CN108464986B - Application of geniposide in relieving skeletal muscle fibrosis - Google Patents

Abstract

The invention discloses an application of geniposide in relieving skeletal muscle fibrosis, and belongs to the field of natural product treatment. The geniposide is used for stimulating C2C12 myoblasts, and the result shows that the geniposide can obviously reduce the fibrosis degree of fibroblasts. Further, a mouse model of skeletal muscle injury caused by contusion is established, and geniposide standard substance is injected into the abdominal cavity to treat the mouse, so that the geniposide is proved to play a role in resisting fibrosis in the injured skeletal muscle. In addition, the degree of fibrosis of the C2C12 myoblasts stimulated with TGF-beta and Smad4 is significantly increased, while the level of fibrosis is significantly reduced compared to that of untreated cells after treatment with geniposide. It can be seen that the action mechanism of geniposide for relieving skeletal muscle fibrosis is probably closely related to TGF-beta/Smad 4 signal pathway.

Description

Application of geniposide in relieving skeletal muscle fibrosis

Technical Field

The invention relates to a new application of geniposide, in particular to an application of geniposide in relieving skeletal muscle fibrosis, belonging to the field of natural product treatment.

Background

Skeletal muscle is the most directly involved organ in human body, accounting for about 40% -50% of the total weight of human body, and is also the largest tissue of human body, playing an important role in human activities, socialization and occupation. Skeletal muscle injury is a soft tissue injury common in sports medicine, while skeletal muscle is a tissue with complete regenerative capacity, which is multipotent to complete regenerative repair after acute traumatic injury. After being damaged, skeletal muscle tissue will go through several processes of inflammation phase, muscle satellite cell proliferation and activation phase and regeneration fiber maturation and remodeling or fibrosis phase. Muscle fibers are the basic structure of skeletal muscle, and fibrosis occurs when muscle fibers exhibit persistent degenerative necrosis and regeneration with chronic inflammation and finally muscle satellite cell depletion or abnormal differentiation, with massive deposition of extracellular matrix. Skeletal muscle hyperfibrosis is manifested by massive proliferation and activation of various cells such as fibroblasts, accompanied by massive collagen production, and failure to be absorbed by the body, leading to a disorder in remodeling of tissues. Skeletal muscle fibrosis causes the formation of scar tissue, is one of pathological signs of skeletal muscle chronic diseases and trauma, seriously affects the healing quality of skeletal muscle and causes the reduction of the exercise contraction capacity. Once the scar is formed, complete repair of the muscle tissue will not be possible.

In the process of skeletal muscle injury repair, transforming growth factor beta (TGF-beta) is a key fibroblast growth factor, can promote the formation of extracellular matrix, promote fibroblasts to synthesize extracellular matrixes such as collagen, fibronectin, proteoglycan and the like, and promote the expression of extracellular matrix protein specific surface membrane receptors. Whereas the fibrotic effects of TGF-. beta.during muscle injury are regulated primarily by the TGF-. beta. -Smad signaling pathway. Smad protein family members play important mediating roles in the TGF-beta signaling pathway as receptor kinases downstream of TGF-beta. To date, 8 Smads family members have been found in mammals and are divided into three subfamilies according to their structure and function: (1) receptor-activated Smads (R-Smads), including Smad1, 2, 3, 5, 8; (2) smads (co-Smad) universal, only Smad4 being currently found in humans; (3) inhibitory Smads (I-Smads), including Smad6, 7. Smad4 is a binding partner for all R-Smads, is a key link of the TGF-beta superfamily signaling pathway, and plays a crucial role in the progression of fibrotic pathology. The TGF- β -Smad signaling pathway involves TGF- β receptor I kinase (TGF- β 1) acting directly on Smad2/3 and phosphorylating Smad2/3, followed by the binding of phosphorylated Smad2/3 to Smad4 to form a stable heteromultimer, and Smad4 promotes the regulation of transcription of target genes after entry of the Smad complex into the nucleus.

For skeletal muscle injuries, the current physical therapies include rest, ice compress, medical ozone, low-intensity laser and pulse ultrasonic, and the chemotherapy includes antioxidants (vitamin C, acetylcysteine, etc.), anti-inflammatory drugs (non-steroids), anti-fibrosis drugs (telmisartan, lipoxin a4, etc.) and growth factor injection (insulin-like growth factor-1, for example), but the physical therapies and the chemotherapy have certain problems in the aspects of effect, side effect, safety, etc., and the treatment and prevention means are still limited. We can explore new ideas starting from natural products formed by long-term evolution and natural screening. However, the research on the aspect of treating skeletal muscle injury of natural products is few, and at present, only on duchenne muscular dystrophy, relevant research finds that dietary natural polyphenol and Kazinol-P in paper mulberry (Broussonetia kazinoki Sieb) have an improving effect.

