CN107243004B

CN107243004B - Application of schisandrin B in preparation of medicine

Info

Publication number: CN107243004B
Application number: CN201710282887.1A
Authority: CN
Inventors: 王怡; 韩集波; 梁广; 黄伟剑; 徐政
Original assignee: Wenzhou Guangcheng Biotechnology Co ltd; Wenzhou Medical University
Current assignee: Wenzhou Guangcheng Biotechnology Co ltd; Wenzhou Medical University
Priority date: 2017-04-26
Filing date: 2017-04-26
Publication date: 2023-01-20
Anticipated expiration: 2037-04-26
Also published as: CN107243004A

Abstract

The invention discloses an application of schisandrin B in preparing a medicament for treating diabetic complications; wherein, the schisandrin B achieves the effect of treating diabetic complications by inhibiting the generation of an action mechanism of chronic inflammation by the myeloid differentiation factor 88 (MyD 88) in a targeted manner. The schisandrin B is preferably orally administered (for preparing powder, tablet, pill, oral liquid and capsule); schizandrin B can effectively relieve the occurrence and development of diabetic cardiomyopathy and diabetic nephropathy, and can be used as a main component of a medicine for treating diabetic complications.

Description

Application of schisandrin B in preparation of medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of schisandrin B in preparation of a medicine for treating diabetic complications. In addition, the invention also relates to application of schisandrin B in preparing Myd88 inhibitor with anti-inflammatory activity.

Background

Diabetes mellitus is a worldwide epidemic that causes serious global health problems, with diabetic patients in china accounting for 9.7% of the entire population. Complications of diabetes can spread throughout the body to vital organs, with most diabetics dying from cardiac, cerebral or renal complications. The organ complications of diabetes mellitus are caused by a multifactorial complex involving a variety of factors such as inflammatory response, RAS activation, insulin resistance, oxidative stress, endoplasmic reticulum stress, and terminal glycosylation products (AGEs), but recent evidence indicates that inflammatory response is closely related to diabetic complications. Hyperglycemia-induced activation of inflammatory signals, inflammatory factor release, increased levels of chemotactic adhesion factors, and inflammatory cell infiltration and accumulation in tissues of various organs subsequently leads to a series of damaging pathological changes of the tissues. It can be seen that the inflammatory response plays an important role in the development of diabetic complications.

Myeloid differentiation factor 88 (MyD differentiation factor 88, myD 88) is a key adaptor molecule in TLRs mediated inflammatory signal transduction pathway, and is a key target molecule for signal transduction to downstream. Studies have shown that signaling by TLR2,3,4,7 and 9 is accomplished through the Myd88 pathway: upon activation of TLRs, the TIR domain interacts with the carboxy-terminal end of Myd88, thereby activating MyD 88-dependent signaling pathways, and subsequently, the MAPKs and NF- κ B signaling pathways are also activated, jointly opening transcription and expression of downstream inflammatory factors 13. As can be seen, myd88 plays an important regulatory role in the LPS-induced inflammatory signaling process.

Schizandrin B (Schisandrin B, sch B) is a lignan extracted from fructus Schisandrae, and has antiinflammatory, antioxidant, hepatoprotective, and cardiovascular diseases protecting effects. Research on the mechanism of the Schisandrin B in relieving metabolic diseases shows that the Schisandrin B mainly treats the diseases through oxidation resistance. However, more and more researches show that schisandrin B can remarkably inhibit inflammatory response induced by LPS besides the antioxidant capacity of schisandrin B.

At present, the direct anti-inflammatory target of schisandrin B is not clear, and in addition, the role of schisandrin B in diabetic complications is not reported. The inventor finds that schisandrin B can exert anti-inflammatory activity by targeting inhibition of an inflammation signal transduction protein Myd88 so as to treat diabetic complications through hard efforts.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a new application of schisandrin B in treating or preventing diabetic complications (especially diabetic cardiomyopathy and diabetic nephropathy) and/or provide a new application of schisandrin B in relieving inflammatory reaction by targeted inhibition of Myd88 protein.

The invention provides an application of schisandrin B in preparation of a medicament, wherein the medicament is used for treating or preventing diabetic complications.

