CN113181196B - Application of crocin in preparation of product for treating rhabdomyolysis syndrome - Google Patents

Abstract

The invention belongs to the fields of traditional Chinese medicine chemistry and molecular biology, and in particular relates to an application of crocin in preparation of a product for treating rhabdomyolysis syndrome. The invention provides an application of crocin in preparing a product with at least one of the following functions of 1) to 6): 1) Preventing and/or treating rhabdomyolysis syndrome; 2) Preventing and/or treating acute renal failure caused by rhabdomyolysis syndrome; 3) Preventing and/or treating acute liver injury caused by rhabdomyolysis syndrome; 4) Reducing the level of inflammatory factors in the serum of animals and/or humans suffering from rhabdomyolysis syndrome; 5) Reducing myoglobin levels in the serum of animals and/or humans suffering from rhabdomyolysis syndrome; 6) Reducing myoglobin levels in the kidneys of animals and/or humans suffering from rhabdomyolysis syndrome. The invention expands the brand new clinical application field for the traditional Chinese medicine crocin total glycoside tablet.

Description

Application of crocin in preparation of product for treating rhabdomyolysis syndrome

Technical Field

The invention belongs to the fields of traditional Chinese medicine chemistry and molecular biology, and in particular relates to an application of crocin in preparation of a product for treating rhabdomyolysis syndrome.

Background

Rhabdomyolysis (RM) is a clinical syndrome in which rhabdomyoblast membrane is damaged, and intracellular toxic substances are released into extracellular fluid and blood circulation, resulting in biochemical disorders and tissue tracheal injury. The main clinical manifestations are myalgia, limb weakness, dark brown urine, frequent concurrent electrolyte disturbance, acute renal failure (acute renal failure, ARF), disseminated intravascular coagulation (disseminated intravascular coagulation, DIC), and multiple organ failure in severe cases. Common causes include hyperkinesia, hyperthermia, crush injury, poisoning, medication, infection, alcoholism, seizures, and the like. The rhabdomyolysis caused by various causes has a common pathogenesis, namely, ATP depletion causes intracellular calcium overload, leading to myofiber necrosis. After muscle cell destruction, a large amount of potassium, phosphate, myoglobin, creatine kinase, lactate dehydrogenase, aspartate transferase and uric acid are released into the blood, causing secondary damage to other tissues and organs.

Acute Renal Failure (ARF) is a serious complication resulting from rhabdomyolysis, and up to 15% to 33% of patients may develop acute renal failure due to low blood volume, myoglobin/uric acid tubular blockage of tubular and direct toxic effects of myoglobin on the kidneys. Under the acidic environment, myoglobin is easily decomposed into heme and globin, and the heme can induce the generation of oxygen free radicals and the removal of nitric oxide, so that the lipid peroxidation damage of tubular epithelial cells is caused. Increased creatine kinase levels, hypoalbuminemia, prolonged thrombin time, metabolic acidosis, etc. are all risk factors for rhabdomyolysis with concomitant acute renal failure.

RM therapy mainly includes three aspects: firstly, the disease causes are treated, the primary disease is actively treated, and factors which destroy muscle cells, such as controlling infection, relieving muscle extrusion, stopping using related medicines and the like, are removed. And then treating rhabdomyolysis, correcting water electrolyte disorder, alkalizing urine during acidosis to keep internal environment stable, promoting urination and dehydration, promoting toxin excretion and the like. Finally, the treatment of complications is directly and effectively carried out by blood purification treatment, and continuous blood purification treatment (CBP) is combined with plasma exchange, continuous venous hemofiltration (CVVH) and continuous venous hemodiafiltration (CVVHDF) modes to remove myoglobin, improve symptoms of patients in a short period, prevent renal function from deteriorating, improve cure rate and reduce clinical mortality.

Catechin (catechins) is a natural non-enzymatic antioxidant with various phenols, and has antimutagenic, anticancer, cardiovascular and cerebrovascular resisting, antidiabetic, antiinflammatory, antibacterial, antiviral, antiaging, optical injury resisting, free radical scavenging, blood lipid reducing, and weight reducing effects. It has been found by researchers that the natural antioxidant theanine can prevent myoglobin uremic acute renal failure caused by RM (Pharmacol Res.2003,48 (5): 503-509). However, the polyhydroxy structure also renders catechin structurally unstable under neutral and alkaline conditions. The catechin has the defects of low fat solubility, poor stability, low human body utilization rate and the like, so that the development and application of the catechin are greatly limited.

