CN117653647A - Application of protopanoxadiol in preparing medicine for preventing or treating lupus nephritis - Google Patents

Abstract

The invention provides an application of protopanaxadiol in preparing a medicine for preventing or treating lupus nephritis by inhibiting PTX3 and regulating a complement pathway. The new application provided by the invention creatively takes the protopanaxadiol as a medicament for preparing the medicament for improving the kidney inflammation of lupus nephritis, and the protopanaxadiol reduces the levels of serum anti-ds-DNA antibodies, blood creatinine and urea nitrogen by improving proteinuria and relieves the symptoms of lupus nephritis. By lowering the level of PTX3, complement factor C3, the effect of kidney inflammation is inhibited.

Description

Application of protopanoxadiol in preparing medicine for preventing or treating lupus nephritis

Technical Field

The invention belongs to the field of medicines, and in particular relates to an application of protopanaxadiol in preparing a medicine for preventing or treating lupus nephritis by inhibiting PTX3 and regulating a complement pathway.

Background

Lupus Nephritis (LN) is Nephritis with obvious immune kidney injury caused by autoimmune disease systemic Lupus erythematosus (Systemic Lupus Erythematosus, SLE), is one of the most serious organ manifestations of SLE and main causes of mortality, and is mainly clinically manifested as hematuria, proteinuria, renal insufficiency and the like. Epidemiology shows that about 60% of SLE patients develop LN in five years, and 10% to 15% of LN patients develop end-stage renal disease (End stage renal disease, ESRD), severely jeopardizing the patient's life and health.

At present, the main clinical therapeutic drugs for LN include glucocorticoids, immunosuppressants, monoclonal antibodies (belimumab) and the like, and although the therapeutic drugs have a certain curative effect on LN patients, the therapeutic drugs also have serious toxic and side effects (such as inhibition of reproductive system functions, increase of tumor incidence, damage of hematopoietic function, damage of normal body immune function and the like) and partial ineffective patients and the like, so that survival and prognosis of the patients are further influenced. Therefore, a safer and more effective LN therapeutic drug is developed, and an effective target spot of LN therapy is found to have important market prospect.

The pathogenesis of LN is closely related to immune complex deposition, local complement activation and the like, wherein complement factor deposition in the kidney membranous region is closely related to LN patient proteinuria, renal interstitial fibrosis and the like, and is also a key link in the progress of LN to ESRD. Long Pentraxin 3 (ptx 3) belongs to the family of pentameric proteins, and the content increases dramatically during inflammatory reactions; and PTX3 is an important factor in the regulation of the complement system, PTX3 can bind to complement factors C1q, MBL, ficolin-1 and-2, etc. in inflammatory kidneys, activating the Classical Pathway (CP) and Lectin Pathway (LP) of the complement system, leading to the formation of complement key factor C3, further leading to the assembly of C5b-9, ultimately leading to the occurrence of renal inflammation and fibrosis.

Ginsenoside is the main active ingredient of traditional Chinese medicine ginseng, and is often used for treating various acute and chronic kidney diseases. Protopanaxadiol is the glucose-removing metabolite of diol type ginsenoside gastrointestinal flora with highest content in ginsenoside, the exposure amount of protopanaxatriol in vivo is higher than that of protopanaxatriol metabolite, and the protopanaxadiol has higher bioavailability than that of other monomers, and is easier to be absorbed into blood to serve as an active compound.

According to the invention, the protopanaxadiol is firstly discovered to have obvious improvement effect on LN mouse disease states by inhibiting PTX3 expression and regulating complement pathway activation.

The invention uses MRL/lpr mice as lupus nephritis models, and the effect of the protopanoxadiol on improving lupus nephritis is verified by administering the protopanoxadiol. The dosage of the protopanoxadiol is 50mg/kg.

Disclosure of Invention

The invention aims to solve the problems in the prior art, provides application of protopanoxadiol in treating and inhibiting lupus nephritis, develops novel application of the protopanoxadiol, and has obvious improvement effect on lupus nephritis mice by inhibiting PTX3 expression and regulating complement pathway activation through the protopanoxadiol.