In the present invention, we first discovered the therapeutic effect of schisandrin b on Diabetic Cardiomyopathy (DCM). We created a mouse model of type 1 diabetes using STZ and perfused mice with Schizandrin B (20 and 40 mg/kg/day) for 16 weeks. The result shows that the schisandrin B can remarkably relieve STZ-induced fibrosis and apoptosis of heart and kidney tissues and cardiac and renal insufficiency of the diabetic mice (see example 1 for details). Meanwhile, the high blood sugar level of mice with type 1 diabetes mellitus is not influenced by orally taking schisandrin B, which indicates that the effect is not caused by reducing blood sugar.

Diabetic complications include, but are not limited to, one or more of diabetic cardiomyopathy, diabetes-related atherosclerosis, diabetic nephropathy, diabetic retinopathy, and diabetic foot disease. Further preferably, the diabetic complication is diabetic cardiomyopathy and/or diabetic nephropathy.

Preferably, the schisandrin B is used for improving the fibrosis of heart and kidney tissues, reducing the apoptosis of heart and kidney cells and improving the heart and kidney functions.

Preferably, the schisandrin B plays a role in treating or preventing diabetic complications by inhibiting or reducing chronic inflammation induced by hyperglycemia at the lesion site of the complications. In the invention, schizandrin B is found to inhibit high sugar (HG 33mM glucose) -induced activation of inflammatory signaling pathways (MAPKs and NF-kB) and release of inflammatory factors (TNF-alpha and IL-6) in H9c2 and rat primary myocardial cells.

Preferably, the schisandrin B plays a role in treating or preventing diabetic complications by directly targeting and inhibiting inflammatory signal transduction protein-myeloid differentiation factor 88 (Myd 88). TLR4 is the major receptor for LPS in the classical inflammatory signaling pathway stimulated by LPS. LPS binds to TLR4 to form a cell membrane complex that further recruits downstream intracellular adaptor proteins, such as: myd 88-dependent inflammatory factor transcription pathway. We first found through experiments that schizandrin b can specifically inhibit the complexation of TLR4 and Myd88 proteins in this inflammatory signaling pathway (see example 3 for details). In order to further confirm the selectivity of schisandrin B on TLR4 and Myd88, the interaction relationship between schisandrin B and the two proteins is detected by using recombinant human TLR4 protein, recombinant human Myd88 protein and SPR experimental technology. The result shows that Schisandrin B can directly interact with Myd88 protein, but the combination of the Schisandrin B and TLR4 is weak. Meanwhile, a bis-ANS competitive displacement experiment is used for investigating the binding rate of Schisandrin B and Myd88, so that the fluorescence absorption intensity of Myd88 protein is reduced in a dose-dependent manner by the Schisandrin B, and the direct binding effect of the Schisandrin B and the Myd88 protein is suggested (see example 4 for details).

In specific application, the medicament is a preparation prepared from schisandrin B and pharmaceutically acceptable auxiliary agents. Among them, powders, tablets, pills, capsules and oral liquids for oral administration are preferable. The pharmaceutical composition of the present invention may be various formulations prepared by combining schisandrin b and pharmaceutically acceptable adjuvants, preferably solid formulations and liquid formulations. The formulation of the present invention may be in unit dosage forms such as tablets, pills, capsules (including sustained release or delayed release forms), powders, suspensions, granules, tinctures, syrups, emulsions, suspensions, injections, etc., and various sustained release forms, so as to be suitable for various administration modes such as oral, parenteral injection, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal, transdermal, etc. (among which powders, tablets, pills, capsules (including sustained release or delayed release forms) and oral liquids for oral administration are preferred).

The invention also provides application of schisandrin B in preparation of a medicament, wherein the medicament is an Myd88 inhibitor, and the medicament can be used for all Myd 88-related diseases.

The invention has the following beneficial effects: schizandrin B is found to have excellent effects of inhibiting inflammation signal transduction protein Myd88 and relieving hyperglycemia-induced inflammatory reaction, so that the diabetes complications can be effectively treated.

For the purpose of facilitating understanding, the invention will be described in detail below with reference to specific drawings and examples. It is specifically intended that the specification and drawings be considered as exemplary only, and not as limiting the scope of the invention. It will be apparent to those skilled in the art from this disclosure that various modifications and variations can be made in the present invention within the scope of the invention, which is also encompassed within the scope of the invention. In addition, where publications are cited herein for purposes of clarity in describing the invention, they are incorporated by reference in their entirety as if fully set forth herein.

Drawings

FIG. 1 Effect of Schizandrin B on STZ-induced cardiac complications in type 1 diabetic mice.

FIG. 2 Effect of Schizandrin B on STZ-induced renal complications in type 1 diabetic mice.

