CN110538323A - Application of anti-LCN 2 antibody in preparation of medicine for treating lupus nephritis - Google Patents

Abstract

The invention belongs to the field of biological diagnosis and medicine, and particularly relates to application of an anti-LCN 2 antibody in preparation of a medicine for treating lupus nephritis. The invention takes MRL/lpr mice of a control group (rat immunoglobulin) and a control group (anti-LCN 2 antibody) as research objects, and the survival rate, the urinary protein level, the spleen index, the kidney inflammation, the immune complex and the complement deposition detection are carried out on the MRL/lpr mice, and the results show that the anti-LCN 2 antibody can improve the survival rate of the mice, reduce the urinary protein and the spleen index of the MRL/lpr mice, reduce the kidney inflammation, the immune complex and the complement deposition, prove that the anti-LCN 2 antibody has good treatment effect on lupus nephritis, is an effective target spot for treating the lupus nephritis, can be developed as a new medicine or medicine target for treating the lupus nephritis, and provides a new way and means for treating the lupus nephritis.

Description

Application of anti-LCN 2 antibody in preparation of medicine for treating lupus nephritis

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of an anti-LCN 2 antibody in preparation of a medicine for treating lupus nephritis.

Background

systemic Lupus Erythematosus (SLE) is a common autoimmune disease involving multiple organs and systems, and is typically characterized by activation of autoreactive lymphocytes, production of autoantibodies, and deposition of immune complexes. Lupus Nephritis (LN) is one of important organ complications affecting the prognosis of SLE, the nephropathy morbidity of SLE patients is up to 50% -60%, and the pathological changes mainly include mesangial cell hyperplasia, mesangial stroma increase, and obvious lymphocyte and macrophage infiltration in glomerulus and renal interstitium; crescent fibrosis, glomerular capillary intravascular hyperplasia, renal interstitial sclerosis, renal tubular atrophy and the like also occur, and the phenomena are the main causes of death of the SLE patient; LN can increase the kidney burden, the progression is rapid, the fatality rate is high, about 70% of LN patients have kidney injury, 10% -15% of LN patients finally develop terminal renal failure, no complete cure method exists at present, and the SLE is one of the most serious complications; therefore, it is a great challenge to control and delay the progression of the disease and improve the survival rate of the patients.

at present, large-dose glucocorticoid combined immunosuppressant such as cyclophosphamide, mycophenolate mofetil and the like is commonly used for clinically treating LN, but the enhanced immunosuppressive therapy is easy to reduce the resistance of patients and bring side effects such as infection and the like, and cyclophosphamide can also cause premature ovarian failure and increase the tumor risk; furthermore, the above treatment is only effective in 80% of LN patients, with 55-80% of patients taking effect for up to 12-24 months, 10-20% remaining ineffective, 10-20% developing end-stage renal failure within 10 years, and 50% experiencing relapse. In order to reduce recurrence, a series of adverse reactions can be generated by further adopting a small-dose glucocorticoid and immunosuppressant for long-term treatment, such as suppression of autoimmunity, easy induction or aggravation of infection, suppression of reproductive system function, increase of tumor occurrence, destruction of hematopoietic function, and damage of normal immune function of the organism. In addition, in order to improve LN prognosis, it is necessary to perform remission maintenance therapy for preventing relapse in addition to acute phase remission induction therapy, and to perform chronic renal failure progression inhibition therapy. Therefore, the search for a new target drug with significant efficacy and high safety is a problem to be solved urgently by the current LN treatment.

Disclosure of Invention

the technical problem to be solved by the invention is to research the effect of the anti-LCN 2 antibody in LN, provide a new target drug for treating LN and provide a new way and means for detecting and treating lupus nephritis, aiming at the defects of the prior art.

in order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: the anti-LCN 2 antibody is applied to the preparation of a medicine for treating LN.

Specifically, spontaneous MRL/lpr lupus mice are used as test objects, and after the mice are administered with anti-LCN 2 antibody for intraperitoneal injection, the proteinuria level of the lupus mice is reduced, the spleen index is reduced, the kidney inflammation is reduced, and immune complexes are reduced, so that the anti-LCN 2 can obviously improve lupus nephritis, is an effective target for treating LN, can be developed as a novel anti-LN medicament or medicament target, and provides a novel way and means for detecting and treating LN.

