CN112972533A - Application of compound agrimony - Google Patents

Abstract

The invention discloses application of compound hairyvein agrimony. Experiments prove that the compound hairyvein agrimony can effectively improve the histopathological changes of lungs, kidneys and colon of mice with invasive mycosis, and plays a role in treating the invasive mycosis by increasing the expression quantity of neutrophil-associated gene lymphocyte antigen complexes and antimicrobial peptide LL-37 in the lung tissues and the colon tissues of the mice. The compound agrimony has an application prospect in treating invasive mycosis.

Description

Application of compound agrimony

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of compound hairyvein agrimonia herb and bud.

Background

Invasive mycosis (IC) refers to a systemic infectious disease caused by invasion of tissues, organs and blood by various pathogenic candida species. It has become the most common fungal infection among hospitalized patients with low immunity and serious underlying disease. The most common pathogenic bacteria of IC are candida albicans, candida tropicalis, candida glabrata, and candida parapsilosis. Most life-threatening fungal infections occur in immunocompromised trauma, high immunosuppressive usage, neutropenia, medical device implantation and organ transplant patients with a mortality rate of about 40%. Such high mortality rates are largely closely related to early diagnosis of IC and prognosis of treatment, but there is now a clinical lack of an ideal rapid diagnostic method that can lead to a loss of optimal treatment opportunities for patients. The original blood culture diagnosis often has the limitations of low sensitivity and long cycle time, and modern medicine takes serology (mannan, mannan antibody and beta-glucan) and molecular (fungus-specific PCR in blood and serum) as a series of screening ranges for high-risk patients, and although these techniques are quite sensitive and highly specific, the technologies are still in research stage to a great extent, and the clinical effectiveness needs to be determined. Clinically, up to 70% of IC patients do not receive any antifungal therapy within 24 hours of blood culture, and the mortality rate of IC patients also increases with the delay of treatment. However, with the intervention treatment of antibacterial drugs such as fluconazole, the clinical isolates generate drug resistance genes and form cross resistance with other azole drugs. Amphotericin B and its lipid formulations are now the broad spectrum of drugs in IC therapy, which are well tolerated, but their hepato-renal toxicity remains the major drawback, and the drugs and methods for clinical treatment of IC are limited.

Herba et Gemma Agrimoniae (herba Agrimoniae) from RosaceaeAgrimonia Pilosa Ledeb. var. japonica (Miq). Nakai) Dried aerial parts of (1). It is recorded in ancient Chinese medical books such as Ben Cao Tu Jing and Yi nationality medical books such as Shi little molehill Yi Shu, from the book of pseudo-drugs, the book of dialectics. Yi nationality thinks that the medicine is bitter and astringent in taste and cold in nature, and enters liver, stomach, lung and large intestine, and the product is decocted with water for treating diarrhea, abdominal pain, diaphragmatic food and dysentery. Later, the medicine is discovered in the long-term medical application processWhen patients with heavy disease conditions and complicated and variable disease conditions occur, the single-medicine treatment often causes the situations of poor efficacy and incomplete treatment, and the large-scale use of the traditional Chinese medicine is easy to cause toxic and side effects. Therefore, according to the medical experience, coptis chinensis, costus root, grassleaf sweelflag rhizome, platycodon grandiflorum and cicada slough are added to enhance the drug effect and reduce the toxic and side effects of curbing by the famous-national old traditional Chinese medicine of Yunnan province. The traditional Chinese medicine is a treasure in the traditional culture of China, is clear in target spot and is different from western medicine which is concentrated on local parts, and the traditional Chinese medicine mainly strengthens the body resistance to eliminate pathogenic factors in treatment and regulates the balance of the body so as to restore and improve the autoimmune function of the body to prevent and treat the body integrally. We find in the research that the compound agrimony has good treatment and prevention effects on invasive mycosis. The invention relates to the action and the efficacy of compound hairyvein agrimony on the aspect of resisting invasive mycosis, and related reports are not found.