FIG. 3 the relieving effect of Schisandrin B on HG-induced myocardial cell inflammatory response.

FIG. 4 Schisandrin B specifically inhibits the complex of TLR4 and Myd88 proteins in LPS-induced inflammatory signaling pathway.

FIG. 5 Schisandrin B has direct binding effect with Myd88 protein.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Example 1 Effect of Schizandrin B on STZ-induced organ complications in type 1 diabetic mice

Male C57BL/6 mice were obtained from the animal Experimental center, wenzhou college of medicine. Mice were housed in a constant temperature animal house with a circadian rhythm of 12-12h with standard rodent chow and water. Animals took at least one week to grow acclimatically before the start of the experiment. Protocols involving animal use were approved by the animal policy and welfare committee of the wenzhou medical college (approval document: 2009/APWC/0031). Schisandrin B used in the experiment was prepared as a water-soluble formulation. The solution had a pH of 7.36 and was filtered through a 0.22 Millipore filter. Male C57BL/6 mice 8-10 weeks old were randomized into 6 groups: normal control group, schizandrin B simple treatment group (Sch B), diabetes group (DM), diabetes group and Schizandrin B (20, 40mg/kg/d two doses) treatment group, each group contains 8. The low dose 50mg/kg/d urostreptozocin (STZ, sigma) is injected intraperitoneally, a model of type 1 diabetes is modeled after 5 days of continuous injection, after 72 hours, fasting blood sugar (fasting for 4-6 hours) is measured by a glucometer, and the model of type 1 diabetes is considered to be established when the blood sugar value is more than or equal to 12 mmol/L. Blood glucose and body weight were monitored weekly for 4 months. Before each group of mice died, cardiac function indices were recorded using a doppler ultrasound detector. Cardiac tissue was paraffin embedded, morphological changes in heart and kidney were detected by masson and HE staining, apoptotic changes in cardiomyocytes were detected by TUNEL technique. The experimental data are shown in fig. 1 and fig. 2. The result shows that the schisandrin B can obviously relieve the STZ-induced fibrosis and apoptosis of heart and kidney tissues and the cardiac and renal insufficiency of the diabetic mice.

FIG. 1A shows the results of TUNEL staining test, with staining pictures on the top and the statistics of the percentage of TUNEL positive cells in each group on the bottom, where blue fluorescence spots localized the nuclei and green fluorescence spots localized at the nuclei were TUNEL positive spots. The results in fig. 1A show that cardiac cells in the diabetes group (DM) were apoptotic in a large amount, whereas cardiac cell apoptosis in the diabetes group and schisandrin b-treated group was significantly improved, which is close to that in the normal control group, and thus schisandrin b could significantly improve cardiac cell apoptosis.

FIG. 1B shows the results of the massson staining test, with the upper panel showing the stained image and the lower panel showing the statistical chart of the proportion of fibrosis in the heart tissue, wherein the myocardial fibers are red and the interstitial medium blue is the collagen fiber accumulation. The results in fig. 1B show that the fibrosis of the heart tissue was severe in the diabetic group (DM), and the fibrosis of the heart tissue was significantly improved in the diabetic group and schisandrin B-treated group, which was solved in the normal control group, and it was seen that schisandrin B could significantly improve the fibrosis of the heart tissue.

FIGS. 1C-1D show that cardiac function index was recorded by Doppler ultrasound detector before each group of mice died, and FIGS. 1C and 1D show that cardiac function was deteriorated in diabetes group (DM) and good in diabetes group and schisandrin B treatment group.

In which, FIGS. 2A-C show the urea nitrogen (BUN) content and the serum Creatinine (CR) content in urine and blood, respectively, and the results indicate that the renal function of the group with diabetes (T1 DM) is poor, while the group with diabetes and schisandrin B treatment has good renal function. FIG. 2D shows the results of the HE staining experiments, which shows that the glomerular swelling of the T1DM group is not significantly improved in the glomerular size of the diabetes combined and schisandrin B treated group, and is close to that of the normal control group, and thus, schisandrin B can significantly improve the glomerular swelling. Fig. 2E shows results of Siriusred staining test, wherein pink is kidney tissue, and deep red is collagen fiber accumulation, and the results indicate that the kidney tissue fibrosis of T1DM group is severe, and the kidney tissue fibrosis of diabetes group and schizandrin b treatment group is obviously improved, which is close to that of normal control group, and thus, it can be seen that schizandrin b can obviously improve the kidney tissue fibrosis. FIG. 2F shows that the western blot experiment detects renal fibrosis marker proteins COL-4 and TGF-beta, and the result shows that the result is similar to that in FIG. 2E.

