阅读说明：本技术 柴胡皂苷b2在制备预防或治疗肾纤维化药物中的应用 (Application of saikosaponin B2 in preparing medicine for preventing or treating renal fibrosis ) 是由 辛宏 谭文福 张雪梅 任大堆 骆佳 杨佳鸿 于 2019-10-12 设计创作，主要内容包括：本发明属于医药技术领域,尤其涉及柴胡皂苷B2对于肾纤维化的预防或治疗作用。柴胡皂苷B2包括以下应用：a1：制备预防和/或治疗肾组织纤维化的药物；a2：预防和/或治疗肾组织纤维化；a3：抑制Hedgehog信号通路；a4：建立肾组织纤维化动物医学模型。本发明首次公开了SSB2的新用途,为SSB2抑制肾纤维化的药理作用提供了理论依据。(The invention belongs to the technical field of medicines, and particularly relates to a prevention or treatment effect of saikosaponin B2 on renal fibrosis. The saikosaponin B2 comprises the following applications: a 1: preparing a medicament for preventing and/or treating fibrosis of kidney tissue; a 2: preventing and/or treating fibrosis of kidney tissue; a 3: inhibiting the Hedgehog signaling pathway; a 4: establishing a kidney tissue fibrosis animal medical model. The invention discloses the new application of SSB2 for the first time, and provides a theoretical basis for the pharmacological action of SSB2 in inhibiting renal fibrosis.)

1. The application of saikosaponin B2 is a1, a2, a3 or a 4:

a 1: preparing a medicament for preventing and/or treating fibrosis of kidney tissue;

a 2: preventing and/or treating fibrosis of kidney tissue;

a 3: inhibiting the Hedgehog signaling pathway;

a 4: establishing a kidney tissue fibrosis animal medical model.

2. The use as claimed in claim 1, wherein the active ingredient of the medicament comprises saikosaponin B2.

3. The use of claim 1, wherein the agent that causes fibrosis in renal tissue is an agent that treats chronic kidney disease.

4. The use according to claim 1, wherein the method for establishing the renal tissue fibrosis animal medical model comprises the following steps:

s1, grouping: dividing animals into a sham operation group and a unilateral ureter ligation group;

s2: administering saikosaponin B2 at different concentrations to two groups of animals;

s3: molding:

s4: after 6-9 days of drug administration after model building, the left kidney is taken and analyzed for renal pathology.

5. The use of claim 4, wherein in S3, the unilateral ureteral ligation group is treated by: anaesthetizing the animal and fixing; cutting off hair of abdomen, sterilizing abdomen skin, cutting 1.5-2.5cm in the center of abdomen, fixing hemostatic forceps, separating left ureter, double ligating, suturing incision, and sterilizing.

6. The use as claimed in claim 4, wherein in S2, the concentrations of saikosaponin B2 are 15mg/kg and 30mg/kg, respectively.

7. A medicament for the prevention and/or treatment of fibrosis in kidney tissue, wherein the active ingredient of the medicament comprises saikosaponin B2.

8. The medicament of claim 7, further comprising a pharmaceutically acceptable excipient selected from one or any combination of a binder, a filler, a plasticizer, a glidant, a disintegrant and a lubricant.

9. The medicament of claim 7, wherein the medicament is an injection or an oral preparation.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of saikosaponin B2 in preparation of a medicine for preventing or treating renal fibrosis.

Background

Chronic kidney disease (chronic kidney disease) has a global prevalence rate of 10-12% and is on an increasing trend year by year, chronic glomerulonephritis, hypertensive nephropathy, diabetic nephropathy, renal interstitial disease, tubular disease, congenital acquired kidney disease, and improper administration of nephrotoxic drugs can all cause chronic kidney disease, and finally End-stage renal failure (End-stage renal disease) becomes a high risk factor for death. Renal fibrosis (renal fibrosis) is a critical and common pathological change in the early stage of chronic kidney disease progression to end-stage renal failure caused by various causes, and seriously affects kidney function. Renal fibrosis is a very complex disease cause and pathogenesis, and no effective method exists for prevention and treatment at present. Therefore, the search for ideal drug targets capable of intervening and reversing renal fibrosis and the development of effective drugs for reversing renal fibrosis have important significance for preventing the occurrence of terminal renal failure caused by various chronic diseases at an early stage.