Disclosure of Invention

The invention aims to provide application of compound agrimony.

Application of compound herba et Gemma Agrimoniae in preparing medicine for preventing/treating invasive mycosis is provided.

The fungi is Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Cryptococcus or Marneffea vulgaris.

Compound herba et Gemma Agrimoniae can be used for treating invasive mycosis by increasing mouse neutrophil and promoting LL-37 release.

The compound agrimony is an oral preparation or an intravenous injection.

The oral dosage form is tablet, hard capsule, soft capsule, powder, pill, or granule.

The intravenous injection is solution injection, suspension injection, emulsion injection, or sterile powder for injection.

The dosage of compound herba et Gemma Agrimoniae is 3 times per day, 1-3g each time.

The invention has the advantages that:

the invention has the advantages and positive effects that the biological research shows that the compound hairyvein agrimony has obvious treatment effect on experimental invasive mycosis, has no drug-resistant side effect and light toxic and side effect, and prompts that the compound hairyvein agrimony has the prospect of developing new drugs for resisting invasive mycosis.

Drawings

FIG. 1 shows a chromogenic medium for Candida albicans;

FIG. 2 is the appearance of mice;

FIG. 3 shows the pathological changes of mouse organs;

FIG. 4 shows the effect of compound Agrimonia pilosa enteritidis capsules on the pathology of lung tissue in IC mice (H & E staining and PAMS staining, 400X);

FIG. 5 shows the effect of Compound Agrimonia pilosa enteritis Capsule on the histopathology of the kidney of IC mice (H & E staining and PAMS staining, 400X);

FIG. 6 shows the effect of Compound Agrimonia pilosa enteritidis Capsule on Colon histopathology in IC mice (H & E staining and PAMS staining, 200X);

FIG. 7 shows the effect of Compound Agrimonia pilosa capsules on Ly6G and LL-37 expression in IC mouse lung tissue (immunofluorescence, 400X);

FIG. 8 shows the effect of Compound Agrimonia pilosa capsules on the expression of Ly6G and LL-37 in intestinal tissue of IC mice (immunofluorescence, 200X);

FIG. 9 shows the weight change of mice in each group after successful modeling;

fig. 10 is a survival curve for each group of mice.

Detailed Description

The present invention is further illustrated but not limited in any way by the following examples, and any modifications made thereto are intended to fall within the scope of the present invention.

Application of compound herba et Gemma Agrimoniae is provided.

Application of compound herba et Gemma Agrimoniae in preparing medicine for preventing/treating invasive mycosis is provided.

The fungi is Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Cryptococcus or Marneffea vulgaris.

Compound herba et Gemma Agrimoniae can be used for treating invasive mycosis by increasing mouse neutrophil and promoting LL-37 release.

The compound agrimony is an oral preparation or an intravenous injection.

The oral dosage form is tablet, hard capsule, soft capsule, powder, pill, or granule.

The intravenous injection is solution injection, suspension injection, emulsion injection, or sterile powder for injection.

The dosage of compound herba et Gemma Agrimoniae is 3 times per day, 1-3g each time.

Example 1

Research on in vivo efficacy of compound agrimony enteritis capsule against candida albicans infection

1 Experimental materials and methods

1.1 Experimental materials and instruments

1.1.1 test strains

Candida albicans sensitive strain ATCC10231 was purchased from American Type Culture Collection (ATCC) and was awarded by snow Engineer, Kunming plant institute, China academy of sciences.

1.1.2 Experimental animals

SPF grade C57BL/6 mice, 50 at 6 weeks of age, purchased from totso great laboratory animals ltd, animal certification No.: SCXK (Chuan) K2015-030, the weight of the animal is 20 +/-2 g, the male and female parts are respectively half, the experimental conditions and the method are examined and qualified by an ethical committee of an animal experiment center of Yunnan university of traditional Chinese medicine, and the animal ethical examination document number is as follows: r-082018074.

