CN107715105B

CN107715105B - Application of IL-34 in preparation of medicine for treating or preventing sepsis

Info

Publication number: CN107715105B
Application number: CN201711021222.1A
Authority: CN
Inventors: 曹炬; 林雪; 颜星星; 张莉萍
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2017-10-26
Filing date: 2017-10-26
Publication date: 2020-11-24
Anticipated expiration: 2037-10-26
Also published as: CN107715105A

Abstract

The invention provides an application of IL-34 in preparing a medicament for treating and/or preventing sepsis, wherein the IL-34 can remarkably improve the recruitment capacity of macrophages and neutrophils, and the purpose of treating and/or preventing sepsis is achieved.

Description

Application of IL-34 in preparation of medicine for treating or preventing sepsis

Technical Field

The invention relates to the technical field of sepsis treatment, in particular to application of IL-34 in preparing a medicament for treating or preventing sepsis.

Background

Sepsis (sepsis) is newly defined as life-threatening multiple organ failure due to a dysregulated host response to infection. Sepsis is extremely dangerous, death rate is high, more than five million deaths occur globally, and morbidity and mortality are still on the trend of increasing year by year. Despite the great advances in anti-infective therapy and organ function support technologies, the mortality rate from Sepsis is still higher than 25% to 30%, even as high as 40% to 50% when septic shock occurs. Sepsis treatment cost is high, medical resource consumption is large, the life quality of human beings is seriously influenced, and great threat is already caused to human health. In this regard, the european severe society, the american severe society, and the international sepsis forum for sepsis rescue have initiated the "life-saving sepsis battle" (SSC), and in 2002, many organizations in europe and america have initiated and signed the "barcelona declaration" together, and further established evidence based on studies on sepsis, and international health organization has established worldwide sepsis as 9/13/annually in order to improve the social concerns and understanding of sepsis.

However, the sepsis is very complicated in generating mechanism, and relates to a series of physiological and pathological processes such as infection, inflammation, immunity, blood coagulation and tissue damage, and inflammatory factors released by infection mobilize various cells and organ systems of the whole body through a nerve-endocrine-immune system to form a complex network, cascade amplification and mutual restriction. Thus. Clinical treatment is very difficult, the treatment method mainly comprises antibacterial treatment and comprehensive treatment aiming at various system changes, and an effective medical method for increasing the bacterial clearance rate of sepsis and improving the survival rate of patients with sepsis is not available at present.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of IL-34 for the preparation of a medicament for the treatment or prevention of sepsis, for solving the lack of an effective medical treatment in the prior art to increase the bacterial clearance of sepsis and improve the survival of patients with sepsis.

To achieve the above and other related objects, the present invention provides the use of IL-34 in the manufacture of a medicament for the treatment and/or prevention of sepsis.

The invention also provides the use of IL-34 in the manufacture of a medicament for the treatment and/or prevention of sepsis-associated organ dysfunction.

In some embodiments of the invention, the sepsis-associated organ dysfunction comprises cardiac, spleen, liver, renal insufficiency.

The invention also provides application of the IL-34 in preparing a medicament for improving macrophage recruitment capacity.

The invention also provides the use of IL-34 in the manufacture of a medicament for increasing neutrophil recruitment.

The invention also provides the application of the expression vector in preparing a medicament for treating and/or preventing sepsis, wherein the expression vector comprises the coding sequence of the IL-34.

The invention also provides the use of an expression vector in the manufacture of a medicament for the treatment and/or prevention of sepsis by gene therapy, wherein the expression vector comprises a coding sequence for IL-34.

The invention also provides the use of a cell, which has been genetically modified to produce IL-34, in the manufacture of a medicament for the treatment and/or prevention of sepsis.

As described above, the invention provides the application of IL-34 in the preparation of the medicine for treating and/or preventing organ dysfunction related to sepsis, and IL-34 can remarkably improve the recruitment capacity of macrophages and neutrophils, thereby achieving the purpose of treating and/or preventing sepsis.