Example 2 Schisandrin B can alleviate HG-induced inflammatory reaction of cardiac muscle cells

The in vitro anti-inflammatory activity of schisandrin B is tested by adopting a method for inhibiting HG (HG) stimulated H9c2 and release of inflammatory factors (TNF-alpha and IL-6) in rat primary myocardial cells by schisandrin B, and the specific method is as follows: 1.2X 10 ^{^6} Culturing H9c2 or rat primary myocardial cells in DMEM culture solution at 37 deg.C for 24 hr, renewing the culture solution, adding schisandrin B (final concentration of 2.5, 5 and 10 μ M) for pretreatment for 1 hr, further treating with high sugar (HG 33mM glucose) for 24 hr, collecting the culture solution, and detecting TNF-alpha and IL-6 content by ELISA; collecting cells to detect total protein concentration, dividing ELISA result by corresponding total protein concentration, and calibrating TNF-alpha and IL-6 content of HG control group as 100; each compound was tested in duplicate 3 times and the mean and error values were calculated. Schisandrin B inhibition of TNF-alpha and IL-6 releaseThe preparation activity is shown in FIG. 3.

Wherein, FIG. 3A shows the inhibitory activity of schisandrin B on TNF-alpha, and it can be seen from FIG. 3 that schisandrin B can reduce the content of TNF-alpha factor, and the content of TNF-alpha factor is gradually reduced with the increase of schisandrin B dosage, indicating that it has better dose-dependent tolerance.

FIG. 3C shows the inhibitory activity of Schisandrin B on IL-6, and it can be seen from FIG. 3 that Schisandrin B can reduce the content of IL-6 factor, and as the dosage of Schisandrin B increases, the content of IL-6 factor gradually decreases, indicating better dose-dependent tolerance.

Meanwhile, the in vitro anti-inflammatory activity of schisandrin B is tested by adopting a method for stimulating the activation and inhibition of inflammatory signal pathways (MAPKs and NF-kappa B) in H9c2 and rat primary myocardial cells by schisandrin B, and the specific method is as follows: culturing 1.2 × 10^ 6H 9c2 or rat primary myocardial cells in a DMEM culture solution at 37 ℃ for 24 hours, then updating the culture solution, adding tested schisandrin B (the final concentration is 2.5, 5 and 10 mu M) for pretreatment for 1 hour, then continuously treating for 1 hour by using high sugar (HG 33mM glucose), collecting the culture solution, and detecting the conditions of MAPK (ERK, P38 and JNK) activation and IkappaB-alpha degradation by using a Western blot method; each compound was tested in duplicate 3 times and the mean and error values were calculated. The inhibitory activity of schisandrin B on the activation of inflammatory signaling pathways (MAPKs and NF-. Kappa.B) is shown in FIG. 3. Experiments show that schisandrin B can effectively relieve HG-induced myocardial cell inflammatory reaction.

Example 3 Schisandrin B specifically inhibits the complexation of TLR4 and Myd88 proteins in LPS-induced inflammatory signaling pathways

TLR4 is the major receptor for LPS in the classical inflammatory signaling pathway stimulated by LPS. However, the binding of TLR4 to LPS requires the involvement of myeloid differentiation protein 2 (MD 2), forming the LPS-TLR4-MD2 complex, which further recruits downstream intracellular adapter proteins, such as: the Myd 88-dependent inflammatory factor transcriptional pathway and the TRIF-dependent interferon-beta (IFN- β) transcriptional pathway. Wherein, MD2 and TLR4 are compounded, TLR4 recruits downstream adaptor protein Myd88 to be an important mark for the activation of the inflammation pathway, and the in vitro inhibitory activity of schisandrin B on the two protein complexes is detected by using a co-immunoprecipitation experiment. The experimental data are shown in FIG. 4. Experiments show that schisandrin B inhibits recruitment of downstream Myd88 by TLR4 induced by LPS in RAW 264.7 macrophage, but has weak inhibitory activity on the compounding of MD2 and TLR 4. In addition, we also examined the effect of schisandrin b on another street protein, TRIF, downstream of TLR 4. The experimental data are shown in FIG. 4. Experiments show that schisandrin B has weak inhibitory activity on the combination of TLR4 and TRIF, and has no obvious influence on the increase of IFN-gamma induced by TRIF dependent by LPS.