The Gardenia fruit is mature fruit of Gardenia jasminoides Ellis of Gardenia of Rubiaceae, is mainly distributed in tropical and subtropical areas, and accounts for about 90% of the total world production in China. The gardenia fruit is the first medical and edible resource issued by the Ministry of health, is commonly used as a traditional Chinese medicine, a food additive, an edible pigment, oil pressing and the like, and has the effects of diminishing inflammation, easing pain, protecting liver and benefiting gallbladder, resisting oxidation and the like. The main active ingredients of the medicine comprise iridoid and crocin, wherein the iridoid mainly takes geniposide as the main component, and the crocin mainly takes crocin as the main component. Geniposide is one of the basic substances with pharmacological activity of fructus Gardeniae, the content is 3-8% according to the production area, and its structural formula is shown in figure 1. Within a certain dosage range, geniposide has the effects of resisting inflammation, protecting nerves, reducing blood sugar and the like. In addition, geniposide has been shown to alleviate liver fibrosis by inhibiting TGF- β/Smad and ERK-Mitogen Activated Protein Kinase (MAPK) signaling pathways, but so far, geniposide has not been reported to have a alleviating effect on skeletal muscle fibrosis.

Disclosure of Invention

The invention provides a compound or a composition for relieving skeletal muscle fibrosis, wherein the compound is geniposide, and the composition is a composition containing geniposide.

The skeletal muscle fibrosis is a process that when skeletal muscle is subjected to acute trauma for regeneration and repair, muscle fibers are continuously degenerated, necrotized, regenerated, inflamed or/and a great deal of extracellular matrix is deposited so as to block the remodeling of tissues.

The relieving refers to that the stimulation effect of geniposide obviously reduces the expression of fibrosis promoting genes collagen I (Col I), Vimetin (Vim), alpha-smoothened act (alpha-SMA) and Smad4 in fibroblasts and injured gastrocnemius tissues compared with the condition of no treatment in a certain dosage and time range, and also obviously reduces the content of collagen fibers and Col I increased due to injury in skeletal muscle cells.

The geniposide may be derived from: gardenia dregs, gardenia fruit, eucommia bark, oldenlandia diffusa, coptis detoxifcation soup and the like.

The composition also comprises pharmaceutically acceptable auxiliary materials, such as a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an adhesive, a disintegrating agent, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a flavoring agent, an anti-adhesive, an integrating agent, an osmotic accelerator, a pH value regulator, a buffering agent, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, an encapsulating agent, a humectant, an absorbent, a diluting agent, a flocculating agent and a deflocculating agent, a filter aid, a release retardant and the like.

The compound or the composition can also be used for preparing a medicament for treating skeletal muscle chronic diseases, or preparing a medicament for treating or removing scar tissues caused by skeletal muscle fibrosis, or preparing a medicament for treating muscle injuries, or preparing a cosmetic for removing skin scar tissues.

The invention uses gardenia oil extraction and alcohol extraction to process C2C12 myoblast, which can restrain the fibrosis process. The geniposide standard substance is used for stimulating C2C12 myoblasts, and the result shows that geniposide can obviously reduce the fibrosis degree of fibroblasts. The invention establishes a mouse model of skeletal muscle injury caused by contusion, and treats the mouse by injecting geniposide standard substance into the abdominal cavity, thereby proving that geniposide plays a role in anti-fibrosis in the injured skeletal muscle. We found that C2C12 myoblasts stimulated with TGF- β and Smad4 showed a significant increase in the degree of fibrosis, whereas the level of fibrosis was significantly reduced after treatment with geniposide compared to that without treatment. It can be seen that the mechanism of action of geniposide in relieving skeletal muscle fibrosis is probably closely related to the TGF-beta/Smad 4 signaling pathway.

Drawings

FIG. 1 chemical structural formula of geniposide.

FIG. 2 shows the concentration and content of geniposide in the untreated gardenia dregs and the concentration and content after water extraction or alcohol extraction.

FIG. 3C 2C12 shows the expression change of protein and mRNA level of the gene related to cell fibrosis under the action of aqueous and alcohol extracts of gardenia dregs.