Drawings

Fig. 1 is a diagram showing a CLP modeling process in an embodiment of the present invention.

FIG. 2 is a graph showing the results of an IL-34 assay in an animal sepsis model according to an embodiment of the present invention.

FIG. 3A, FIG. 3B and FIG. 3C are graphs showing the results of IL-34 expression in blood, lung and peritoneal perfusate, respectively, in an animal sepsis model according to an embodiment of the present invention.

FIGS. 4A, 4B and 4C are graphs showing bacterial load in heart blood, spleen and Peritoneal Lavage Fluid (PLF) of mice according to the example of the present invention.

FIG. 5-1 is a graph showing lung injury in mice injected with sterile PBS control group in the examples of the present invention.

FIG. 5-2 shows a graph of lung injury in mice injected with recombinant IL-34 proteome in an example of the present invention.

FIGS. 5-3 are graphs showing kidney injury in mice injected with sterile PBS control group in the examples of the present invention.

FIGS. 5-4 are graphs showing kidney injury in mice injected with recombinant IL-34 proteome in examples of the present invention.

FIGS. 5-5 are graphs showing liver injury in mice injected with sterile PBS control group in the examples of the present invention.

FIGS. 5-6 are graphs showing liver damage in mice injected with recombinant IL-34 proteome in the examples of the present invention.

FIGS. 5-7 are graphs showing spleen injury in mice injected with sterile PBS control groups in the examples of the present invention.

FIGS. 5-8 are graphs showing spleen injury in mice injected with recombinant IL-34 proteome in examples of the present invention.

FIG. 6A is a graph showing a statistical plot of survival rates of mice in the rIL-34-injected group in the examples of the present invention.

FIG. 6B is a graph showing a statistical chart of survival rates of mice injected with IL-34 antibody in the example of the present invention.

FIG. 7 is a graph showing the detection of impairment of liver and kidney function in mice according to the example of the present invention.

FIG. 8 is a graph showing the content of CCL2 and CXCL1 in mice according to an embodiment of the present invention.

FIG. 9A is a graph showing a statistical plot of mouse survival rates for macrophage knock-out experiments in accordance with an embodiment of the present invention.

FIG. 9B is a graph showing a statistical plot of mouse survival rates for neutrophil knockout experiments in examples of the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It should be understood that the processing equipment or devices not specifically mentioned in the following examples are conventional in the art; all pressure values and ranges refer to absolute pressures.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Interleukin 34(IL-34) belongs to the interleukin family, originally discovered by large-scale screening of human endocrine-type proteins, and is also widely present in chimpanzees, mice, rats, and chickens. IL-34 is present in various tissues in the human body, such as thymus, liver, small intestine, colon, prostate, lung, heart, brain, kidney, testis, ovary, etc. IL-34 (interleukin 34) is a homodimer of 241 amino acids that increases monocyte activity by binding to colony stimulating factor 1(CSF-1) receptor, accelerating its growth rate or survival rate. Receptors for IL-34 have M-CSFR and PTP-zeta, and macrophages have potent immunosuppressive properties.

IL-34 in rheumatoid arthritis patients fibroblast-like synovial cells and synovial fluid high expression, and has a correlation with rheumatoid arthritis patients inflammation conditions in IL-34 in inflammatory bowel disease patients colon high expression, TNF-alpha dependence NF-kappa B induced colon epithelial cells in IL-34 expression. When the kidney is subjected to acute injury and ischemia reperfusion, the IL-34 level is remarkably increased, and the preparation is favorable for relieving the symptoms of renal tubular atrophy and interstitial infiltration after the acute injury of the kidney.

Example 1

Establishment of animal model of sepsis (cecal ligation and puncture, CLP)

Experimental animals: the mice used in the experiment were male, with a body weight of 18-22 g, and were about 6-8 weeks old. The animals were housed in SPF (specific Pathologen free) class laboratories, the university of Chongqing pharmaceutical laboratories. The mice used in the experiment are all SPF-level experimental animals. Wild type C57BL/6 mice (Wild type, WT) were purchased from Beijing Huafukang Biotech GmbH.