Disclosure of Invention

According to the defects existing in the prior art, the invention provides the application of crocin in preparing the product for treating rhabdomyolysis syndrome in combination with the current research front edge, and expands the brand-new clinical application field for the traditional Chinese medicine crocin total glycoside tablet. According to the invention, a model of rhabdomyolysis combined with acute kidney injury is constructed by intramuscular injection of 50% glycerol solution into bilateral hind limbs of a rat, and the drug effect of the crocus sativus total glycoside tablet for inhibiting rhabdomyolysis and acute kidney injury is proved by a preventive gastric lavage administration mode. The invention also predicts the key components and action targets of the crocin tablet for treating rhabdomyolysis by using a network pharmacology related method, and confirms the action of the crocin tablet on rhabdomyolysis syndrome from a molecular level.

The invention is realized by adopting the following technical scheme:

the invention provides an application of crocin in preparing a product with at least one of the following functions of 1) to 6):

1) Preventing and/or treating rhabdomyolysis syndrome;

2) Preventing and/or treating acute renal failure caused by rhabdomyolysis syndrome;

3) Preventing and/or treating acute liver injury caused by rhabdomyolysis syndrome;

4) Reducing the level of inflammatory factors in the serum of animals and/or humans suffering from rhabdomyolysis syndrome;

5) Reducing myoglobin levels in the serum of animals and/or humans suffering from rhabdomyolysis syndrome;

6) Reducing myoglobin levels in the kidneys of animals and/or humans suffering from rhabdomyolysis syndrome.

Specifically, the crocin comprises at least one of crocin I, crocin II, crocin III, crocin IV, crocin V and crocin.

Wherein the product comprises a medicament and/or a pharmaceutical formulation;

the animal is a rat;

the inflammatory factor includes at least one of IL-1β, IL-6, IL-10, TNF- α, TGF- β1 and MCP-1.

Further, as a preferred embodiment of the present invention, the pharmaceutical preparation is any pharmaceutically acceptable dosage form including at least one of a tablet, a capsule, an injection, a granule, a suspension and a solution.

The invention also provides a medicine or a medicine preparation, and the active ingredient of the medicine or the medicine preparation is crocin;

the medicament or the pharmaceutical preparation has at least one of the following functions 1) to 6):

1) Preventing and/or treating rhabdomyolysis syndrome;

2) Preventing and/or treating acute renal failure caused by rhabdomyolysis syndrome;

3) Preventing and/or treating acute liver injury caused by rhabdomyolysis syndrome;

4) Reducing the level of inflammatory factors in the serum of animals and/or humans suffering from rhabdomyolysis syndrome;

5) Reducing myoglobin levels in the serum of animals and/or humans suffering from rhabdomyolysis syndrome;

6) Reducing myoglobin levels in the kidneys of animals and/or humans suffering from rhabdomyolysis syndrome.

Specifically, the crocin comprises at least one of crocin I, crocin II, crocin III, crocin IV, crocin V and crocin.

Further, as a preferred embodiment of the present invention, the pharmaceutical preparation is any pharmaceutically acceptable dosage form, including at least one of a tablet, a capsule, an injection, a granule, a suspension and a solution;

the animal is a rat;

the inflammatory factor includes at least one of IL-1β, IL-6, IL-10, TNF- α, TGF- β1 and MCP-1.

Compared with the prior art, the invention has the beneficial effects that:

the feasibility of treating rhabdomyolysis syndrome by the crocin is verified through experiments for the first time, the crocin is applied to clinically treating the rhabdomyolysis syndrome caused by various causes, effectively improves clinical symptoms, relieves muscle and kidney injury, protects liver function and kidney function, inhibits inflammatory reaction and oxidative stress, and provides a new treatment mode for patients of the type.

The invention constructs a medicine-disease target network through a network pharmacological method for the first time, and confirms that the crocin has a treatment effect on rhabdomyolysis syndrome from a molecular level by using target prediction.

The above as well as additional features, aspects, and advantages of the present application will become more readily apparent with reference to the following detailed description.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.