compared with the prior art, the invention has the beneficial effects that: the anti-LCN 2 antibody can reduce urine protein and spleen index of LN, reduce kidney inflammation and immune complex deposition, and provide experimental data and theoretical basis for the anti-LCN 2 antibody to obviously improve lupus nephritis; the anti-LCN 2 antibody can be used as a new intervention target of an anti-LN medicament, provides a brand-new choice and idea for the current treatment of LN medicaments, and widens the selection field of the treatment of LN medicaments.

drawings

FIG. 1: effect of anti-LCN 2 antibody injection on survival of MRL/lpr mice;

FIG. 2: effect of anti-LCN 2 antibody injection on MRL/lpr mouse urinary protein levels, wherein: 0.01< P <0.05 indicates significant difference, marked with ×; p <0.01 indicates a very significant difference, marked by;

FIG. 3: effect of anti-LCN 2 antibody injection on spleen index of MRL/lpr mice, wherein: 0.01< P <0.05 indicates significant difference, marked with ×;

FIG. 4: effect of anti-LCN 2 antibody injection on kidney inflammation in MRL/lpr mice, wherein: a is the result of hematoxylin-eosin staining of the kidney of the MRL/lpr mouse of the control group (rat immunoglobulin) and the control group (anti-LCN 2 antibody) in example 4, B is the result of pathological scoring of the glomerulonephritis of the MRL/lpr mouse of the control group (rat immunoglobulin) and the control group (anti-LCN 2 antibody) in example 4, C is the result of scoring of inflammatory cell infiltration around the kidney blood vessel of the MRL/lpr mouse of the control group (rat immunoglobulin) and the control group (anti-LCN 2 antibody) in example 4, 0.01< P <0.05 indicates a significant difference and is marked with [;

FIG. 5: effect of anti-LCN 2 antibody injection on MRL/lpr mouse kidney immune complex and complement deposition, wherein: a is the result of immunofluorescence staining of MRL/lpr mouse kidney cryosections IgG and C3 in the control group (rat immunoglobulin) and control group (anti-LCN 2 antibody) in example 5, B is the result of fluorescence intensity scoring of MRL/lpr mouse kidney IgG in the control group (rat immunoglobulin) and control group (anti-LCN 2 antibody) in example 5, C is the result of fluorescence intensity scoring of MRL/lpr mouse kidney C3 in the control group (rat immunoglobulin) and control group (anti-LCN 2 antibody) in example 5, 0.01< P <0.05 indicates a significant difference, and is marked with.

Detailed Description

in order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described below by using specific examples.

The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.

example 1: LCN2 antibody injection increases survival in MRL/lpr mice

16-week-old female MRL/lpr mice were divided into two groups, 10 in the control group (rat immunoglobulin), 8 in the control group (anti-LCN 2 antibody):

Control group (rat immunoglobulin): female MRL/lpr mice were given a weekly intraperitoneal injection of 100 μ g of a control antibody (rat immunoglobulin) for 4 weeks and the survival of the MRL/lpr mice during administration was recorded;

Control group (anti-LCN 2 antibody): female MRL/lpr mice were given a weekly intraperitoneal injection of 100 μ g of anti-LCN 2 antibody for 4 weeks and the survival of MRL/lpr mice during administration was recorded;

wherein the anti-LCN 2 antibody is Lipocalin-2/NGAL (mouse) antibody, purchased from R & D company of America, and has a clone number of # 228418; MRL/lpr mice are a genetically mutated mouse strain (lpr is an abbreviation for lymphocyte proliferation) in which a transposon inserted into the Fas gene intron causes Fas transcription to be abnormally expressed without inducing apoptosis, thereby causing lymphocyte proliferation to be out of control, female MRL/lpr mice are a commonly used murine model of lupus.

At the end of the week 21 trial, the cumulative survival of the mice was counted and the effect of the anti-LCN 2 antibody treatment on MRL/lpr mice was tested using a log-rank test for statistical analysis.

as shown in FIG. 1, after administration of MRL/lpr mice with continuous injections of anti-LCN 2 antibody for 4 weeks, the survival rate of MRL/lpr mice reached 100% (anti-LCN 2 antibody), while the survival rate of MRL/lpr mice in the control group (rat immunoglobulin) was 80%, indicating that anti-LCN 2 antibody increased the survival rate of MRL/lpr mice.