Protopanaxadiol is the most important active ingredient of ginsenoside. Ginsenoside can be classified into three types according to the sapogenins: (1) the protopanaxadiol mainly comprises ginsenoside Ra1, ra2, rb1, rb2, rc, rd, rg3, etc.; (2) the protopanaxatriol type mainly comprises ginsenoside Re, rf, rg1, rg2, rh1, etc.; (3) olea fruit type, such as ginsenoside Ro. Wherein the chemical name of protopanoxadiol (20 (S) -PPD): dammar-24-ene-3,12,20-diol, (3 beta,12beta, 20R) -; (3 beta,5xi,12beta,14beta, 20R) -dammar-24-ene-3,12,20-diol, molecular weight 460.70, molecular formula C30H5203, structural formula:

for the above applications, the protopanoxadiol is used alone.

For the above application, the amount of protopanoxadiol was 50mg/kg.

For the application described above, the therapeutic administration of protopanoxadiol was by intraperitoneal injection.

The protopanaxadiol is used as a raw material for preparing the medicines for improving the symptoms of lupus nephritis, kidney inflammation, proteinuria and the like.

In the application, in the mouse experiment, the protopanaxadiol is injected into the abdominal cavity to inhibit the expression of PTX3, so as to regulate the activation of complement pathway, improve the symptoms of mouse proteinuria and the like, and play a role in relieving nephritis.

For the above application, female MRL/lpr mice were selected for use in the mouse experiments

The invention has the beneficial effects that:

the invention develops a new application of protopanoxadiol, innovatively takes protopanoxadiol as a medicament for preparing and improving lupus nephritis kidney inflammation, and reduces the levels of serum anti-ds-DNA antibodies, blood creatinine and urea nitrogen by improving proteinuria. Specifically, it was found that protopanaxadiol can reduce the level of PTX3, complement factor C3, and inhibit the effect of kidney inflammation in a mouse model of spontaneous lupus nephritis. Due to the above-mentioned effects of protopanoxadiol, protopanoxadiol contributes to the study and treatment of lupus nephritis.

Drawings

FIG. 1 is a graph showing the measurement of the proteinuria level in mice of each group in example 1 of the present invention

FIG. 2 is a graph showing the serum creatinine levels of mice in each group according to example 1 of the present invention

FIG. 3 is a graph showing the serum urea nitrogen levels of mice in each group measured in example 1 of the present invention

FIG. 4 is a graph showing the determination of serum anti-ds-DNA antibody levels in each group of mice in example 1 of the present invention

FIG. 5 is a graph showing the measurement of PAS staining of kidney pathology in mice of example 1 according to the present invention

FIG. 6 is a Western Blot image of the determination of groups of mice PTX3 and C3 in example 1 of the present invention

Detailed Description

The invention is further illustrated by the examples/test examples below, but is not limited in any way.

EXAMPLE 1 therapeutic Effect of Protopanaxadiol on mice with idiopathic lupus nephritis

Experimental instrument

Full-automatic biochemical analyzer (Mindray, BS-350S)

Experimental medicine and reagent

PTX3 antibody (ab 90806), C3 antibody (ab 181147), protopanaxadiol (homemade), sterile water, rodent feed (Beijing KeAoXieLi), BCA protein concentration assay kit (P0010S, beyotime)

Experimental animal

10 healthy SPF-grade female C57BL/6 mice, 30 MRL/lpr mice, and 12 weeks old were used. Experimental animal science research institute (Institute of LaboratoryAnimal Science) from Beijing CAMS of the people's republic of China

Experimental grouping and administration

Experimental animals were randomly divided into 4 groups of 10 animals each, group 1: normal group, 10C 57BL/6 mice, were normally given diet; group 2: model group, 10 MRL/lpr mice were given normal diet; group 3: physiological saline group, 10 MRL/lpr mice, were intraperitoneally injected with an equal volume of physiological saline; group 4: PPD group, MRL/lpr mice 10, PPD (1 mg/ml) was intraperitoneally injected with 50mg/kg. PPD or physiological saline was administered once daily, continuously to 16 weeks of age. The BCA protein was quantitatively detected by collecting 24h urine from mice at each 7 day interval after dosing and the day before the end of the experiment, respectively, before starting dosing. After the end of the administration, serum, kidney tissue, etc. of each group of mice were collected for subsequent detection.