In the drawings:

FIG. 1 is a graph showing the HE staining results of right hind limb musculature of different groups of rats in example 1; 1 (a) -normal control group; 1 (B) -rhabdomyolysis model group; 1 (C) -crocin tablet low dose group; 1 (D) -crocin tablet dosage group; 1 (E) -crocin tablet high dose group; 1 (F) -catechin groups; n=6/group (random choice), scale bar=50 μm, x 200-fold magnification.

FIG. 2 is a graph showing the result of PTAH staining of right hind limb musculature in each group of rats in example 1; 2 (a) -normal control group; 2 (B) -rhabdomyolysis model group; 2 (C) -crocin tablet low dose group; 2 (D) -crocin tablet dosage group; 2 (E) -crocin tablet high dose group; 2 (F) -catechin groups; n=6/group, scale bar=50 μm, x 200-fold magnification.

FIG. 3 is a graph showing the results of PTAH staining of right kidney tissue from each group of rats in example 2; 3 (a) -normal control group; 3 (B) -rhabdomyolysis model set; 3 (C) -crocin tablet low dose group; dose group in 3 (D) -crocin tablet; 3 (E) -crocin tablet high dose group; 3 (F) -catechin groups; n=6/group, scale bar=50 μm, x 200-fold magnification.

FIG. 4 shows the levels of serum inflammatory factors IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta 1 and MCP-1 in each group of rats in example 3; n=10/group; * p <0.05, < p <0.01vs. normal control group; #p <0.05, #p <0.01vs. model group.

FIG. 5 shows the amounts of serum ALT, AST, BUN, scr and CK from each group of rats in example 4; ALT and AST, n=6/group (random choice); BUN, cr and CK, n=10/group; * P <0.01vs. normal control group; #p <0.05, #p <0.01vs. model group.

FIG. 6 is the levels of serum and renal myoglobin expression levels for each group of rats in example 5; n=10/group; * P <0.01vs. normal control group; #p <0.05, #p <0.01vs. model group.

FIG. 7 is a graph of immunohistochemical detection of the expression of myoglobin in kidney of rats in each group of example 5, 7 (A) -normal control group; 7 (B) -rhabdomyolysis model group; 7 (C) -crocin tablet low dose group; 7 (D) -crocin tablet dosage group; 7 (E) -crocin tablet high dose group; 7 (F) -catechin groups; n=6/group (random choice), scale bar=50 μm, x 200-fold magnification.

FIG. 8 shows the levels of the serum oxidative stress-related indicators LDH, MDA and SOD of each group of rats in example 6; n=10/group; * P <0.01vs. normal control group; #p <0.05, #p <0.01vs. model group.

FIG. 9 is a Wen diagram of the crocin total glycoside tablet acting target rhabdomyolysis-related target gene in example 7.

FIG. 10 is a network diagram of drug-chemical-target protein interactions-disease interactions in example 8.

FIG. 11 is GO enrichment analysis of potential targets in example 9.

FIG. 12 is a KEGG pathway enrichment analysis of potential targets in example 9.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, in order to make the objects and technical solutions of the present invention more apparent. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to product specifications; the reagents and materials, unless otherwise specified, are commercially available.

1. The materials used in the following examples are as follows:

(1) Experimental objects

SPF-class male SD rats, body weight (200-220) g, purchased from Peking Vitre Liwa laboratory animal technologies Co., ltd., license number SCXK (Beijing) 2016-0006, laboratory animal quality eligibility number No.110011201107399932.

(2) Animal feeding

During the experiment, rats were kept in the animal house of Chinese medical institute, chinese medical college, and the use license SCXK (Beijing) 2019-0003 for experimental animals. The animal house is a barrier system, artificial illumination is carried out, the light and shade period is 12 hours, the temperature is controlled within the range of (20-22), the relative humidity is (40-70)%, and the ventilation times are 15 times/h. Rats were housed in plastic cages, 5 per cage. The plastic drinking bottle with 100ml is used for containing purified water for drinking at will, and fresh drinking water and padding are replaced every day.

(3) Experimental medicine

The crocin tablet is produced by Ruiyang pharmaceutical Co., ltd, chinese medicine standard character Z20163079, product batch number 19080702, content of each tablet of 12mg, production date 2019.8.7 and validity period of 2021.7. Catechin (Catechin) is produced by Beijing Soy Bao technology Co., ltd., 100mg, purity not less than 98.0%, product lot number SC8160, and expiration date up to 20200915.