1.1.3 Experimental drugs

Compound agrimony enteritis capsule, yunnan jinbi pharmaceutical limited, approved article: national medicine standard character Z53021622, product batch number: 171202.

fluconazole dispersible tablets, Nanchang Hongyun pharmaceutical Co Ltd, approved Wen No: national drug standard H20051376, product batch number: 161105.

cyclophosphamide for injection, Baxter Oncology GmbH, imported drug registration No.: h20160467, product batch number: 0a 355A.

1.1.4 Experimental reagents

1.1.5 reagent preparation

Sodium carboxymethylcellulose (CMC-Na) solution: 5g of sodium carboxymethylcellulose powder is weighed and added into 1000mL of ultrapure water to be fully and uniformly mixed, and the mixture is prepared into 0.5 percent sodium carboxymethylcellulose solution by using an autoclave at the temperature of 121 ℃ for 30 min.

Cyclophosphamide (CTX) solution: the cyclophosphamide injection prepared from cyclophosphamide for injection by using 0.9% normal saline is kept to be operated in the dark and stored in the dark during the dissolving process, and the cyclophosphamide injection is prepared for use on site to ensure the stability of the cyclophosphamide injection in aqueous solution.

Compound agrimony enteritis capsule working solution: 0.4g of compound agrimony enteritis capsule content medicine is taken for each capsule, 3 capsules are taken, the content is ground into fine powder by using a mortar, and 40mLCMC-Na solution is added in parts to prepare 600mg/kg compound agrimony enteritis capsule suspension.

Fluconazole working solution: grinding two tablets into fine powder by using a mortar, adding 2mL of LCMC-Na solution to prepare a mother solution of 50mg/mL, and adding 1.2mL of the mother solution to CMC-Na solution to prepare a suspension of 15mg/kg and 40mL when in use.

Saxifrage liquid medium: 50.1g of the culture medium powder is accurately weighed and fully mixed with 1000mL of ultrapure water, sterilized in an autoclave at 115 ℃ for 15min, cooled to room temperature and stored in a refrigerator at 4 ℃.

Sakheira agar medium: weighing 65.1g of culture medium powder accurately, using 1000mL of ultrapure water to mix fully, sterilizing for 15min at 115 ℃ in an autoclave, pouring the liquid into a culture dish when cooling to about 50 ℃, and placing the culture dish in a refrigerator at 4 ℃ for later use after solidification.

Candida chromogenic medium: 47.7g of culture medium powder is accurately weighed and fully mixed with 1000mL of ultrapure water, the mixture is heated by a microwave oven to boiling and then taken out, the mixture is gently shaken and then placed into the microwave oven for heating, the small bubbles are observed to become large bubbles, the large bubbles are observed, the large bubbles are cooled to about 50 ℃, the liquid is poured into a culture dish, and the culture dish is placed in a refrigerator at 4 ℃ for standby after solidification.

1.1.6 Experimental instruments

1.2 Experimental methods

1.2.1 activation and identification of Candida albicans

Taking out the strain from a refrigerator at minus 80 ℃, sucking a proper amount of the strain suspension, dropwise adding the strain suspension into a 50mL centrifuge tube containing a Sabouraud's liquid culture medium, sealing the centrifuge tube by using a sterile air-permeable sealing film, and then placing the centrifuge tube into a constant-temperature shaking table at 37 ℃ for shaking for 24 hours. After 24h, the Candida albicans was collected by centrifugation at 3000rpm after 3 washes with 0.9% physiological saline for 5 min. Diluting the collected candida albicans by using 0.9% physiological saline, dropwise adding 10 mu L of bacterial suspension to a Sabouraud's agar culture medium, coating by using a coater, then placing in a constant-temperature and constant-humidity incubator for culture, after 24h, selecting a single colony by using an inoculating loop, performing plate streaking on the Sabouraud's agar culture medium and the candida albicans chromogenic culture medium, and after culturing in the constant-temperature and constant-humidity incubator for 48h, taking the chromogenic culture medium for observation; the Sabouraud agar medium is taken out after being cultured for 24h and is stored in a refrigerator at 4 ℃, and 0.9% physiological saline is used for washing and preparing before injection.