(A) The C2C12 myotube cells were treated with aqueous and alcoholic extracts of Gardenia jasminoides Ellis for 12h (the concentration of geniposide in the aqueous and alcoholic extracts was 0.4mg/mL), and Dimethylsulfoxide (DMSO) was used as a control. Protein levels of α -SMA, ColI and Vim were determined by immunoblot analysis (western blot), GAPDH being the internal control. (B-D) detection of mRNA levels of ColI, Vim and α -SMA by real-time quantitative fluorescent PCR (RT-qPCR). Data are expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test).

FIG. 4 expression changes of genes related to C2C12 cell fibrosis at protein and mRNA levels by geniposide with different concentrations.

(A-C, E) C2C12 myotube cells were stimulated with geniposide at various concentrations (0,0.05,0.1,0.2,0.4,0.8mg/mL) for 12h and mRNA levels of α -SMA, Col I, Vim and Smad4 were analyzed by RT-qPCR. Data are expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test). (D, F) protein levels of α -SMA, ColI, Vim and Smad4 were determined by western blot at the same concentration gradient, and GAPDH was the internal control.

FIG. 5 change in expression of genes associated with C2C12 cell fibrosis in protein and mRNA levels after geniposide treatment for various periods of time.

(A-C, E) C2C12 myotube cells were treated with 0.4mg/mL geniposide for 0, 1, 2, 4, 8, 12h, respectively, and analyzed by RT-qPCR for mRNA levels of α -SMA, ColI, Vim, and Smad 4. Data are expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test). (D, F) protein levels of α -SMA, ColI, Vim and Smad4 were determined by western blot at the same time gradient, and GAPDH was the internal control.

Figure 6 improvement of geniposide in fibrosis caused by skeletal muscle injury in mice. (A) Gastrocnemius muscles (time points 0, 7, 14 and 21 days) of skeletal muscle acute injury model mice were stained with hematoxylin and surong (HE) (n ═ 3) (microscope 20 fold, 20X). (B) Gastrocnemius muscles (time points 0, 7, 14 and 21 days) of skeletal muscle acute injury model mice were stained with sirius red (n ═ 3) (20X). (C) Gastrocnemius muscles (time points 0, 7, 14 and 21 days) of mice, a model of acute injury to skeletal muscle, were immunohistochemically stained (Col i antibody) (n ═ 3) (20X). (D) Gastrocnemius muscles (time points 0, 7, 14 and 21 days) of skeletal muscle acute injury model mice were HE stained (n ═ 3) (20X), Con being a blank control group, untreated injury group; geni is geniposide treatment group at a dose of 25mg/kg/d (based on mouse body weight). (E) sirius vinifera staining (n ═ 3) (20X) of gastrocnemius muscles (time points 0, 7, 14 and 21 days) of skeletal muscle acute injury model mice, Con being a blank control group, untreated injury group; geni is geniposide treated group at a dose of 25mg/kg/d (based on mouse body weight). (F) Gastrocnemius muscle from mice, a model of acute injury to skeletal muscle (time points 0, 7, 14 and 21 days) was immunohistochemically stained (with Col i antibody) (n ═ 3) (20X), Con being a blank control group and an untreated injury group; geni is geniposide treated group at a dose of 25mg/kg/d (based on mouse body weight). (G, I) protein levels in α -SMA, ColI, Vim and Smad4 were analyzed by Western blot in tibialis anterior muscle tissue of acutely injured mice (14 days), with GAPDH being the internal control. Con is a blank control group and an untreated injury group; geni is geniposide treated group at a dose of 25mg/kg/d (based on mouse body weight) (n-3). (H) The mRNA levels of α -SMA, Vim and ColI in gastrocnemius of acutely injured mice (14 days) were analyzed by RT-qPCR, and the data were expressed as mean. + -. standard error, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test). Con is a blank control group and an untreated injury group; geni is geniposide treated group at a dose of 25mg/kg/d (based on mouse body weight) (n-3).

FIG. 7 change in expression of genes associated with C2C12 cell fibrosis stimulated by TGF- β with geniposide in protein and mRNA levels.