The method comprises the following steps: adult C57 mice were anesthetized by intraperitoneal injection of 100 microliters of 1.5% sodium pentobarbital, fixed to the operating plate, shaved at the abdomen with an electric pet clipper, disinfected of the skin, made a 1cm long incision in the center of the abdomen, ligatured to the cecum, and punctured with a 26 gauge syringe needle, as shown in fig. 1. Finally, the wound is sutured and the skin is disinfected. (see, in particular, Daniel Rittirsch, Peter A Ward, et al. immunological design of experimental reactions by local differentiation and pure. Nat. Protoc. 2009; 4(1): 31-36). The modeling method is a classical modeling method of a sepsis animal model, and the modeling method is a gold standard animal model for researching sepsis at present.

Detection experiment of IL-34 in blood of sepsis patient

The results of collecting blood samples of healthy subjects and blood samples of sepsis patients, counting the sex, age, and onset of disease (the results are shown in table 1), centrifuging the samples, and measuring the expression level of IL-34 using an ELISA kit (purchased from RD corporation), are shown in fig. 2, and it was found from the results that the expression level of IL-34 in sepsis patients was significantly improved.

TABLE 1 data statistics for sepsis patients and healthy persons

Characteristics	Sepsis patients(n＝68)	healthycontrols(n＝20)
			Male sex	37	11
Age,years	68(46‐89)	23‐75(49)
			WBC	18(1‐27)	NA
CRP	159(60‐258)	NA
			Infection site,no.of patients
Respiratory	28	NA
			Abdominal	21	NA
Vascular
		5	NA
Urinary				7	NA
	Other	5	NA
Unknown
		5	NA
Bacteremia				52	NA
	Isolates,no.of patients
Gram positive				21	NA
	Gram negative	27	NA
Fungus				3	NA
	Miscellaneous	9	NA
Unknown				8	NA
	APACHE II score	20(15‐31)	NA
SOFA score				7(4‐12)	NA
	ICU stay	7(4‐15)	NA
Died/survived				12/56	NA

IL-34 detection experiment in animal sepsis model

After the mice were modeled for sepsis, samples of heart blood, lung, and Peritoneal Lavage (PLF) were taken at three time points of 12 hours, 24 hours, and 48 hours, respectively, and the expression level of IL-34 was measured using an ELISA kit (purchased from RD corporation), and the results are shown in fig. 3A, 3B, and 3C, from which it was found that IL-34 expression was significantly increased in animals with sepsis.

Determination of bacterial load:

respectively modeling WT mice and mice exogenously given IL-34 protein, after 24 hours, anaesthetizing the mice, fixing, respectively taking Peritoneal Lavage Fluid (PLF), heart blood and spleen, carrying out 10-fold serial dilution on the blood, then taking 100 microliter planking, carrying out 100-fold and 10000-fold serial dilution on the Peritoneal lavage fluid, then taking 100 microliter planking, homogenizing the spleen, then taking 100 microliter planking after 10-fold serial dilution, and counting the colony number on a blood agar plate after 18-24 hours.

As shown in FIGS. 4A, 4B and 4C, the exogenous IL-34 protein administered to mice had lower bacterial load in PLF, blood and spleen and higher bacterial clearance in the body after the sepsis model was established, compared to WT mice, indicating that IL-34 facilitates bacterial clearance.

Pathological detection of each organ injury:

and (3) establishing a sepsis model for the WT mouse and the mouse given with the exogenous IL-34 protein, anaesthetizing the mouse after 24 hours, killing the mouse, taking the lung, the liver, the spleen and the kidney, fixing the mouse in 4% paraformaldehyde for 24-48 hours, dehydrating, dipping wax, embedding, slicing, and performing microscopic examination after HE staining.