(4) Experimental reagent and consumable

The rat TNF- α ELISA kit (lot number Z20037304), the rat IL-1β ELISA kit (lot number Z20037306), the rat IL-6ELISA kit (lot number Z18037302), the rat IL-10ELISA kit (lot number Z19037303), the rat TGF- β1 (transforming growth factor β1) ELISA kit (lot number Z23037307) and the rat monocyte chemotactic protein 1 (monocyte chemotactic protein 1/monocyte chemotactic and activating factor, MCP-1/MCAF) ELISA kit (lot number Z21037305) were purchased from the Wohan Huamei bioengineering Co., ltd;

lactate dehydrogenase assay kit (lactic dehydrogenase, LDH, lot No. 20201109), superoxide dismutase assay kit (superoxide dismutase, SOD, lot No. 20201126), malondialdehyde assay kit (malondialdehyde, MDA, lot No. 20201126), creatine kinase assay kit (creatine kinase, CK, lot No. 20201109), glutamate pyruvate transaminase assay kit (glutamic pyruvic transaminase, ALT/GPT, lot No. 20201106), glutamate transaminase assay kit (glutamic oxalacetic transaminase, AST/GOT, lot No. 20201106), creatinine assay kit (creatine, cr, lot No. 20201109), urea nitrogen assay kit (blood urea nitrogen, BUN, lot No. 20201106), rat serum myoglobin (lot No. 20201116), and rat tissue hemoglobin (lot No. 2020116) are all purchased from the south-establishment engineering institute;

BCA protein concentration assay kit (lot No. RK 240545) was purchased from Pierce, usa; sodium pentobarbital (lot 20191119) was purchased from Sigma in the united states (split charging); glycerol (lot 306D039 SX), chloral hydrate (lot T8590) and paraformaldehyde (lot 20200728) were purchased from beijing soeba technologies limited; normal saline (lot number 1807023205) was purchased from Shijia four-drug company, inc.; rat myoglobin (myogenin) antibodies (lot ab 77232) were purchased from Abcam, usa; HRP-labeled goat anti-rabbit secondary antibody (lot number K5007) was purchased from danish DAKO; red conventional capped vacuum blood collection tube (lot number 9291629) was purchased from Shanghai BD medical instruments limited; disposable intravenous blood taking needle (lot 2018041016) was purchased from Hebei Xinle medical instruments and technology Co., ltd, model size AF0.7X25TW.

(5) Experimental instrument

German Sartorius Quintix3102-1CN precision electronic balance; germany Eppendorf 5452R low temperature ultra high speed centrifuge; becton-Dickinson SpectraMax M5 multifunctional enzyme labeling instrument in U.S.A.; SCNTZ-48 high-flux tissue grinder of Ningbo Xinzhi biotechnology Co Ltd; japanese NIKON ECLIPSE TI-SR fluorescence microscope; NIKON DS-U3 imaging System in Japan.

2. The experimental procedure used in the following examples is as follows:

(1) Model making

On the day of the experiment, pentobarbital sodium (1%, 0.55ml/100 g) was intraperitoneally anesthetized, and rats were single-point injected with 50% glycerol solution (diluted with physiological saline) at a dose of 2ml (10.0 ml/kg), with reference to Tsai JP et al (Int J Med Sci.2017,14 (7): 680-689.).

(2) Dose of experiment

The total crocin glycoside tablet clinical human (60 kg) uses 12 mg/tablet, 4 tablets/time, 3 times/day, and total 144 mg/day, which is equivalent to 2.4mg/kg/day, and the corresponding rat dose (200 g) is 15.0mg/kg/day, which is used as the medium dose of the experiment, and is increased by 2 times, and 7.5mg/kg/d and 30.0mg/kg/d are respectively used as the low dose and the high dose of the experiment.

The catechin reference dose of the positive control drug was 80.0mg/kg/day (Pharmacol Res.2003,48 (5): 503-509).

(3) Administration mode

And adopting a preventive administration mode, starting molding on 8 th day after 7 days of continuous gastric lavage, and carrying out subsequent drug effect experiments after 24 hours.