(A-C, E) mRNA expression of α -SMA, ColI, Vim and Smad4 was measured on qPCR by stimulating C2C12 myotube cells with TGF- β (20ng/mL) and then 0.4mg/mL geniposide for 12 h. Con is a blank control group treated by DMSO, TGF-beta is a TGF-beta stimulation group, and TGF-beta + Geni is a TGF-beta and geniposide co-stimulation group. Data are expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test). (D, F) stimulation of C2C12 myotube cells with TGF-. beta.20 ng/mL followed by 0.4mg/mL geniposide for 12h detected protein level expression of alpha-SMA, Col I, Vim and Smad 4. GAPDH is an internal control. For different treatment groups, "+" indicates present and "-" indicates absent.

FIG. 8 change in expression of genes associated with C2C12 cell fibrosis stimulated by geniposide on Smad4 at protein and mRNA levels.

(A) Smad4 was transfected into C2C12 myotube cells, stimulated with geniposide at 0.4mg/mL for 24h, and protein expression of Smad4, Col I, α -SMA and Vim was analyzed by Western blot with GAPDH as an internal control. For the different treatment groups, "+" represents present and "-" represents absent. (B) Smad4 was transfected into C2C12 myotube cells, stimulated with 0.4mg/mL geniposide for 24h, and analyzed by RT-qPCR for Col I, α -SMA and Vim mRNA expression. Control + Vector was DMSO blank Control, Geni + Vector was untransfected Smad4 geniposide treated group, and Geni + Smad4 was transfected Smad4 and geniposide treated group. Data are expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test).

Detailed Description

Example 1 extraction of geniposide and validation of Annellar skeletal muscle fibrosis

1 method of experiment

1.1 preparation method of aqueous extract and alcoholic extract of fructus Gardeniae dregs

(1) Preparation of aqueous extract of gardenia dregs

Grinding fructus Gardeniae dregs, and sieving with 60 mesh sieve. Weighing 15g of gardenia meal powder and adding 150mL of deionized water to leach for 1h in a water bath at 60 ℃. After filtration, the supernatant was retained, and 100mL of deionized water was added to the residue, followed by water bath at 60 ℃ for 1 hour. Similarly, the third leach is made by adding 50mL of deionized water to the residue. After the water bath was over for 1h, all supernatants were mixed, cooled, centrifuged at 4000rpm for 10min and the supernatants collected. Purifying with macroporous adsorbent resin HPD-100A. Firstly, pre-treating HPD-100A macroporous resin: dynamic eluting with 95% ethanol at 4BV (bed volume), passing through the column at 2BV/h (if bubbles are generated, the bubbles are removed), rinsing the resin with distilled water at 2BV/h until the effluent liquid has no obvious ethanol smell in the beaker, and keeping the water level above the resin layer to avoid drying the column. Taking a part of pretreated HPD-100A resin, carrying out static adsorption on the supernatant for 12h, then carrying out dynamic continuous elution by using 2BV of deionized water, 2BV of 5% ethanol, 2BV of 10% ethanol and 5BV of 20% ethanol, wherein the elution flow rate is 1BV/h, collecting 20% ethanol eluent, and carrying out rotary evaporation at 50 ℃ until all ethanol is removed. Then freeze-drying to obtain powder, namely the gardenia dreg water extract, and putting the gardenia dreg water extract into a refrigerator at the temperature of 20 ℃ below zero for later use.

(2) Preparation of alcohol extract of gardenia dregs

Grinding fructus Gardeniae dregs, and sieving with 60 mesh sieve. Weighing 40g of gardenia meal powder, adding 400mL of 60% ethanol, leaching for 8h at room temperature, filtering and collecting supernatant. And adding 200mL of 60% ethanol into the filter residue, leaching for 8h, and filtering to collect supernatant. A third addition was made of 100mL of 60% ethanol. All filtrates were mixed, centrifuged at 4000rpm for 10min, the supernatant collected and then rotary evaporated at 50 ℃ until all ethanol was removed. Purifying with macroporous adsorbent resin HPD-100A. Taking a part of pretreated HPD-100A resin, carrying out static adsorption on the supernatant for 12h, then carrying out dynamic continuous elution by using 2BV of deionized water, 2BV of 5% ethanol, 2BV of 10% ethanol and 5BV of 20% ethanol, wherein the elution flow rate is 1BV/h, collecting 20% ethanol eluent, and carrying out rotary evaporation at 50 ℃ until all ethanol is removed. Then freeze-drying to obtain powder, namely the gardenia dreg alcohol extract, and placing the gardenia dreg alcohol extract into a refrigerator at the temperature of-20 ℃ for later use.