1.2.2 mouse immunosuppressive model establishment

50 mice in each half of four-week-old C57BL/6 mice are subjected to adaptive feeding for one week, 40 mice in each half of the four-week-old C57BL/6 mice are subjected to intraperitoneal injection of Cyclophosphamide (CTX) solution once a day for 3 days continuously, and after Candida albicans inoculation, cyclophosphamide is subjected to intraperitoneal injection once every two days to form a mouse immunosuppressive model, wherein the dosage of the first three days is 100mg/kg, the dosage of the second two subsequent days is 75mg/kg, the administration volume is 10mL/kg, and the remaining 10 mice are used as a Blank group (Blank).

1.2.3 Candida albicans invasive infection model establishment and administration

The mice successfully established by 40 immunosuppressive models are randomly divided into 4 groups, namely a Control group (Control), a Model group (Model), a fluconazole administration group (FLC) and a compound agrimonia pilosa enteritis capsule administration group (FFXHC). Each group was 10, each group was randomly numbered. The experimental period is 7 days, and each group of mice is fed with normal diet and drinking water freely, and are raised in clean animal rooms with 24 +/-2 ℃ and 12h alternating light and shade. The following table shows the administration doses of each group obtained by taking the daily dose of the Chinese patent drug by the person in the commercial drug manual according to the conversion relationship of the administration dose of the human and the mouse specified in the pharmacological test methodology. The administration mode is intragastric administration, the administration is carried out once a day, and the intragastric volume is 20 mL/kg.

TABLE 1-1 groups of mice, drugs and dosages administered

1.2.4 sample Collection

After 24 hours of last administration, the mice are anesthetized by pentobarbital sodium, samples are collected, the organs with serious lesion of the model group mice are taken, the organs are cleaned by normal saline, then the water is absorbed by filter paper, and the organs are preserved by 4% paraformaldehyde universal stationary liquid, wherein the main organs are the lung, the kidney and the colon of the mice.

1.2.5 index detection

1.2.5.1 hematoxylin-eosin staining (H & E) method for observing histopathological changes of lung, kidney and intestine of mice

a. Paraffin section dewaxing to water: taking the embedded tissue slices with the thickness of 5-8 mu m, and washing the slices with tap water after putting the slices into dimethylbenzene I (20 min), dimethylbenzene II (20 min), absolute ethyl alcohol I (5 min), absolute ethyl alcohol II (5 min) and 75% ethyl alcohol (5 min) in sequence;

b. hematoxylin staining: coloring the slices in hematoxylin staining solution for 3-5 minutes, washing with tap water, adding differentiation solution for differentiation, washing with tap water, returning blue with blue returning liquid, and washing with running water;

c. eosin staining: placing the slices in 85% -95% gradient alcohol for 5min respectively for dehydration, and then dyeing in eosin dye solution for 5min;

d. dewatering and sealing: the sections were placed in the following systems, respectively: carrying out transparency for 5min on absolute ethyl alcohol I, absolute ethyl alcohol II, absolute ethyl alcohol III, xylene I and xylene II, and then sealing the film by using neutral resin;

e. microscopic examination, image acquisition and observation of tissue morphology and inflammatory cell infiltration.