(4) Experimental grouping

A total of 6 groups of 12 were assigned, each group being as follows:

normal control group: continuously irrigating the same amount of normal saline for 7 days, injecting the same amount of normal saline into the external muscles of the hind limbs of the two sides of the rat on the 8 th day by single point injection, ending the experiment after 24 hours, and taking materials;

model group: continuously irrigating the stomach with normal saline with the same amount for 7 days, injecting 50% glycerol solution into the external muscle of the hind limb of the two sides of the rat on the 8 th day, ending the experiment after 24 hours, and taking materials;

saffron total glycosides tablet high, medium and low dose group: continuously performing gastric lavage administration according to 30.0mg/kg/d, 15.0mg/kg/d and 7.5mg/kg/d for 7 days, molding on 8 th day, and finishing experiments and obtaining materials after 24 hours;

positive control group (Catechin): the medicine is continuously infused in the stomach according to 80.0mg/kg/d for 7 days, the mould is built on the 8 th day, and the experiment is ended after 24 hours and the materials are obtained.

(5) Drug efficacy index

At the end of the experiment, the mixture was weighed, chloral hydrate (10%, 0.35ml/100 g) was anesthetized, the abdominal aorta was bled, and the rat hindlimb lateral muscles and kidneys were dissected out.

(5.1) muscle and renal pathology

The hind limb muscles were subjected to hematoxylin-eosin staining (HE) and phosphotungstic acid hematoxylin staining (phsphotungstic atid, hamatoxylin, PTAH), and the extent of skeletal muscle injury was assessed; kidneys were HE stained to assess the extent of damage to the kidneys. Dissecting and taking out hind limb muscles of a glycerol injection part, fixing in 10% paraformaldehyde solution, embedding paraffin, preparing pathological sections, and staining HE and PTAH; dissecting kidney, fixing and embedding, preparing pathological sections and HE staining; the pathological changes of the muscles and kidneys of each group are observed under the light microscope.

(5.2) serum Biochemical index

The abdominal aorta is subjected to blood taking, 3000rpm/min and centrifugation for 10min, and the supernatant is taken, and indexes such as glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), creatinine (Scr), urea nitrogen (BUN), creatine Kinase (CK) and the like are detected by a biochemical kit in combination with an enzyme-labeled instrument.

(5.3) serum and renal myoglobin (Mb)

The abdominal aorta was bled, centrifuged at 3000rpm/min for 10min, and the supernatant was collected and assayed for serum myoglobin (Mb) levels by ELISA.

Weighing the kidney with equal weight according to the weight (g): volume (ml) =1:5, 5 volumes of homogenate medium (physiological saline) was added, ultrasonic homogenate was performed, rat kidney homogenate was prepared, 12000g/min, centrifugation was performed for 15min, and the supernatant was taken and protein concentration of each sample was measured using BCA protein concentration measurement kit. The absorbance was then measured at 450nm, following ELISA kit instructions.

(5.4) Kidney myoglobin (Mb)

Kidneys were dissected out, fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, immunohistochemical staining was performed, and renal myoglobin (Mb) expression was measured.

(5.5) serum inflammatory factors

The abdominal aorta was bled, centrifuged at 3000rpm/min for 10min, and the supernatant was assayed for the expression of serum inflammatory factors IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta 1 and MCP-1 by ELISA.

(5.6) serum oxidative stress level

The abdominal aorta was bled, centrifuged at 3000rpm/min for 10min, and the supernatant was assayed by a biochemical kit in combination with a microplate reader for the levels of superoxide dismutase (SOD), lactate Dehydrogenase (LDH) and Malondialdehyde (MDA).

(6) Data processing

All data were taken as mean ± standard deviation

The SPSS13.0 software performs statistical treatment, the comparison among multiple groups adopts single-factor analysis of variance, the comparison among any two groups adopts t test, and p <0.05 is the difference, so that the method has statistical significance.

1. Inhibition effect of crocin tablet on rhabdomyolysis syndrome of rat model

EXAMPLE 1 influence of crocin tablet on rhabdomyolysis of rat hind limb muscle tissue

The 6 groups of rats are grouped, the preventive administration mode is adopted, the medicine is continuously infused for 7 days by adopting different treatments, the modeling is started on the 8 th day, 6 rats are randomly selected, and the subsequent muscle pathology experiment is carried out after 24 hours, wherein the HE staining result is shown in figure 1, and the PTAH staining result is shown in figure 2.