As shown in FIGS. 5-1 to 5-8, the control mice showed significantly more severe damage to their organs, and the pathological sections showed marked inflammation aggravation, inflammatory cell infiltration, proteinic inflammatory exudation, tissue hyperemia and edema, and cell necrosis, as compared to the IL-34 experimental group.

Survival rate experiments:

a sepsis model is built on a WT mouse, the large part of the caecum is ligated to make a severe sepsis model, 500ng of recombinant mouse IL-34 protein (purchased from R & D company in the United states) is injected into the abdominal cavity of an experimental group mouse after the skin is sewn, sterile PBS with the same volume is injected into the abdominal cavity of a control group mouse, the survival condition of the mouse is observed and recorded, the observation is carried out twice a day, and the observation is carried out for 2 weeks continuously until the mouse does not die any more. The results are shown in FIG. 6A, the survival rate of the mice injected with rIL-34 is 66.7%, the survival rate of the mice injected with PBS is 40%, and the survival rate of the rIL-34 is obviously higher than that of the PBS and can be kept for a long time, which indicates that IL-34 is beneficial to the survival of the mice in sepsis.

To further verify the above conclusions, mice were used as a model of severe sepsis, experimental mice were intraperitoneally injected with 10ug of IL-34 antibody (purchased from R & D in the united states), control mice were intraperitoneally injected with an equal volume of IgG, and the survival of the mice was observed and recorded, twice daily for 2 weeks, until the mice no longer died. As shown in FIG. 6B, the survival rate of the mice in the experimental group injected with the antibody is significantly reduced and is significantly lower than that of the control group, which inversely indicates that IL-34 is beneficial to the survival of the mice in sepsis.

And (3) determining the liver and kidney functions of the mouse:

ALT and AST are indexes reflecting liver injury, and the increase of ALT and AST indicates that stem cells are seriously injured. LDH is an index reflecting kidney, lung and myocardial damage, and an increase in LDH indicates severe damage.

The method comprises the steps of establishing a sepsis model for a mouse, injecting 500ng of recombinant IL-34 protein (purchased from R & D company in the United states) into the abdominal cavity of an experimental group mouse, injecting sterile PBS with the same volume into the abdominal cavity of a control group mouse, collecting a heart blood sample after 24 hours, carrying out centrifugal treatment, and detecting ALT, AST, LDH, UREA and CREA, wherein the result is shown in figure 7, the damage of the liver and kidney functions of the recombinant IL-34 protein injection group mouse is obviously weakened compared with that of the control group, and the IL-34 is prompted to be beneficial to protecting the liver and kidney functions of the mouse.

Total cell count:

the method comprises the steps of establishing a sepsis model for a mouse, injecting 500ng of exogenous recombinant IL-34 protein (purchased from R & D company in the United states) into the abdominal cavity of an experimental group of mice, injecting sterile PBS with the same volume into the abdominal cavity of a control group of mice, collecting an abdominal cavity lavage fluid specimen after 24 hours, counting the total cell number of two groups by using a Bowden counting plate after the specimen is processed, and finding out that the total cell number in the mouse is increased, the recruitment capacity of neutrophils and macrophages is enhanced and bacteria removal is facilitated after the recombinant IL-34 protein is injected according to the experimental results shown in Table 2.

TABLE 2

	24h IL-34+CLP	24h PBS+CLP
			Total number of cells	67.2(32.3-118.6)	32.25(12.4-43.6)
Neutrophils	41.3(19.8-72.8)	22.6(8.7-30.5)
			Macrophage cell	7.7(3.7-13.6)	2.6(1.0-3.4)
Lymphocytes	13.3(16.4-23.5)	3.12(1.19-4.2)

Five mice in each group are tested, the data in brackets shows the maximum value and the minimum value interval of each group after a plurality of tests, the median of each group is taken to obtain the table, and the numerical order in the table is 10⁵An order of magnitude.