(1) BCA assay detects 24h proteinuria:

urine collected each time was centrifuged at 3000rpm for 20min, and the supernatant was used for detecting urine protein concentration. The specific operation method is as follows:

1) Standard protein was formulated as a 25mg/mL solution and diluted to 0.5mg/mL with PBS;

2) Adding 200 mu L of BCA working solution into each hole, and preparing BCA working solution according to the number of detected samples, wherein the ratio of A to B is 50:1;

3) 0, 1, 2, 4, 8, 12, 16 and 20 mu L of protein standard solution are sequentially added into a 96-well plate, then 20, 19, 18, 16, 12, 8, 4 and 0 mu L of PBS are sequentially added immediately, 1 mu L of urine sample to be detected is added into each sample hole, and then 19 mu L of PBS is sequentially added immediately.

4) 200 mu L BCA working solution is added to each well, and incubated for 20-30 min at 37 ℃.

5) Measuring the absorbance OD value of each hole at 562nm wavelength by using an enzyme-labeled instrument, drawing a standard curve to obtain a standard curve equation,

6) And calculating the protein concentration in the sample solution according to the OD value of the sample and the labeling curve equation.

(2) Detection of anti-ds-DNA antibodies, creatinine and urea nitrogen:

after the mice of each experimental group are anesthetized, adopting a method of taking blood from eyeballs, and centrifuging at 3000rpm for 10min after blood is coagulated to obtain supernatant, and detecting indexes of anti-ds-DNA antibodies, blood creatinine and urea nitrogen by using a full-automatic blood biochemical analyzer.

(3) Detection of renal histopathological changes in mice

The kidney tissue of the mice remaining at the end of the experiment was treated as follows:

periodic Acid (PAS) staining method

1) Baking slices to be dyed at 62 ℃ for 1h;

2) Dewaxing with xylene, double steaming, rehydrating, soaking with aqueous solution of periodic acid for 5min, and washing with distilled water for 3 times;

3) Dyeing for 15min by using Schiff's solution, and flushing for 5min in flowing water;

4) Hematoxylin dye liquor is used for dyeing for 3-5 min, and the dyeing is carried out in flowing water for 5min; color separation is carried out by using an ethanol solution containing 1 percent hydrochloric acid;

5) Dehydrating, transparentizing and sealing the sheet, wherein the operation steps are the same as HE dyeing;

6) And observing and collecting images under a microscope.

(4) Western blot detection of expression levels of PTX3 and C3 related proteins in kidney tissues of mice in each group

Extracting total protein of kidney tissue:

1) Preparing RIPA and PMSF into a lysate according to the proportion of 100:1, and preparing the lysate in situ;

2) Cutting 25mg of kidney tissue, placing into a 1.5mL centrifuge tube, adding 250 mu L of tissue lysate, and shearing under ice bath;

3) Homogenizing the tissue to be in suspension state by using an electric homogenizer under ice bath condition, rapidly swirling, and swirling for 1 time every 1min for 3 times;

4) Centrifuging at 12000rpm at 4deg.C for 10min, and collecting supernatant to obtain total protein of renal tissue;

5) Detecting the protein concentration of the extracted total protein by using a BCA protein concentration determination kit;

6) According to the sample Loading amount of 30 mug per hole in electrophoresis and the volume of 15 mug, preparing an electrophoresis sample by using a protein sample, a 5 xLoading buffer and PBS under ice bath condition, rapidly and uniformly mixing on a vortex instrument, sealing by using a sealing film, placing the mixture on a protein sample boiling device for denaturation at 100 ℃ for 5min, cooling to room temperature, and sub-packaging to prepare a protein electrophoresis sample;