HE staining results show that the normal control group has normal striated muscle structure, the model group has pathological change of striated muscle solubility, the striations disappear, most of the striations are cavitation-like, and local inflammatory cells infiltrate; after the crocin tablet is applied, the rhabdomyolysis injury is obviously reduced, and the rhabdomyoline of the high-dose group is close to normal (figure 1).

The PTAH dyeing result shows that the normal control group striated muscle fiber is purple blue or light purple blue, has complete structure and obvious striation, and the model group striated muscle fiber texture is not clear, and most of the striation muscle fiber has cavitation or gaps and is changed in a honeycomb shape; the crocin tablet administration group had reduced striated muscle fiber injury, the structure gradually recovered to normal, and the improvement was most evident in the high dose group (fig. 2).

Example 2 influence of crocin tablet on rhabdomyolysis rat kidney tissue

The kidney pathology experiment was performed on each group of rats identical to example 1, and HE staining results are shown in fig. 3.

The kidney tissue structure of the normal control group is normal, and the glomerulus and the tubular are not obviously pathologically changed; the glomerulus of the model group is obviously engorged with blood and swelled, the saccule is expanded, the epithelial cells of the proximal tubular are swelled, the lumen is expanded, the arrangement is disordered or collapsed, the interstitial is engorged with blood capillaries, a large amount of inflammatory cells infiltrate, and myoglobin tubes are formed in part of glomeruli; the saffron glycoside tablet administration group had reduced glomerular and tubular epithelial cell engorgement swelling, reduced intraglomerular tubular, reduced interstitial capillary engorgement and inflammatory cell infiltration, significantly alleviated renal pathology, and most significant in the high dose group (fig. 3).

Example 3 Effect of crocin tablet on rhabdomyolysis of serum inflammatory factors in rats

10 rats after 24h of each model were sampled, and the abdominal aorta was bled, and the expression of serum inflammatory factors IL-1β, IL-6, IL-10, TNF- α, TGF- β1 and MCP-1 was measured by ELISA, and the results are shown in FIG. 4.

The serum inflammatory factors IL-1β, IL-6, IL-10, TNF- α, TGF- β1 and MCP-1 levels were significantly elevated in the model group compared to the normal control group. Compared with the model group, the IL-10 level of the crocin tablet low dose group, the IL-10 level of the medium dose group and the IL-1 beta, the IL-6, the TNF-alpha, the TGF-beta 1, the MCP-1 and other inflammatory factors of the medium dose group and the high dose group are obviously reduced. The catechin as positive control agent can reduce the level of inflammatory factors IL-1β, IL-10 and TGF- β1. The results indicate that the crocin tablet antagonizes acute inflammation caused by rhabdomyolysis (fig. 4).

EXAMPLE 4 Effect of crocin tablet on rhabdomyolysis rat liver and renal function

The rats after 24h of molding were randomly sampled, and blood was collected from abdominal aorta, and content indexes of glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), creatinine (Scr), urea nitrogen (BUN) and Creatine Kinase (CK) were detected by using a kit and an enzyme-labeled instrument, and the results are shown in FIG. 5.

Compared with the normal control group, the levels of biochemical indexes such as ALT, AST, BUN, scr, CK and the like of the model group are obviously increased. The levels of both the dose and high dose groups ALT, AST, BUN, scr and CK in the crocin tablet were significantly reduced compared to the model group (fig. 5). The catechin as a positive control agent can also reduce the level of the five biochemical indexes. The result shows that the crocin tablet has protective effect on acute liver injury and kidney injury caused by rhabdomyolysis.

Example 5 influence of crocin tablet on the expression of rhabdomyolytic rat serum and renal myoglobin

After 24h of each model, 6 rats were randomly sampled, and the aorta was sampled or kidneys were sampled, and serum and renal myoglobin (Mb) levels were measured by ELISA, respectively, as shown in FIG. 6; after another 24h molding, 6 rats were subjected to immunohistochemical detection experiments, and the staining results are shown in FIG. 7.

The serum and renal myoglobin expression levels were significantly elevated in the model group compared to the normal control group. Serum and renal myoglobin expression levels were significantly reduced in both the dose and high dose groups in the crocin tablet compared to the model group (fig. 6). The catechin as positive control can reduce the expression level of serum and renal myoglobin. Immunohistochemical staining results show that the normal control group kidney has no myoglobin; the expression of the myoglobin of the kidney of the model group is obviously increased, the positive is brown yellow, the strong positive is brown, and the glomerulus, the proximal tubular and the distal tubular are distributed; compared to the model group, the crocin tablet-administered group showed gradually decreased myoglobin expression, most significantly in the high dose group (fig. 7). The positive control agent catechin has reduced coloring rate, and can inhibit the expression of renal myoglobin.