The molecular mechanisms currently involved in renal fibrosis are complex and not well defined. Some recent studies have shown that aberrant activation of the hedgehog signaling pathway is involved in renal fibrosis. The hedgehog (hh) signaling pathway is an evolutionarily conserved developmental pathway that plays an important role in regulating the development of mammalian embryos, including kidney development. It has been found that there are at least 3 homologous genes in mammals: sonic hedgehog (SHH), desert hedgehog (DHH), indian hedgehog (indian hedgehog, IHH), in the mammalian HH signaling pathway, are composed mainly of HH ligands SHH, IHH, membrane receptors Patched (PTCH1, PTCH2), smoothened (smo), and downstream transcription factors Gli family (Gli1, Gli2, and Gli 3). The classical HH signal pathway activation pathway is that an HH ligand synthesizes a propeptide in advance, an N-terminal segment (ShhN) is generated through automatic catalytic cracking, then the propeptide is transferred to a plasma membrane after being modified by cholesterol and isopentenyl lipid and is released to the outside of cells, HH is combined with PTCH to release inhibition on SMO, SMO releases processes such as Gli phosphorylation and the like, so that Gli enters a cell nucleus in a full-length form to start transcription of a target gene, when ligand HH is deficient, receptor PTCH and SMO are combined to form a compound, SMO activity is inhibited, transcription factor Gli is phosphorylated, ubiquitinated and proteasome truncated, and Gli cannot enter the cell nucleus in the full-length form, so that transcription of the target gene is inhibited. Recent studies have shown that there is abnormal activation of the Hedgehog signaling pathway in fibrotic kidney tissue and plays a very important role in the development and progression of renal fibrosis. Studies have shown that Gli1 ablated mice are able to fight UUO-induced renal interstitial fibrosis and that Cyclopamine (Cyclopamine), an inhibitor of SMO, is able to reduce UUO-induced renal fibrosis, GDC0449 is a second generation Cyclopamine derivative, approved by the FDA for marketing, acting by binding directly to SMO.

The Saikosaponin B2(Saikosaponin B2, SSB2) belongs to one of pentacyclic triterpenoid oleanane type derivatives, and no report on the effect of inhibiting renal fibrosis is found at present.

Disclosure of Invention

One of the purposes of the invention is to provide an application of saikosaponin B2, which is a1, a2, a3 or a4 as follows:

a 1: preparing a medicament for preventing and/or treating fibrosis of kidney tissue;

a 2: preventing and/or treating fibrosis of kidney tissue;

a 3: inhibiting the Hedgehog signaling pathway;

a 4: establishing a kidney tissue fibrosis animal medical model.

Preferably, the active ingredient of the medicament comprises saikosaponin B2.

Preferably, the medicament for renal tissue fibrosis is a medicament for treating chronic renal disease.

Preferably, the method for establishing the renal tissue fibrosis animal medical model comprises the following steps:

s1, grouping: dividing animals into a sham operation group and a unilateral ureter ligation group;

s2: administering saikosaponin B2 at different concentrations to two groups of animals;

s3: molding:

s4: after 6-9 days of drug administration after model building, the left kidney is taken and analyzed for renal pathology.

It should be understood that other steps may be included before step S1, between steps S1 and S2, between steps S2 and S3, between steps S3 and S4, and after step S4, and are within the scope of the present invention.

Preferably, in S3, the unilateral ureter ligation group is processed by the following steps: anaesthetizing the animal and fixing; cutting off hair of abdomen, sterilizing abdomen skin, cutting 1.5-2.5cm in the center of abdomen, fixing hemostatic forceps, separating left ureter, double ligating, suturing incision, and sterilizing.

Preferably, in S2, the concentrations of saikosaponin B2 are 15mg/kg and 30mg/kg, respectively.

It should be understood that the concentration of saikosaponin B2 in the present invention is not limited to the above two, and one skilled in the art can select any concentration of saikosaponin B2 as required and all fall within the protection scope of the present invention.

Preferably, in S3, the abdominal median incision is 2 cm.

In some preferred embodiments of the present invention, the mouse renal fibrosis model is established by unilateral ureteral ligation, and the experiment is divided into 4 groups: normal control group, model group, low-dose SSB2 medication group, high-dose SSB2 medication group.

The fibrosis degree and pathological changes of the kidney tissues are detected by HE staining, Sirius red staining and Masson staining, and mRNA and protein expression changes of alpha-SMA, fibrinectin, collagen I and Gli1 in the kidney tissues are detected by fluorescent quantitative PCR and Western blot. The SSB2 dose in the low and high dose groups was 15, 30mg/kg mice.