Example 4 Schizandrin B has direct binding effect with Myd88 protein

The optical Surface Plasmon Resonance (SPR) technology is used for detecting that the schisandrin B and Myd88 protein have direct binding effect: myd88 protein was immobilized on the surface of CM5 sensor chip by standard amino chemical coupling, HBS-EP was used as flow buffer (0.01M Hepes (pH 7.4), 0.15M NaCl,3mM EDTA,0.005% surfactant P20), and a constant flow rate of 5. Mu.l/min was continued through the CM5 sensor chip, which was first activated with 35. Mu.l of a mixed solution of 0.05M NHS and 0.2M EDC, and reacted for 7min to activate surface carboxyl groups. Myd88, formulated to 1mM with an appropriate pH,10mM acetic acid buffer solution, was injected onto the activated sensor chip surface for immobilization, and finally unreacted active carboxyl groups were blocked with 50. Mu.l of 1M ethanolamine and non-covalently adsorbed species were washed away. HBS-EP buffer was washed until baseline was stable. And taking out the CM5 chip for standby. During kinetic measurement, schisandrin B is diluted into different concentrations (the concentration containing DMSO is not higher than 5%) by HBS-EP, and is respectively injected into a CM5 sensor chip with immobilized Myd88, 20 μ l of the solution is injected each time, and the measurement is carried out 3 times. After each measurement, the sensor chip was regenerated with a 1M NaCl solution (pH 4.5) containing 10mM sodium acetate, and then the next measurement was performed. The results of the experiment were evaluated and calculated for kinetic constants using evaluation software BIA evaluation, and their binding constants and kinetic parameters. The experimental data are shown in FIG. 5A. The result shows that Schisandrin B can directly interact with Myd88 protein, but the combination of the Schisandrin B and TLR4 is weak.

A bis-ANS competitive displacement experiment is utilized to investigate the binding rate of schisandrin B and Myd 88: rhMyd88 (5 Nm) was mixed with 1,1' -Bis (ignali) -4,4' -Bis (naphthalene) -8,8' -disulphonate (Bis-ANS, 5 μm) in PBS, and the mixture was measured at 385Nm for excitation light and 430-590Nm for emitted light intensity with a fluorometric detector. A bis-ANS group, a bis-ANS + Myd88 group, a bis-NS + Myd88+ Sch B (2.5 Um) group, a bis-ANS + Myd88+ Sch B (5 Um) group, a bis-ANS + Myd88+ Sch B (10 Um) group, a bis-ANS + Myd88+ Sch B (20 Um) group, and a bis-ANS + Myd88+ Sch B (40 Um) group are set. The wavelength of the rh Myd88 and bis-ANS mixture was first detected as it stabilized, and then incubated for 5min with varying concentrations of Sch B. The excitation light is detected again at 385nm and the emitted light has an emission light intensity of 430-590 nm. The experimental data are shown in FIG. 5B. The result shows that the Schisandrin B dose-dependently reduces the fluorescence absorption intensity of Myd88 protein, and the direct binding effect of the Schisandrin B and the Myd88 protein is prompted.

Claims

1. An application of schisandrin B in the preparation of medicine is characterized in that the medicine is used for treating type 1 diabetes complications;

the type 1 diabetes complication is diabetic cardiomyopathy and/or diabetic nephropathy.

2. The use of schisandrin b in the preparation of a medicament according to claim 1, wherein schisandrin b is used for improving fibrosis of heart and kidney tissues, reducing apoptosis of heart and kidney cells, and improving heart and kidney function.

3. The use of schisandrin B in the preparation of a medicament according to claim 1, wherein schisandrin B is used for treating or preventing type 1 diabetes complications by inhibiting or reducing chronic inflammation induced by hyperglycemia at the site of the complication focus.

4. The use of schisandrin B in the preparation of a medicament according to claim 3, wherein schisandrin B is used for treating or preventing type 1 diabetes complications by direct target inhibition of inflammatory signal transduction protein-myeloid differentiation factor 88.

5. The use of schisandrin B in the preparation of a medicament according to claim 1, wherein the medicament is a formulation of schisandrin B and pharmaceutically acceptable adjuvants.

6. The use of Schizandrin B in the preparation of a medicament according to claim 1, wherein the Schizandrin B comprises powder, tablet, pill, capsule and oral liquid for oral administration.