Example 2: LCN2 antibody significantly reduced the urinary protein concentration in MRL/lpr mice

2.1) test reagents and equipment: a Bradford protein content detection kit (Nanjing Kaikyi organism), a SynergyHT multifunctional enzyme-linked immunosorbent assay (Biotek company, USA);

2.2) test subjects and treatments: 16 week old female MRL/lpr mice were divided into two groups, 10 control (rat immunoglobulin) and 8 control (anti-LCN 2 antibody);

2.21) control group (rat immunoglobulin): female MRL/lpr mice were administered a weekly intraperitoneal injection of 100 μ g of a control antibody (rat immunoglobulin) for 4 weeks, starting two weeks prior to administration and stopping one week after administration, and urine was collected weekly;

2.22) control (anti-LCN 2 antibody): female MRL/lpr mice were administered a weekly intraperitoneal injection of 100 μ g of anti-LCN 2 antibody for 4 weeks, starting two weeks prior to administration and stopping one week after administration, and urine was collected weekly;

By the end of the 21 week test, urine protein concentrations in the urine of mice from control group (rat immunoglobulin) and control group (anti-LCN 2 antibody) were measured separately using Bradford protein content detection kit (in combination with the Bradford protein content detection kit instructions);

in the examples, the specific steps for determining the urine protein concentration are:

Components of kit

drawing a standard curve: an elisa plate was taken and reagents were added as follows:

Number of holes	0	1	2	3	4	5
							Coomassie brilliant blue G-250 solution (uL)	195	195	195	195	195	195
deionized water (mu L)	5	4	3	2	1	0
							protein Standard solution (μ L)	0	1	2	3	4	5
protein content (μ g)	0	1	2	3	4	5

thirdly, placing the ELISA plate on an oscillator to vibrate for 30s, standing for 2min, then vibrating for 30s again, and measuring the OD value at 595nm through a SynergyHT multifunctional ELISA reader within 1 h. Drawing a standard curve by taking the protein content as an abscissa and the OD value as an ordinate;

Diluting the sample to be detected to a proper concentration, adding 5 mul of the diluted sample to be detected and 195 mul of Coomassie brilliant blue G-250 solution into each hole, fully mixing uniformly, standing for 2min, using a mark No. 0 hole as a blank hole, and using a microplate reader with a rated OD value at 595 nm;

calculating the protein concentration of the sample according to the OD value, the dilution times and the standard curve of the sample.

2.3) analysis of results: in the course of onset of lupus nephritis, the kidney is damaged, resulting in the decrease of glomerular filtration function and reabsorption capability, and the increase of urine protein level, which will gradually increase with the development of lupus nephritis in MRL/lpr mice; as shown in fig. 2, the urine protein concentration of MRL/lpr mice in the control group (rat immunoglobulin) gradually increased with the increase of the week age, and the urine protein concentration of MRL/lpr mice in the control group (anti-LCN 2 antibody) decreased at the beginning of week 19, compared to the urine protein concentration of control group (rat immunoglobulin) at weeks 20 and 21, indicating that the urine protein concentration of MRL/lpr mice was significantly decreased by the LCN2 antibody injection.

example 3: anti-LCN 2 antibody significantly reduced the spleen index in MRL/lpr mice

3.1) test subjects and treatments: 16 week old female MRL/lpr mice were divided into two groups of 8 mice each:

3.11) control group (rat immunoglobulin): female MRL/lpr mice were given a weekly intraperitoneal injection of 100 μ g of a control antibody (rat immunoglobulin) for 4 weeks to the end of the 21 week trial, and the mice were weighed;

3.12) control (anti-LCN 2 antibody): female MRL/lpr mice were given a weekly intraperitoneal injection of 100 μ g of anti-LCN 2 antibody for 4 weeks to the end of the 21 week trial, and the mice were weighed;

3.13) body weight and spleen weight: placing the mice in a tray, placing the mice on an electronic balance (Shanghai Heng, MP2001) for weighing, fixing the mice after sacrifice, separating the skin, opening the abdominal cavity, first pulling up the abdominal muscles at the center of the abdomen with forceps, then cutting out a small incision with scissors, cutting the peritoneum of the mice upward from the midline with scissors until the xiphoid process of the mice, in order to expose the organs in the abdominal cavity, cutting open the abdominal muscles along the ribs, pulling up the stomach with forceps, so that the spleen can be seen, cutting the spleen down, placing the mice on an electronic balance (BS 124S, Sartorius, Germany) for weighing;

3.2) data processing and result analysis: observing the therapeutic effect of an anti-LCN 2 antibody on MRL/lpr mice as spleen weight per gram (g) of animal body weight (mg) as the animal's corresponding spleen index; MRL/lpr mice are a commonly used lupus mouse model, have obvious symptoms of systemic splenomegaly, and at the end of a 21-week test, the spleen of the MRL/lpr mice in a control group (anti-LCN 2 antibody) is obviously reduced compared with the spleen of the control group (rat immunoglobulin), and as can be seen from the statistical results in figure 3, the anti-LCN 2 antibody can obviously reduce the spleen index of the MRL/lpr mice and reduce the proliferation of the spleen of the mice.