EXAMPLE 6 Effect of crocin tablet on serum oxidative stress levels in rhabdomyolyzed rats

The rats after 24h of molding were sampled, and the abdominal aorta was sampled and tested for the effects of serum oxidative stress related indicators LDH, MDA and SOD using a kit and a microplate reader, and the results are shown in FIG. 6.

Compared with the normal control group, the serum LDH and MDA expression level of the model group is obviously increased, and the SOD expression level is obviously reduced. The low, medium and high dose groups of safflor tablet showed reduced levels of serum LDH and MDA expression and increased levels of SOD expression compared to the model group (fig. 8). The catechin as positive control can also reduce the expression level of serum LDH and MDA and increase the expression level of SOD.

The above results indicate that:

1. the rats were single-point injected with 50% glycerol solution (10.0 ml/kg) in the lateral muscle of the bilateral hind limb, blood was collected from the abdominal aorta after 24h, the supernatant was centrifuged, and the injection site muscle was removed. The HE staining result of the muscle shows that the striated muscle is changed in dissolution pathology, the striated muscle disappears, most of the striated muscle is in a vacuole shape, and local inflammatory cells infiltrate; the muscle PTAH staining results showed that the striated muscle fibers were not clearly textured, mostly with vacuoles or gaps, in a honeycomb-like fashion. Renal HE staining showed significant glomerular hyperemia and swelling, balloon dilation, proximal tubular epithelial cell swelling, luminal dilation, disordered alignment or collapse, interstitial visible capillary hyperemia, massive inflammatory cell infiltration, and myoglobin tubular formation in part of glomeruli and tubular. Elevated levels of serum inflammatory factors IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta 1, and MCP-1; the levels of biochemical indexes ALT, AST, BUN, scr, CK and the like are increased; elevated serum and renal myoglobin expression levels; serum LDH and MDA expression levels increased and SOD expression levels decreased.

2. By adopting the mode of preventive gastric lavage to the crocin tablet, the staining results of muscle HE and PTAH both show that the rhabdomyolysis injury of the administration group is obviously reduced, the structure is gradually recovered to be normal, and the improvement is most obvious in the high-dose group. Renal HE staining showed reduced glomerular and tubular epithelial cell engorgement swelling, reduced intraglomerular tubular, reduced interstitial capillary engorgement and inflammatory cell infiltration in the dosing group, most pronounced in the high dose group. The serum IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta 1 and MCP-1; the level of biochemical indexes such as serum ALT, AST, BUN, scr, CK and the like is reduced; serum and renal myoglobin expression levels are reduced; serum LDH and MDA expression levels were reduced and SOD expression levels were increased.

3. The saffron glucoside tablet antagonizes acute inflammation, inhibits oxidative stress, protects liver function and kidney function, and reduces muscle and kidney injury caused by rat striated muscle injury.

2. Prediction of action target of crocin tablet on treating rhabdomyolysis

The Rhabdomyolysis (RM) is used for treating diseases, and key components and action targets of the crocin tablet for treating rhabdomyolysis are predicted by using a network pharmacology related method according to a research mode related to drug target-disease target-pharmacology activity.

Example 7 screening prediction of crocin chemical composition and related target and target related to rhabdomyolysis syndrome

The chemical components of the crocin are collected through a literature, inChI numbers of the chemical components of the crocin are obtained by using a PubCHem database (http:// PubChem. Ncbi. Nlm. Nih. Gov /), and target prediction of the chemical components is carried out through BATMAN-TCM (http:// bionet. Ncpsb. Org/BATMAN-TCM /).

The crocin comprises crocin I (crocin I), crocin II (crocin II), crocin III (crocin III), crocin IV (crocin IV), crocin V (crocin V) and crocin acid (crocetin). The chemical components are subjected to target spot query through a BATMAN-TCM database and then are subjected to duplication removal, and the total number of related targets is 172.