Chemokine detection in mice

A sepsis model was established for mice, 500ng of exogenous recombinant IL-34 protein (purchased from R & D company in the United states) was intraperitoneally injected into experimental mice, sterile PBS of the same volume was injected into control mice intraperitoneally, after 6 hours, heart blood and peritoneal lavage fluid samples were collected, and the content of CCL2 and CXCL1 in vivo was detected by using an ELISA kit, as shown in FIG. 8, it was found from experimental results that the CCL2 and CXCL1 levels in mice were significantly increased after 500ng of exogenous recombinant IL-34 protein was injected, indicating that IL-34 increased the recruitment of neutrophils and macrophages in vivo.

Mouse macrophage knockouts

And (2) injecting 200 mu l of Clophosome into the abdominal cavity of the experimental group mouse, injecting an equal volume of Clophosome control reagent into the control group mouse, taking PLF samples of the experimental group mouse and the control group mouse after 48 hours, checking, establishing a sepsis model after confirming that macrophages in the mouse are knocked out, then, injecting exogenous recombinant IL-34 protein into the experimental group mouse, observing and recording the survival condition of the mouse by using sterile equal volume of PBS of the control group mouse. The observations were made twice daily for 2 weeks until the mice no longer died. As a result, as shown in fig. 9A, the survival rate of the mice in the experimental group after knocking out macrophages was significantly reduced compared to WT. The results show that the mice in the experimental group all died within three days after the sepsis model is established, the survival rate is reduced to 0, and the WT mice can still survive for a long time after the sepsis model is established. Indicating that IL-34 in sepsis model in beneficial role and increased macrophage recruitment is related to.

Mouse neutrophil knockout:

the experimental mice are injected with 125 mul RB6-8C5 (purchased from DNAX Research Institute, Palo Alto, CA) into the abdominal cavity, the control mice are injected with isovolumetric IgG, after 48 hours, the PLF samples of the experimental mice and the control mice are taken for examination, after the removal of neutrophils in the mice is confirmed, a sepsis model is established, then the experimental mice are given with exogenous recombinant IL-34 protein, the control mice are given with isovolumetric sterile PBS, and the survival condition of the mice is observed and recorded. The observations were made twice daily for 2 weeks until the mice no longer died. As a result, as shown in fig. 9B, the survival rate of the mice in the experimental group after knockout of neutrophils was significantly reduced compared to WT. The results show that the mice in the experimental group all died within three days after the sepsis model is established, the survival rate is reduced to 0, and the WT mice can still survive for a long time after the sepsis model is established. Indicating that IL-34 in sepsis model in beneficial role and increased in vivo neutrophil recruitment is related.

In conclusion, after a large number of experimental studies, the invention discovers that IL-34 can increase the recruitment of neutrophils and macrophages in organisms, researches the treatment effect of the IL-34 through zoology experiments, and proposes the application of the IL-34 in preparing the medicine for treating or preventing sepsis.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

Use of IL-34 in the manufacture of a medicament for the treatment and/or prevention of sepsis, characterized in that: the medicine can improve the recruitment capacity of macrophages and neutrophils in an organism, and the IL-34 is recombinant IL-34 protein.
Use of IL-34 for the preparation of a medicament for the treatment and/or prevention of sepsis-related organ dysfunction, characterized in that: organ dysfunction related to sepsis comprises heart, spleen, lung, liver and renal insufficiency, and the IL-34 is recombinant IL-34 protein.
3. Use of an expression vector in the manufacture of a medicament for the treatment and/or prevention of sepsis, wherein said expression vector comprises a coding sequence for IL-34, and said IL-34 is a recombinant IL-34 protein.
4. Use of an expression vector comprising a coding sequence for IL-34, wherein IL-34 is a recombinant IL-34 protein, in the manufacture of a medicament for the treatment and/or prevention of sepsis by gene therapy.
5. Use of a cell in the manufacture of a medicament for the treatment and/or prevention of sepsis, wherein said cell is genetically modified to produce IL-34, and said IL-34 is a recombinant IL-34 protein.