In some preferred embodiments of the present invention, an ex vivo cell fibrosis model was constructed by inducing NRK-49F in rat fibroblasts to activate HH pathway using conditioned medium containing ShhN (ShhN-CM), and experiments were divided into 6 groups: the normal control group, the model group, the three drug groups with SSB2 dosage and the GDC0449 drug group detect the content change of the alpha-SMA and the fibrinectin of the cells by an immunofluorescence method, and detect the mRNA and the protein expression change of the alpha-SMA, the fibrinectin, the collagen I and the Gli1 in the kidney tissues by fluorescence quantitative PCR and Western blot. The dosage of SSB2 in the three dosage medication groups is 5, 10 and 20 mu mol/L; the dosage of GDC0449 in the GCD0449 medicine composition is 0.1 mu mol/L.

In some preferred embodiments of the invention, the effects of SSB2 on TNF-. alpha.stimulated NF-. kappa.B transcriptional activity, PEG2 stimulated Wnt/β -catenin transcriptional activity, and Gli 2. delta.N and Glo D473H plasmid transfection-induced Gli transcriptional activity were examined using the Dual-Luciferase reporter assay (DR).

The invention also aims to provide a medicament for preventing and/or treating kidney tissue fibrosis, wherein the active ingredient of the medicament comprises saikosaponin B2.

Preferably, the medicament further comprises a pharmaceutically acceptable excipient, wherein the excipient is selected from one or any combination of a binder, a filler, a plasticizer, a glidant, a disintegrant and a lubricant.

Preferably, the medicament is an injection or an oral preparation.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

The invention discloses the new application of SSB2 for the first time, and provides a theoretical basis for the pharmacological action of SSB2 in inhibiting renal fibrosis.

Drawings

FIG. 1 is a graph of SSB2 intervention UUO mouse renal HE staining, Sirius red staining and Masson staining (200X).

Fig. 2 is a schematic diagram of SSB2 reducing UUO-induced renal fibrosis marker protein production and reducing the expression level of Gli1 protein on the Hedgehog signaling pathway.

Fig. 3 is a graphical representation of SSB2 reducing the expression levels of the UUO-induced renal fibrosis marker protein and Gli1 mRNA on the Hedgehog signaling pathway.

FIG. 4 is a schematic diagram of SSB2 intervention on Shh induced expression of the NRK-49F cell fibrosis marker protein and expression of the Gli1 protein.

FIG. 5 is a schematic representation of SSB2 reducing Shh-induced expression of NRK-49F cell fibrotic protein and Gli1 mRNA.

FIG. 6 is a schematic representation of SSB2 reducing Shh-induced expression of Fibronectin, α -SMA in NRK-49F cells.

Fig. 7 is a schematic diagram of SSB2 selective inhibition of the Hedgehog signaling pathway.

Fig. 8 is a schematic representation of the inhibition of Smo in the Hedgehog signaling pathway by SSB 2.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

It should be noted that, if the specific conditions are not indicated, the process is carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the reagents used are not indicated by the manufacturer, and are all conventional products available on the market.

The invention carries out animal experiments and cell experiments, carries out intervention by applying SSB2 to the established animal models and cell models, and uses experimental methods such as histomorphology analysis, western blot experiments, fluorescent quantitative PCR, dual-luciferase reporter gene detection method and the like. In the embodiment of the invention, animal experiment results show that SSB2 can inhibit renal fibrosis induced by pathological environment and reduce damage to nephrons in the fibrosis process; cell experiment results show that SSB2 inhibits the activation of fibroblasts to myofibroblasts by selectively inhibiting a Hedgehog signaling pathway, the increase of a fibrosis marker protein induced by ShhN is reduced, the action target point of the SSB2 is likely to be SMO, and SSB2 can be further used for preparing medicines for treating or preventing renal fibrosis.

In the embodiment of the invention, an intervention experiment comprising the following steps is specifically carried out:

(1) mouse unilateral ureter obstruction surgery; (2) analyzing the tissue morphology; (3) ex vivo cell studies; (4) performing a western blot experiment; (5) real-time fluorescent quantitative PCR experiment; (6) performing cell immunofluorescence experiments; (7) detecting a dual-luciferase reporter gene; (8) and (3) data analysis: the results are expressed as Mean ± SD, and all data were analyzed using GraphPad Prism software One-Way ANOVA method, with P > 0.05 considered no significant difference.