The contents of geniposide in the aqueous extract and the alcohol extract of the gardenia dregs are respectively 674mg/g and 421mg/g through high performance liquid chromatography. The chromatographic conditions were as follows:

c18 column (4.6 mm. times.250 mm, 5 μm); mobile phase: methanol: 0.05% phosphoric acid water solution (gradient elution 0-20min:10: 90; 20-30min: 50: 50; 30-40min:20: 80; 40-45min:10:90) at 30 deg.C; the detection wavelength is 238 nm; the flow rate is 1.0 mL/min; the amount of the sample was 10. mu.L. Geniposide standards were purchased from Sigma.

1.2 cell culture and transfection

The myoblasts of the C2C12 mouse were placed at 37 ℃ in 5% CO₂Cultured in a DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% double antibody (P/S). When the C2C12 cells and primary myoblasts were induced to differentiate into myofibers, the FBS content in the cell culture medium was replaced with 2% Horse Serum (HS).

C2C12 cells were treated as follows: (1) stimulating the cells with a certain concentration of aqueous extract (112.4mg/mL) and alcoholic extract (154.0mg/mL) for 12h respectively after the C2C12 cells are differentiated on the 4 th day, and adding DMSO into a blank control; (2) stimulating the cells with geniposide of 0,0.05,0.1,0.2,0.4,0.8mg/mL for 12h respectively; (3) stimulating the cells with 0.4mg/mL geniposide for 0, 1, 2, 4, 8 and 12 hours respectively; (4) stimulating cells with 20ng/mL TGF-beta, and stimulating with 0.4mg/mL geniposide for 12 h; (5) cell transfection was performed with Lipofectamine LTX and Plus reagents, and plasmid Smad4 was transferred into the cells, which were then stimulated with 0.4mg/mL geniposide for 24 h.

1.3 animal experiments

The 2-3 week males used in the experiment were housed in SPF (specific pathogen free) grade animal houses, where the animals had free access to food and water (provided by the Shanghai Slek laboratory animal center). The animal room is kept at a constant temperature of 22 +/-3 ℃ and a relative humidity of 35 +/-5%, and 12 hours is a periodic day-night cycle. The use and handling of model animals were performed strictly in accordance with the protocols of the animal care and use committee of the institute of food science and technology, university of south China.

After the males had acclimatized to the new environment, they were randomly assigned to seven groups. One group of mice was used as a blank control, and neither treatment by crushing nor injection of geniposide was performed, and six groups were used to establish acute injury models of gastrocnemius: after anesthesia by intraperitoneal injection of chloral hydrate (0.7ml/100g), the left and right hind limbs of the mouse were fixed on a flat plate, respectively, and the hind limb muscle of the mouse was injured by pounding 15g of a stainless steel ball (diameter 2cm) from a height of fixed 1m, twice in succession. Three groups of the drug were injected with geniposide standard (25mg/kg) per day according to body weight, and the other three blank controls were not administered for 21 consecutive days. On days 7, 12, and 21 of geniposide treatment, one group of drug-treated groups and one blank group were sacrificed, wherein all mice were sacrificed at 21 days, gastrocnemius muscles, soleus muscles, and tibialis anterior muscles in the left and right hind limbs were taken, the right leg gastrocnemius muscles were fixed in 10% formalin for omics analysis, and the remaining tissues were immediately frozen with liquid nitrogen and stored at-80 ℃.

1.4 immunoblot analysis (western blot)

Expression of Col I, Vim, α -SMA and Smad4 in harvested C2C12 cells and gastrocnemius tissue was assayed according to standard protocols.

1.5RNA extraction and real-time quantitative fluorescent PCR

Total RNA was extracted from C2C12 cell and tissue samples using Trizol reagent and RNA concentration was determined using Nanodrop. First strand cDNA was synthesized using Prime Script RT System (Takara). Real-time quantitative PCR was performed on the ABISTEP-ONE7900RT-PCR system. Three replicates were made for each sample. The specific primer sequences used are shown in the table below.

1.6 omics analysis

Mouse GAS tissue was fixed in 10% formalin for 72h, then GAS was dehydrated by an alcohol gradient (30-100%), washed, finally embedded in paraffin, and then vertically cut into muscle fiber sections with a thickness of 5 μm using a microtome. GAS samples harvested 7, 14 and 21 days post-contusion were observed using Hematoxylin and Eosin (HE) and Sirius Red (Sirius Red) staining. GAS sample sections collected 4 days after electrotransfection were subjected to immunohistochemical staining using Col 1 antibody. The sections were observed with an inverted optical microscope.

