inhibit the methicillin-resistant staphylococcus aureus infection, and the effect of the salidroside is equivalent to that of antibiotics.
1 animals
C57/BL6 mice, male, 10-12 weeks old, 20-25 g in weight, purchased from the center of third-force medical laboratory animals, and normally kept in clean animal rooms.
2 experimental grouping
PBS group: administration of PBS therapy
Salidroside group: administration of Salidroside 40mg/kg treatment
Antibiotic group: administration of 50mg/kg vancomycin treatment
3 Experimental methods
Mice were given a dorsal subcutaneous injection of 2ml of air to create a cavity, 3 days later at which a further injection of 2ml of air was made. After 3 days, mixing required amount of methicillin-resistant staphylococcus aureus, salidroside or vancomycin in 0.5ml of PBS, anesthetizing the experimental animal with ethyl ether, injecting methicillin-resistant staphylococcus aureus (2x105c.f.u) into the skin capsule cavity, and after 2 hours, injecting 0.5ml of PBS into the skin capsule cavity to obtain a PBS group; administering salidroside (40mg/kg) as salidroside group; the antibiotic group was treated with 50mg/kg vancomycin.
In order to examine whether salidroside has a treatment effect on methicillin-resistant staphylococcus aureus infection or not, mice are sacrificed 24 hours after treatment, 1ml of PBS is used for irrigating the skin capsule cavity of the mice to obtain lavage fluid, the total cell number is calculated and centrifuged, Ly6G antibody which is fluorescently labeled is used for labeling neutral granulocytes, F4/80+ and Ly6G + cell populations are detected through flow cytometry, the neutral granulocytes are calculated, the supernatant after centrifugation is used for detecting the levels of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta in peritoneal lavage fluid through flow cytometry, the methicillin-resistant staphylococcus aureus colony number is calculated through a coating plate method, and the inflammatory infiltration degree of skin is observed through HE staining. To test the therapeutic effect of different methods for treating methicillin-resistant staphylococcus aureus infections.
4. Statistical method
Data are presented as mean ± standard deviation, and one-way variance analysis (indexing one-way ANOVA) was performed using GraphPad prism8.0, P ≦ 0.01, P ≦ 0.001, and P ≦ 0.0001.
The results are shown in fig. 14-20, compared with the PBS group, the numbers of neutrophils and colonies and inflammatory factors MCP-1, IL-6, TNF- α and IL-1 β in the salidroside group are all significantly reduced, the number of bacterial colonies 278 in the salidroside group is reduced by about 64% compared with the PBS blank group 770, HE staining shows that the degree of inflammatory cell infiltration is low, indicating that salidroside has a therapeutic effect on methicillin-resistant staphylococcus aureus infection; compared with the antibiotic group, the salidroside group has no obvious difference in the number of neutrophils, colony number, inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta, and HE skin staining shows that the infiltration degree of inflammatory cells has no obvious difference, which indicates that the effect of salidroside and vancomycin is basically equivalent.
Example 6 Salidroside promotes macrophage phagocytosis of methicillin-resistant Staphylococcus aureus in vitro
PBS or salidroside is added into macrophages cultured in vitro for pretreatment, methicillin-resistant staphylococcus aureus marked by a molecular probe BacLightgreen is added, and the average fluorescence intensity of BacLightgreen in the macrophages is detected by flow cytometry, so that the phagocytosis capability of the macrophages on the methicillin-resistant staphylococcus aureus is detected.
1 animals
C57/BL6 mice, male, 10-12 weeks old, 20-25 g in weight, purchased from the center of third-force medical laboratory animals, and normally kept in clean animal rooms.
2 experimental grouping
PBS group: administration of PBS therapy
Salidroside group: administration of salidroside treatment
3 Experimental methods
The experimental animals were anesthetized with ether and then intraperitoneally injected with 1.5 ml of 3% thioglycollic acid medium. After 72 hours, lavage the mouse abdominal cavity with 5ml PBS, centrifugate and get peritonitis cell, put the above-mentioned cell into low adhesion culture dish of DMEM cell culture solution already containing 10% fetal calf serum to culture for 12 hours, remove the suspension cell and leave and is the primary mouse abdominal cavity macrophage. Macrophages given PBS 24 hours of incubation were the PBS group; the macrophages incubated with salidroside (60uM) were given as the salidroside group.