7) Western blot detection is carried out on protein samples

Experimental results

MRL/lpr mice had significantly elevated 24h proteinuria, serum creatinine, urea nitrogen, and anti-ds-DNA antibody levels (P < 0.01) compared to the normal group; compared with a model group and a normal saline group, after the MRL/lpr lupus mice are subjected to PPD intervention, indexes such as proteinuria, serum creatinine, urea nitrogen, anti-ds-DNA antibodies and the like are obviously improved (P is less than 0.05). The result of the kidney tissue pathology PAS examination shows that the pathology of the model group and the physiological saline group is characterized by thickening of basement membrane, narrowing or occlusion of capillary vessel cavity, even double-track sign of basement membrane is visible, and inflammatory cell infiltration is visible around glomerulus; after PPD drug treatment, MRL/lpr mice glomerular inflammatory cell infiltration is reduced, the basal lamina thickness is even, and the kidney disease is obviously improved. Western blot results show that compared with a normal group and a normal saline group, the expression of the kidney PTX3 and complement C3 proteins of a model group mice are obviously increased (P < 0.0001); after PPD drug treatment, both mouse kidney PTX3 and complement C3 levels were significantly reduced (P < 0.05). The above results all indicate that PPD has therapeutic effects on LN mice, and can be used as potential drug for LN therapy, and that the therapeutic effects are associated with inhibition of PTX3 expression and modulation of complement pathway activity.

The relevant experimental result data have been further illustrated by the accompanying drawings, the specific data of which are illustrated in the following figures:

FIG. 1 mice of each group were 24h proteinuria; experimental data are expressed as ± s (n=10); * P <0.05, < P <0.01vs. blank; # P <0.01vs. model group.

Serum creatinine levels in mice of each group of fig. 2; experimental data are expressed as ± s (n=10); * P <0.0001vs. blank; # # P <0.0001vs.

FIG. 3 serum urea nitrogen levels for each group of mice; experimental data are expressed as ± s (n=10); * P <0.05, < P <0.0001vs. blank; # # P <0.0001vs.

FIG. 4 serum anti-ds-DNA levels for each group of mice; experimental data are expressed as ± s (n=10); * P <0.0001vs. blank; # # P <0.0001vs.

FIG. 5A photograph of kidney tissue of mice (x 400) observed under a microscope after PAS staining

FIG. 6 expression levels of PTX3 and C3 in mouse kidney tissue; experimental data are expressed in ± s (n=3). * P <0.0001vs. normal group; # P <0.05, # P <0.01vs.

Claims (8)

1. Application of protopanaxadiol saponins in preparing medicines for preventing or treating lupus nephritis diseases is provided.

2. The use according to claim 1, wherein the use in the medicament for lupus nephritis comprises the use of protopanoxadiol alone.

3. The use according to claim 2, wherein the amount of protopanoxadiol used in the medicament for treating lupus nephritis is 50mg/kg.

4. The use according to claim 3, wherein the protopanoxadiol is administered by intraperitoneal injection.

5. The use according to claim 4, wherein protopanaxadiol is administered by intraperitoneal injection to inhibit the expression of PTX3, thereby regulating the activation of the complement pathway and improving the symptoms such as proteinuria, and is useful for preventing or treating lupus nephritis.

6. The use according to claim 4, wherein the protopanoxadiol is used as a raw material for preparing a medicament for preventing or treating symptoms such as lupus nephritis, kidney inflammation and proteinuria.

7. The use according to any one of claims 1 to 6, wherein the pharmaceutical dosage form comprises injectable formulations such as liquid formulations, suspension formulations, semi-solid formulations, microsphere formulations and the like.

8. The use according to claim 7, wherein the medicament comprises a pharmaceutically acceptable adjuvant of protopanaxadiol saponin.