1.2.5.2 Hexamine silver staining (PAMS) for observing the distribution of Candida albicans in lung, kidney and intestine tissues of mice

a. Paraffin section dewaxing to water: taking the embedded tissue slices with the thickness of 5-8 mu m, and washing the slices with tap water after putting the slices into dimethylbenzene I (20 min), dimethylbenzene II (20 min), absolute ethyl alcohol I (5 min), absolute ethyl alcohol II (5 min) and 75% ethyl alcohol (5 min) in sequence;

b. dyeing with hexa-amino-silver: the slices are acted for 10min in 1% periodic acid water solution, washed by tap water, then washed twice by distilled water, each time for 3min, the hexammine silver solution is heated to 65 ℃, the slices are placed in the solution for dip dyeing for 75min, and washed twice by distilled water, each time for 3min, the color of the solution is adjusted by 0.1% gold chloride for 5min, and the solution is washed twice by distilled water, each time for 3min, the solution is treated by 5% sodium thiosulfate for 5min, and the solution is washed twice by distilled water, each time for 3min, and cytoplasm is counterdyed by 0.5% brilliant green;

c. dewatering and sealing: dewatering and sealing: the sections were placed in the following systems, respectively: carrying out transparency for 5min on absolute ethyl alcohol I, absolute ethyl alcohol II, absolute ethyl alcohol III, xylene I and xylene II, and then sealing the film by using neutral resin;

d. microscopic examination, image acquisition and observation of colony distribution in tissue.

1.2.5.3 immunofluorescence assay for localization of LL-37 and neutrophils in Lung and Kidney tissues in mice

a. Respectively placing paraffin sections of lung and kidney of mouse in xylene I (15 min), xylene II (5 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), 85% ethanol (5 min), and 75% ethanol (5 min), and washing with distilled water;

b. the sections were placed in EDTA antigen retrieval buffer (pH 8.0) and antigen retrieval was performed by quenching 8min with a microwave donkey medium fire for 8min and then turning the medium fire for 7min (note the excessive evaporation of the buffer leading to dry sections). Naturally cooling the slices, and washing with PBS for 5min for 3 times on a decolorizing shaker;

c. gently spin-drying the slices, circling the tissues by using a organized pen, and dripping BSA (bovine serum albumin) into the circles to incubate for 30min for sealing;

d. gently spin-drying the slices, dripping the first primary antibody diluted by PBS, and placing the slices in a wet box for incubation overnight at 4 ℃;

e. washing with PBS for 3 times (5 min each time) in a shaking table, dripping corresponding secondary antibody labeled with HRP after slicing, and incubating at room temperature for 50 min;

f. washing with PBS 3 times for 5min each time on a shaking table, dripping fluorescent dye CY3-TSA after gently spin-drying the slices, incubating for 10min at room temperature in dark place, and after incubation is finished, placing the slices on a shaking table, and washing with TBST 3 times for 5min each time;

g. microwave treatment: repeating the step b;

h. adding a second primary antibody, a second antibody and a fluorescent dye solution FITC-TSA: the operation steps are the same as those of e and f;

i. spin-drying the slices slightly, dripping DAPI dye liquor into the rings to re-dye cell nuclei, and incubating for 10min at room temperature in a dark place;

j. slightly spin-drying the slices, dripping an autofluorescence quencher into the ring, and washing for about 10min with running water after 5min;

k. washing the slices on a shaking table for 3 times with PBS (phosphate buffer solution) for 5min each time, slightly spin-drying the slices, and sealing the slices with an anti-fluorescence quenching sealing tablet;

microscopic examination: the slice is imaged under a scanner. (DAPI emits blue light, FITC emits green light, CY3 emits red light under UV excitation)

2 results of the experiment

2.1 identification of Candida albicans

Candida albicans ATCC10231 is inoculated on a chromogenic medium and cultured in a constant temperature and humidity incubator for 48 hours in an aerobic way, and the colony of the Candida albicans ATCC10231 strain is found to be green, and the strain is proved to be the Candida albicans by comparing with the specification of the Candida albicans chromogenic medium (shown in the attached figure 1).