2 results of the experiment

2.1 the crude extract of fructus Gardeniae dregs has inhibitory effect on the fibrosis process in C2C12 myocyte

As can be seen from the protein and mRNA levels of fig. 3, treatment of C2C12 cells with aqueous and alcoholic extracts significantly reduced the expression of the profibrotic genes Col I, Vim and α -SMA compared to the blank control.

2.2 geniposide can inhibit the process of fibrosis of C2C12 myotube cells

As shown in FIGS. 4A-D, FIGS. 5A-D, expression of the profibrotic genes Col I, Vim and α -SMA in C2C12 myoblasts was significantly reduced compared to the blank control, and the expression levels of Col I, Vim and α -SMA were dose-and time-dependent decreased under different concentrations and different times of stimulation with geniposide standard.

2.3 geniposide can alleviate skeletal muscle fibrosis

We further validated the therapeutic effect of geniposide on skeletal muscle injured mice. As can be seen in FIGS. 6A-C, after acute skeletal muscle contusion, the cell morphology was severely disrupted. As shown in FIGS. 6D-F, the geniposide-treated group showed better cell morphology, reduced collagen fiber and ColI contents faster than the untreated group, and significantly improved skeletal muscle injury. It can also be seen from the protein and mRNA levels (FIGS. 6G-F) that the expression of Col I, Vim and α -SMA in the tissues was also significantly reduced by geniposide.

2.4 geniposide may play an anti-fibrotic role in skeletal muscle through the TGF-beta/Smad 4 signaling pathway

TGF-beta was used to stimulate C2C12 myoblasts, the expression of Col I, Vim and alpha-SMA was significantly increased (FIGS. 7A-D), and the expression of these profibrosis genes was significantly decreased after stimulation with geniposide, which was shown to inhibit the progress of fibrosis induced by TGF-beta. As can be seen from fig. 7E-F, stimulation of TGF- β significantly increased the expression of its downstream receptor kinase Smad4 compared to the blank, while stimulation of geniposide decreased the expression of Smad4, and the anti-fibrotic effect of geniposide may be correlated with the TGF- β/Samd4 signaling pathway. From E-F in fig. 4, and E-F in fig. 5, it can be seen that geniposide stimulated C2C12 cells, Smad4 expression at both mRNA and protein levels was dose-and time-dependent decreased, and in addition, geniposide treatment also significantly decreased Smad4 expression in vivo experiments (fig. 6I). Subsequently, stimulation of C2C12 cells with Smad4 increased protein and mRNA level expression of Smad4, Col I, α -SMA and Vim, while stimulation with geniposide decreased Smad4, Col I, α -SMA and Vim expression in cells (FIGS. 8A-B). In addition, qPCR levels showed that geniposide treatment rescued the anti-fibrotic phenotype (fig. 8B).

SEQUENCE LISTING

<110> university of south of the Yangtze river

Application of <120> geniposide in relieving skeletal muscle fibrosis

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Claims (6)

1. Use of geniposide in the manufacture of a medicament for alleviating skeletal muscle fibrosis, wherein the skeletal muscle fibrosis is a process in which muscle fibers exhibit persistent degeneration, necrosis, regeneration, inflammation or/and massive deposition of extracellular matrix that hinder remodeling of tissues when skeletal muscle undergoes acute trauma for regenerative repair.

2. The use of claim 1, wherein the relief is a decrease in the expression of the fibrosis promoting genes collagen I (Col I), Vimetin (Vim), alpha-smoothened muscle actin (alpha-SMA) and Smad4 in fibroblasts and damaged skeletal muscle gastrocnemius tissue at a dose and for a time frame that also decreases the level of collagen fibers and Col I in skeletal muscle cells that are increased by damage.

3. The use according to claim 1, wherein the sources of geniposide comprise: fructus Gardeniae dregs, fructus Gardeniae, Eucommiae cortex, herba Hedyotidis Diffusae, and Coptidis rhizoma detoxicating decoction.

4. The use of claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients comprising solvents, propellants, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, sequestering agents, permeation enhancers, buffers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, flocculants and deflocculants, filter aids, release retardants.

5. Use according to claim 1, wherein the medicament is an aerosol or a dressing.

6. Application of geniposide in preparing medicine for treating or removing scar tissue caused by skeletal muscle fibrosis.

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