In order to examine the influence of salidroside on the ability of phagocytizing methicillin-resistant staphylococcus aureus by macrophages in vitro, the macrophages are taken, a molecular probe BacLightgreen marked methicillin-resistant staphylococcus aureus is added for incubation for 30 minutes in vitro (the ratio of cells to bacteria is 1:10), the macrophages are marked by a fluorescence marked F4/80 antibody after being collected, and the average fluorescence intensity of BacLightgreen in the macrophages is detected by flow cytometry so as to reflect the phagocytosis ability of the macrophages to the methicillin-resistant staphylococcus aureus. The results are expressed as relative values and are shown in FIG. 23.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
1. A method for inhibiting bacteria, which comprises allowing salidroside to act on the bacteria.
2. The method according to claim 1, wherein the concentration of salidroside is 60uM and the bacteria are escherichia coli and/or staphylococcus aureus and/or methicillin-resistant staphylococcus aureus.
3. The method of claim 1, wherein the salidroside is exposed to the escherichia coli and/or staphylococcus aureus and/or methicillin-resistant staphylococcus aureus for 24 hours, and the colony count of the escherichia coli is reduced by 37 ± 19% compared to a blank control group; the colony number of the staphylococcus aureus is reduced by 68 +/-20% compared with that of a blank group control group; the colony number of the methicillin-resistant staphylococcus aureus is reduced by 64 +/-10% compared with that of a blank control group, wherein the blank control group is formed by adding PBS (phosphate buffer solution) with the same amount as salidroside into escherichia coli and/or staphylococcus aureus and/or methicillin-resistant staphylococcus aureus.
4. Application of salidroside in preparing medicine for treating bacterial infection is provided.
5. The use of claim 4, wherein the bacteria comprise Escherichia coli and/or Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus, and the salidroside is exposed to the Escherichia coli and/or Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus for 24 hours, the colony count of Escherichia coli is reduced by 37 ± 19%; the colony number of the staphylococcus aureus is reduced by 68 +/-20%; the colony number of the methicillin-resistant staphylococcus aureus is reduced by 64 +/-10%.
6. The use according to claim 4, wherein the salidroside is used for treating the E.coli for 24 hours, the neutrophil count is less than 15 x106(ii) a The MCP-1 contentLess than 40 pg/ml; said IL-6 content is less than 30 pg/ml; said TNF-alpha content is less than 25 pg/ml; the IL-1 beta content is less than 30 pg/ml.
7. The use according to claim 4, wherein the salidroside treats Staphylococcus aureus for 24 hours and the neutrophil count is less than 10 x105(ii) a The content of the MCP-1 is less than 50 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; the IL-1 beta content is less than 15 pg/ml.
8. The use as claimed in claim 4, wherein the salidroside is used to treat said methicillin-resistant Staphylococcus aureus for 24 hours, the number of neutrophils being less than 10 x105(ii) a The content of the MCP-1 is less than 100 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; the IL-1 beta content is less than 5 pg/ml.
9. Application of salidroside in preparing medicines for inhibiting neutrophil proliferation and secretion of inflammatory factors MCP-1, IL-6, TNF-alpha and IL-1 beta is provided.
10. The use of claim 9, wherein the salidroside is administered to E.coli for 24 hours and the neutrophil count is less than 15 x106(ii) a The content of the MCP-1 is lower than 40 pg/ml; said IL-6 content is less than 30 pg/ml; said TNF-alpha content is less than 25 pg/ml; said IL-1 β content is less than 30 pg/ml;
the salidroside can treat staphylococcus aureus for 24 hours, and the number of the neutrophils is less than 10 multiplied by 105(ii) a The content of the MCP-1 is less than 50 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; said IL-1 β content is less than 15 pg/ml;
the salidroside is used for treating methicillin-resistant staphylococcus aureus for 24 hours, and the number of the neutrophils is less than 10 multiplied by 105(ii) a The content of the MCP-1 is less than 100 pg/ml; said IL-6 content is less than 20 pg/ml; said TNF-alpha content is less than 10 pg/ml; the IL-1 beta content is less than 5 pg/ml.
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