Example 4: anti-LCN 2 antibodies can alleviate pathological changes in kidney in MRL/lpr mice

4.1) test subjects and treatments: 16 week old female MRL/lpr mice were divided into two groups of 8 mice each:

4.11) control group (rat immunoglobulin): female MRL/lpr mice were administered a weekly intraperitoneal injection of 100 μ g of a control antibody (rat immunoglobulin) for 4 weeks to the end of the 21 week trial, and mouse kidney tissue was removed;

4.12) control (anti-LCN 2 antibody): carrying out intraperitoneal injection on 100 mu g of anti-LCN 2 antibody once a week on female MRL/lpr mice, continuously carrying out administration for 4 weeks till the end point of 21-week test, and taking kidney tissues of the mice;

4.13) mouse Kidney hematoxylin-eosin (HE) staining:

after the mice were sacrificed, the kidneys were removed and fixed in 10% formaldehyde solution.

Firstly, sequentially treating a specimen with 70% ethanol, 80% ethanol, 90% ethanol and 95% ethanol, wherein the absolute ethanol is dehydrated twice for 1 hour each time for 2 hours each step;

② the sample is transparently processed by dimethylbenzene for 2 times, each time for 30min, and then embedded by paraffin;

Thirdly, placing the paraffin blocks in a paraffin slicer (RM 2235, come card, Germany), continuously slicing the paraffin blocks in the thickness of 2-3 mu m, and then placing the paraffin blocks in water at 45 ℃ for spreading and fishing; baking the slices for 1.5h at 65 ℃, and then standing and drying at normal temperature for later use;

Sequentially carrying out paraffin section dewaxing hydration treatment by using dimethylbenzene, absolute ethyl alcohol, 95% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol and 70% ethyl alcohol for 10min each;

Dyeing with hematoxylin for 7 min;

sixthly, 1 percent hydrochloric acid alcohol is added for a plurality of seconds to make the cell nucleus blue-purple, and the cell nucleus is repeatedly washed by distilled water;

seventhly, treating the tissue slices with 1% ammonia water for several seconds, and repeatedly washing the tissue slices with distilled water after the tissue slices turn blue;

Staining the slices with 1% eosin for 3 min;

Observing the HE section of the kidney under a microscope, and evaluating the pathology of the kidney;

4.2) data processing and result analysis:

4.21) data processing: the kidney tissues of the mice in the control group (rat immunoglobulin) and the control group (anti-LCN 2 antibody) were subjected to sectioning and HE staining in sequence, and then the renal lesions were observed under a light microscope. According to the international accepted literature scoring standard, the degree of inflammatory cell infiltration of the glomeruli and the periphery of renal vessels of lupus mice is classified and evaluated by a kidney pathologist, and the glomerular lesion score: the severity of glomerulonephritis is graded by 1-4 as follows: 1 minute: focal, mild or early hyperplastic; and 2, dividing: multifocal hyperplasia with increased stroma; and 3, dividing: diffuse hyperplasia; and 4, dividing: diffuse sclerosis/extensive crescent; the interstitial and vascular lesions are also graded according to the number/focus of mononuclear cells around the renal vessels in 1-4 points, the degree of infiltration of cells around the renal vessels: 1 minute: mononuclear cell infiltration around the renal pelvis/blood vessel; and 2, dividing: mononuclear cell infiltration is arranged around the aorta, and a small range (10-20) of mononuclear cell infiltration is arranged around the lobular arteria; and 3, dividing: mononuclear cell infiltration is formed around the arteriolar branch, and the mononuclear cell infiltration range is wider; and 4, dividing: monocyte infiltration extends to the surrounding parenchyma/most affected vessels.