2.2 appearance and in vivo organ pathological changes of mouse model infected with Candida albicans

After an invasive infection model is established, the mice are found to have reduced vitality, the phenomenon of aversion to cold and curling the back is caused, the intake amount and the drinking amount are greatly reduced, the hair color is messy, dull and lusterless, the weight and the survival rate of eyes and horns are obviously reduced (figure 2), and some mice are accompanied with diarrhea. After 7 days, the pathological changes of the viscera are observed after the cervical vertebra is removed after anesthesia, and the cortex of the mouse is found to be thinned, the abdominal fat is rare or absent, the pathological changes of the lung and the kidney are serious, a large amount of blood clots appear in the lung, the white bacterial colony protrusion appears in the kidney, the color is dark and grey, the whole color of the small intestine is blue, the content is less, and a large amount of gas exists (figure 3).

2.3 Compound Agrimonia pilosa capsules for treating enteritis and lung pathology in mice model infected by Candida albicans

As shown in fig. 4: the lung tissue of the mice in the blank group is pink in appearance, smooth in surface and good in elasticity, while the lungs of the mice in the control group are edematous after cyclophosphamide injection, the lungs of the mice in the model group are more serious after candida albicans inoculation, the lungs of the mice are edematous, a large number of blood clots are raised on the surface of the mice, and the pulmonary edema and the blood clots are relieved after drug intervention; h & E staining shows that blank lung tissues in a blank group are complete and have a net structure, the lung interstitium does not have a thickening phenomenon and does not have inflammatory cell infiltration, a control group is accompanied by a small amount of inflammatory cell infiltration, compared with the blank group and the control group, the lung interstitium in a model group is thickened and congested, a large amount of inflammatory cell infiltration occurs, improvement appears after drug treatment is given, the lung interstitium thickening and inflammatory cell infiltration phenomena in an FLC group are reduced and are closer to the control group, the lung interstitium thickening in an FFC group is obviously reduced and is closer to the blank group, but the inflammatory cell infiltration condition is more serious than the blank group and is closer to the control group; PAMS staining was seen in the blank and control groups as sterile colonies, and yeast-like fungi were seen in the model, FLC and FFXHC groups, but both FFXHC and FLC groups were decreased after drug intervention. The result shows that the compound agrimony enteritis capsule can effectively improve the pathological change of the lung tissues of the IC mice.

2.4 Compound Agrimonia pilosa capsules for treating kidney disease of mice infected by Candida albicans

As shown in fig. 5: the kidney tissues of mice in the blank group and the control group are dark red in appearance, smooth in surface and good in elasticity, white protrusions appear on the surfaces of the kidneys after candida albicans are inoculated, the color is dull and lusterless, after the mice are subjected to drug drying, the appearance can be greatly improved, the surfaces of the FLC groups are smooth and good in elasticity, the FLC groups are close to the blank group and the control group, the protrusions on the surfaces of the FFXHC groups are reduced, the colors are dark red and have certain luster, and the FFXHC groups have certain elasticity; h & E staining shows that the kidney corpuscle structure and the renal interstitium of mice in a blank group and a control group are complete, no inflammatory cell infiltration condition exists, compared with the blank group and the control group, most of glomerular structures of a model group are necrotic, inflammatory cell infiltration appears around focuses, tubular hemorrhage and other conditions occur, after drug treatment, the inflammatory infiltration of the kidneys of the mice in an FLC group and an FFXHC group is reduced, and the symptom of the glomerular structure necrosis is relieved; PAMS staining was seen in the blank and control groups as sterile colonies, and yeast-like fungi were seen in the model, FLC and FFXHC groups, but both FFXHC and FLC groups were decreased after drug intervention. The result shows that the compound agrimony enteritis capsule can effectively improve the histopathological change of the kidney of an IC mouse.

2.5 Compound Agrimonia pilosa capsules for treating kidney disease of mice infected by Candida albicans

As shown in fig. 6: after cyclophosphamide is given to the control group of mice to establish an immunosuppression model, the colon of the mice is obviously shortened, the intestinal mucosa of the model group is damaged after candida albicans is inoculated, the content is reduced, the length of the colon is obviously increased and the intestinal content is increased after drug treatment is given; h & E staining shows that compared with a blank group, intestinal villus swelling and congestion of a control group, intestinal villus swelling and congestion of a model group mouse, epithelial cells are exfoliated, inflammatory cell infiltration exists in an indigenous layer, the improvement is obvious after the drug is given, intestinal villus edema of an FLC group is reduced, and inflammatory cell infiltration of an FFXHC group is reduced; PAMS staining shows that Candida albicans colonizing the intestinal tract is detected in all groups of mice, but the Candida albicans invading the lamina propria of the intestinal tract is not observed. The result shows that the compound agrimony enteritis capsule can improve the colon pathological change of the IC mice.