The related diseases are searched by using the key words of 'Rhabdomyolysis' through an OMIM database (https:// OMIM. Org /) and a GeneCards database (https:// www.genecards.org /), and the key words are used as targets of Rhabdomyolysis related diseases after duplication removal. 774 disease targets were obtained through OMIM and Genecard databases together, and 18 drug intersection targets were obtained (fig. 9), which could be used as potential targets for the treatment of rhabdomyolysis by crocin.

Example 8 construction of drug-chemical composition-target protein interaction-disease network map

The drug-disease co-target was obtained in example 7, and 18 targets of the intersection of the above disease and drug were imported into a String database (https:// String-db. Org/cgi/input. Pl) to construct a protein-protein interaction (protein-protein interaction, PPI) network. The protein type is set as 'Homo sapiens', the lowest interaction threshold is set as 'medium confidence' (0.400), other default values are kept, the protein interaction relation is obtained, 18 sides and 31 sides (sides represent interaction among targets) of key nodes (nodes represent targets) are obtained, and the average node value is 3.44. A protein interaction network map was constructed using Cytoscape 3.7.1 software, while a drug-chemical-target protein interaction-disease interaction network map was constructed (fig. 10).

Example 9 GO functional enrichment and KEGG pathway enrichment analysis

The intersection target of the crocin for treating rhabdomyolysis is imported into a David database (https:// David. Ncifcrf. Gov /), and GO (gene ontology) functional enrichment analysis and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis are performed.

The results of the GO functional enrichment show that the crocin treatment rhabdomyolysis is correlated with 64 biological processes (biological process, BP), 5 molecular functions (molecular function, MF) and 19 cell compositions (cellular component, CC), BP is screened, CC-lgP is ranked ten first, and the analysis results of MF-lgP is ranked five first are plotted (FIG. 11), the biological processes involve positive regulation of transcription, DNA templates (positive regulation of transcription, DNA-templates), positive regulation of NF-kappa B transcription factor activity (positive regulation of NF-kappaB transcription factor activity), negative triglyceride sequestration regulation (negative regulation of sequestering of triglyceride), and the like. Molecular functions include cytosol (cytosol), protein complexes (protein complexes), plasma membrane outside (external side of plasma membrane), and the like. The molecular functions mainly involve ribonucleic acid polymerase ii transcription factor activity, ligand activated sequence specific DNA binding (RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding), steroid hormone receptor activity (steroid hormone receptor activity), identical protein binding (identical protein binding) and the like.

The KEGG pathway enrichment analysis involved 12 pathways, which were made into a bubble map (fig. 12). Potential pathways associated with rhabdomyolysis are mainly involved in Non-alcoholic fatty liver disease (Non-alcoholic fatty liver disease (NAFLD)), type i diabetes (Type I diabetes mellitus), myocardial contraction (Cardiac muscle contraction), hypertrophic Cardiomyopathy (HCM) (Hypertrophic cardiomyopathy (HCM)), and the like (P < 0.05).

The above results were as follows: the crocin comprises crocin I, crocin II, crocin III, crocin IV, crocin V and crocin acid as main chemical components. 18 targets of FMR1, CAPN3, PYR2, INS, ATP1A, CKM, FASLG, NR H2, TRPV1, PPARA, IL1B, PNPLA2, AR, MYL2, PRDM16, PPARG, ALDH1A, TNF and the like are predicted to be potential targets of crocin for treating rhabdomyolysis.

It should be understood that the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited to the above-described embodiment, but may be modified or substituted for some of the features described in the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (3)

1. Use of saffron total glycosides tablet for preparing a product having at least one of the following functions 1) to 6):

1) Preventing and/or treating rhabdomyolysis syndrome;

2) Preventing and/or treating acute renal failure caused by rhabdomyolysis syndrome;

3) Preventing and/or treating acute liver injury caused by rhabdomyolysis syndrome;

4) Reducing the level of inflammatory factors in the serum of animals and/or humans suffering from rhabdomyolysis syndrome;

5) Reducing myoglobin levels in the serum of animals and/or humans suffering from rhabdomyolysis syndrome;

6) Reducing myoglobin levels in the kidneys of animals and/or humans suffering from rhabdomyolysis syndrome.

2. The use according to claim 1, wherein,

the product comprises a medicament and/or a pharmaceutical formulation;

the animal is a rat;

the inflammatory factor is at least one of IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta 1 and MCP-1.

3. The use according to claim 2, wherein the pharmaceutical formulation is in any pharmaceutically acceptable dosage form.

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