4.22) analysis of results: as shown in fig. 4, the MRL/lpr mice in the control group (rat immunoglobulin) had significantly thickened glomerular basement membrane, increased mesangial cell proliferation, broadened stroma, and segmental necrotic lesions with crescent moon formation, while after treatment with anti-LCN 2 antibody (anti-LCN 2 antibody), the glomerular pathological changes were reduced, the mesangial proliferative lesions were significantly reduced, and the infiltration of inflammatory cells around the small blood vessels within the renal interstitium was reduced, indicating that the anti-LCN 2 antibody injection could alleviate the renal pathological changes in MRL/lpr mice.

example 5: anti-LCN 2 antibodies reduce MRL/lpr mouse renal immune complex (IgG) and complement C3 deposition

5.1) test subjects and treatments: 16 week old female MRL/lpr mice were divided into two groups of 8 mice each:

5.11) control group (rat immunoglobulin): female MRL/lpr mice were administered a weekly intraperitoneal injection of 100 μ g of a control antibody (rat immunoglobulin) for 4 weeks to the end of the 21 week trial, and mouse kidney cryosections were prepared for IgG and C3 immunofluorescent staining;

5.12) control (anti-LCN 2 antibody): carrying out intraperitoneal injection on 100 mu g of anti-LCN 2 antibody to female MRL/lpr mice once a week, continuously administering for 4 weeks till the end point of a 21-week test, and preparing frozen sections of mouse kidneys for IgG and C3 immunofluorescence staining;

5.13) mouse kidney IgG and C3 immunofluorescent staining:

After the mice were sacrificed, the kidneys were removed, rapidly frozen in liquid nitrogen and embedded with OCT embedding medium (SAKURA, USA).

firstly, slicing by a freezing slicer (German come card, CM1950) with the thickness of 2-4 mu m, and adhering on a glass slide;

② drying the mixture at room temperature for 30 min;

putting the slide into precooled ice-cold acetone for fixing for 10min, and washing for 3 times with PBS (phosphate buffer solution) for 3-5 min each time;

(iv) dropping anti-IgG-FITC or anti-C3-FITC fluorescent antibody (Santa Cruz, USA) on the section, and incubating at room temperature

washing for 30min with PBS for 3 times, 3-5 min each time;

Sealing by using a sealing agent;

Fluorescence intensity of glomerular IgG and C3 was observed under a fluorescence microscope.

5.2) data processing and result analysis:

5.21) data processing: the fluorescence intensity was calculated by analyzing the results of immunofluorescence staining of MRL/lpr mouse kidney cryosections IgG and C3 using Image-proclus 6.0 software for control group (rat immunoglobulin) and control group (anti-LCN 2 antibody); immunofluorescence semi-quantitative scoring criteria: scoring more than 10 glomeruli (including capillary walls and mesentery regions) of each kidney section, and dividing the glomeruli into 0 according to the fluorescence intensity degree: no fluorescent signal, 1: invisible fluorescence, 2: fluorescence was clearly visible, 3: the fluorescence signal is strong; 4: the fluorescence is high in brightness and glaring.

5.22) analysis of results: when lupus nephritis is developed, B cells secrete a large amount of autoantibodies, the autoantibodies are combined with autoantigens to form immune complexes, and the immune complexes are deposited on a glomerular basement membrane to cause kidney injury; as shown in fig. 5A, in the control group (rat immunoglobulin) and the control group (anti-LCN 2 antibody) MRL/lpr mouse kidney cryosection IgG and C3 immunofluorescent staining results, IgG and C3 were clearly deposited in the glomerulus in the MRL/lpr mouse kidney of the rat immunoglobulin group, and IgG and C3 were clearly reduced in the MRL/lpr mouse kidney of the anti-LCN 2 antibody group; as shown in fig. 5A and 5B, semi-quantitative post-statistical findings, IgG deposition was decreased in the control group (anti-LCN 2 antibody) relative to the control group (rat immunoglobulin), whereas C3 was statistically significant in the two group comparisons, and the kidney C3 immunofluorescence intensity was significantly decreased in the control group (anti-LCN 2 antibody) relative to the control group (rat immunoglobulin) (fig. 5C), indicating that the anti-LCN 2 antibody was able to reduce mouse kidney immune complex and complement deposition.

in conclusion, compared to the control group (rat immunoglobulin), the survival rate of MRL/lpr mice in the control group (anti-LCN 2 antibody) was 100%, the survival rate was improved, the proteinuria concentration was significantly decreased, the spleen index was significantly decreased, the kidney inflammation was significantly reduced, and the kidney immune complex and complement deposition were reduced; the LCN 2-resisting antibody is preliminarily proved to have good treatment effect on lupus nephritis, is an effective target for treating lupus nephritis, can be developed as a new medicine or a medicine target for treating lupus nephritis, and provides a new way and means for treating lupus nephritis.

the above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.

Claims (1)

1. application of the anti-LCN 2 antibody in preparing a medicament for treating lupus nephritis.