2.6 Effect of Compound Agrimonia pilosa Ledeb enteritis Capsule on Lung LL-37 related neutrophil of mouse model infected by Candida albicans invasively

As shown in FIG. 7, the expression of Ly6G and LL-37 in the lungs of the model group mice was enhanced as compared with that of the blank group; after the compound agrimony enteritis capsule is used for treating, the expressions of Ly6G and LL-37 are obviously enhanced, and the co-localization is obvious; the co-localization of the DAPI and the Ly6G labeled neutrophils and the LL-37 labeled antibacterial peptide shows that the neutrophilic granulocyte quantity in the lung of the mouse is increased by the treatment of the compound agrimony enteritis capsule, and a large amount of antibacterial peptide LL-37 is generated, so that the two are in a large co-localization condition in the lung, and the connection exists. The result shows that the compound agrimonia pilosa ledeb enteritis capsule can increase the number of neutrophils in the lung and release the antibacterial peptide LL-37, but the relationship between the two is not clear.

2.7 Effect of Compound Agrimonia pilosa capsules on intestinal LL-37-associated neutrophils in mice model infected by Candida albicans

As shown in FIG. 8, although Ly6G in the intestine was highly expressed in the blank group and the model group, but was not co-localized with DAPI, LL-37 was expressed in the blank group, the model group and the FFXHC group, and the major expression site was outside the villus, and the co-localization of Ly6G, DAPI and LL-37 in the intestinal villus was observed after the compound Agrimonia pilosa capsules for enteritis treatment, indicating that neutrophils migrated to the mouse intestine and accompanied with the release of the antimicrobial peptide LL-37 after the compound Agrimonia pilosa capsules for enteritis treatment.

3 small knot

The inventor researches and discovers that the compound agrimony enteritis capsule has obvious improvement effect on the survival state of mice infected by candida albicans invasively, the survival rate and the body weight of mice in an administration group are obviously improved, the colony number of the kidney is obviously reduced, the organ coefficient of immunity is also obviously improved, and meanwhile, the inventor also discovers that the quantity of neutrophilic granulocytes in the peripheral blood of the mice in an FFXHC group is obviously higher than that of the mice in a control group and a model group; LL-37 plays a key role in innate immunity and acquired immunity, we dynamically monitored LL-37 homologous antimicrobial peptide CRAMP in invasive Candida albicans infected mice, and found that CRAMP in plasma rises after treatment with compound Agrimonia pilosa enteritis capsule, reaches a peak at day 5, and then falls but is still higher than that in the model group, suggesting that our LL-37 and neutrophils play an important role in compound Agrimonia pilosa enteritis capsule treatment of Candida albicans invasive infection.

Clinically, the major affected organs of IC are lung, kidney, intestine and other organs, and the IC mouse model also has similar symptoms. Neutropenia patients are more susceptible to invasive infections by candida. And the absence of neutrophils is an unavoidable condition for invasive infection by candida albicans, which also suggests that innate immunity plays a very important role in IC. LL-37, as part of innate immunity, has potent chemotactic and immunoregulatory properties in addition to its broad-spectrum antimicrobial action. H & E staining and PAMS staining experimental results show that after the compound agrimony enteritis capsule is used for treating an IC mouse, inflammatory cell infiltration of lungs and kidneys of the mouse can be effectively relieved, and the colony count of the lungs and the kidneys can be effectively reduced; immunofluorescence staining results show that after the compound agrimony enteritis capsule is used for treating, the lung Ly6G and LL-37 of a mouse are increased in expression, the number of neutrophils is increased, a large amount of LL-37 is released, and the co-localization of the two is observed frequently, but the relationship between the two is unclear, and the cleaning is needed urgently. The bactericidal mechanism of the neutrophils mainly depends on the formation of NETs, and researches show that the bactericidal effect and the function of regulating the immunity of the NETs depend on LL-37. Therefore, it is speculated that the mechanism of compound agrimonia pilosa enteritis capsule treatment of IC mice is likely to function by increasing the number of neutrophils and releasing LL-37, and LL-37 is likely to be produced by neutrophils, but the relationship between the two is not so, and further confirmation is needed. The next experiment is mainly to carry out in vitro experimental verification on the influence of the compound agrimony enteritis capsule on LL-37 related neutrophils.

4 conclusion

The compound agrimony enteritis capsule can improve pathological changes of lungs, kidneys and colon of an IC mouse, and can reduce the colony count of the lungs and kidneys of the IC mouse; compound herba et Gemma Agrimoniae enteritis capsule treats IC mice by increasing mouse neutrophil granulocytes and promoting LL-37 release.

Example 2

Compound herba et Gemma Agrimoniae for treating invasive Marneffei basket fungus infection

The method comprises the following steps: the mice were administered with 0.1ml/10g of cyclophosphamide (100 mg/ml) intraperitoneally 3 days before, and the experimental group was administered with 1x10 concentration of Marneffei basket fungus (TM) intraperitoneally 4 days after⁷One/ml, 0.1ml/10 g. The control group was injected with saline intraperitoneally.

Grouping: a control group; compound herba et Gemma Agrimoniae group; amphotericin group B; compound agrimony combined with amphotericin B.

Cyclophosphamide moulding

After cyclophosphamide injection, the weight of the mice gradually decreases, the activity is reduced, and the food intake is reduced. The body weight of the experimental group further decreased after TM injection, especially the body weight decreased most obviously 4 days before TM injection, and the body weight was more stable after 5 days. The body weight gradually recovered after day 4 in the control group (fig. 9).

After the mice are successfully modeled, amphotericin B, compound agrimony and compound agrimony are respectively given for treatment in combination with amphotericin B, so that the weight and survival rate of the mice in the agrimony group are higher than those of the mice in the amphotericin B group and the model group, and the survival curve is shown in attached figure 10. The survival rate of the compound agrimony and amphotericin B treatment is higher than that of the amphotericin B treatment group and the model group. The survival rate of the compound agrimony group is higher than that of the model group, which shows that the compound agrimony group is effective in antifungal treatment, and especially the treatment effect of the compound agrimony group combined with amphotericin B is caused by simple amphotericin B treatment.

The dosage of compound herba et Gemma Agrimoniae is 3 times per day, 1-3g each time.

Claims (8)

1. The application of the compound hairyvein agrimony is characterized in that the compound hairyvein agrimony is applied to a medicine for preventing/treating invasive mycosis.

2. The use according to claim 1, wherein the fungus of an invasive mycosis is Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Cryptococcus or Marneffeta.

3. The use according to claim 1 or 2, wherein said combination of agrimony is for the treatment of invasive mycoses by increasing mouse neutrophils and promoting the release of LL-37.

4. The use of claim 1 or 2, wherein the compound herba Agrimoniae is in the form of oral preparation or intravenous injection.

5. Use according to claim 4, characterized in that the oral dosage form is a tablet, hard capsule, soft capsule, powder, pill, granule.

6. The use according to claim 4, wherein the intravenous injection is a solution injection, a suspension injection, an emulsion injection, or a sterile powder for injection.

7. The use of the compound agrimony as claimed in claim 1, wherein the dose of the compound agrimony is 1-3g for 3 times a day.

8. A pharmaceutical composition, characterized by comprising the compound agrimony as claimed in any one of claims 1 to 7 as an active ingredient or a pharmaceutically acceptable carrier.

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