Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle

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Bioactive Constituents, Metabolites, and Functions

Effects of geniposide from Gardenia fruit pomace on skeletal muscle fibrosis Haiou Pan, Yan Li, Hai feng Qian, Xiguang Qi, Gangcheng Wu, Hui Zhang, Meijuan Xu, Zhiming Rao, Jinlong Li, Li Wang, and Hao Ying J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00739 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Journal of Agricultural and Food Chemistry

Effects of geniposide from Gardenia fruit pomace on skeletal muscle fibrosis Haiou Pan†, #, Yan Li†, ‡, #, Haifeng Qian†, Xiguang Qi†, Gangcheng Wu†, Hui Zhang†, Meijuan Xu§, Zhiming Rao§, Jin-long Li∥, Li Wang†, *, Hao Ying‡, *

†

State Key Laboratory of Food Science and Technology, School of Food Science and

Technology, Jiangnan University, Wuxi 214122, China; ‡

Key Laboratory of Food Safety Research, Institute for Nutritional Sciences,

Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China §

The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory

of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China ∥

School of Pharmacy, Nantong University, Nantong 226001,China

*

Correspondence: [email protected]; [email protected]

#

These authors contribute equally to this work

1

ACS Paragon Plus Environment


Abstract Geniposide is the main bioactive constituent of Gardenia fruit. Skeletal muscle fibrosis is a common and irreversible damage process. Numerous studies have shown that geniposide could improve many chronic diseases including metabolic syndrome and tumor. However, the effects of geniposide on skeletal muscle fibrosis are still poorly understood. Here, we found that crude extracts from Gardenia fruit pomace could significantly decrease the expressions of pro-fibrotic genes in vitro. Moreover, geniposide could also reverse pro-fibrotic gene expression induced by TGF-β and Smad4, a regulator of skeletal muscle fibrosis. In addition, geniposide treatment could significantly downregulate pro-fibrotic gene expression, and improve skeletal muscle injury in a mouse model of contusion. These results together suggest that geniposide has an anti-fibrotic effect on skeletal muscle through suppression of TGF-β/Smad4 signaling pathway. Keywords: Gardenia fruit pomace; geniposide; skeletal muscle fibrosis; TGF-β; Smad4

2


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Introduction Skeletal muscle is a versatile tissue of which motor injury during exercise is common in sports and can be triggered by contusion, stretching, laceration and strain. It is mentioned that regardless of the type and mechanism of injury, the related morphological, biochemical changes and so on are similar.1 The healing process following skeletal muscle injury is complex, including 3 phases: inflammation, regeneration, and fibrosis.2 Acute traumatic injuries, such as contusion cause the rupture of large myofiber bundles then going on muscle regeneration and formation of new myotendinous junctions and connective tissue scar.3 The occurrence of fibrosis, forming scar tissue, is due to the proliferation and activation of myofibroblasts which secrete extracellular matrix (ECM) inducing abnormal and exaggerated deposition in muscular tissue and finally impairs the healing process and predisposes the muscle to recurrent injury.4 Skeletal muscle fibrosis not only makes human hypokinetic, but is a critical important inducement of some muscle deseases (such as Duchenne muscular dystrophy) and affects various tissues or organs.5 Many therapies, such as rest, ice, low-level laser therapy, anti-inflammatory and anti-apoptosis medications are available or developed against this condition at present, which have a limited efficacy in preventing or treating the formation of posttraumatic muscle fibrosis.6-8 Therefore, the development of new therapeutic strategies for the treatment of skeletal muscle fibrosis is urgent. TGF-β, myostatin, connective tissue growth factor (CTGF/CCN2), Notch and Wnt signaling pathways have often been investigated in regulating the occurrence of 3



fibrosis.2,

9-11

Currently, studies show that mechanism on some therapeutic agents

(such as telmisartan, lipoxin A4, sauchinone) is related to block TGF-β signaling pathway.12-14 TGF-β, one of the best studied molecules, is a critical pro-fibrotic cytokine, which facilitates the synthesis of ECM components, such as collagens, laminin and glycoprotein, as well as inhibits the degradation of ECM.15 Besides, the fibrotic effect of TGF-β is achieved mainly through Smads family in the process of healing muscle injury, which are downstream receptor kinases that mediate intracellular signalling of the TGF-β superfamily.2 So far, there are eight Smads family members found in mammals and divided into three groups in terms of their structures and functions: 1) the receptor-regulated Smads (R-Smads) including Smad1, Smad2, Smad3, Smad5, and Smad8; 2) the common Smad (Co-Smad) including only one member, Smad4; 3) the inhibitory Smads (I-Smads) including Smad6 and Smad7.16 Smad4, the common partner for all R-Smads, serves as a pivotal link in the signaling pathway of the TGF-β superfamily, which plays a crucial role in the pathological development of fibrosis.17 TGF-β can up-regulate the expressions of pro-fibrotic genes such as vimentin (Vim), collagen (includes COL1A1, Col I) and α-smooth muscle actin (α-SMA).15,

18

Therefore, searching for the potential and

effective drugs or agents from natural plants to treat injured skeletal muscle will be a valuable exploring direction. Gardenia jasminoides Ellis (G. jasminoides, family Rubiaceae) is distributed widely in many Asian countries. The mature fruit of G. jasminoides, Gardenia fruit, is one of the widely used Chinese herbals and also can be used as edible resource such 4


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as oil, natural food colorant (Gardenia yellow, red) in food dyeing field (noodles, confectioneries, wine, pickles), etc.19 The major bioactive constituents in G. jasminoides are mainly iridoids (such as geniposide), carotenoids (such as crocin, crocetin and its derivatives), flavonoids, organic acids, sterols, volatile oil, polysaccharide and others.20 A large number of research has shown that the efficacy of Gardenia fruit includes anti-inflammatory, anti-diabetic, neuroprotective, analgesic, etc.19-21 More than 20 kinds of iridoid compounds such as geniposide and gardenoside have been separated in Gardenia fruit, and many studies have found multiple positive functional activities of geniposide.19, 20 Geniposide exhibits beneficial effects on many aspects including anti-inflammatory, hepato-protective and hypoglycemic properties and neuroprotection.22-25 Many researchers have studied the anti-inflammatory property of geniposide from G. jasminoides. Geniposide could play anti-inflammatory and anti-apoptotic roles in decreasing the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17

and Bax and increasing the levels of

anti-inflammatory cytokines (IL-4, TGF-β1) and Bcl-2.26-28 Moreover, geniposide could

alleviate

hepatic

fibrosis

by

inhibiting

the

TGF-β/Smad

and

ERK-mitogen-activated protein kinase (MAPK) signaling pathways.29 Therefore, it’s potential that geniposide might suppress skeletal muscle fibrosis. However, studies on the anti-fibrotic effect of geniposide or crude extracts from Gardenia fruit and the underlying mechanisms of anti-fibrotic effect have not been performed. In this study, in order to make the best of resource, the material was Gardenia fruit pomace, from which we could obtain crude extracts mainly containing 5



geniposide. We aimed to analyze whether the aqueous extract and ethanol extract from Gardenia fruit pomace and geniposide played an inhibitory role in the fibrotic process using C2C12 myotubes and mice model with injured skeletal muscle caused by acute contusion assays. Then based on the in vitro and in vivo results, our conjecture of the anti-fibrotic effect of crude extracts from Gardenia fruit pomace and standard material geniposide would be tested. Furthermore, the underlying mechanism was also explored and we speculated that geniposide was likely to achieve the effect of reducing muscle fibrosis by inhibiting the TGF-β/Smad4 signaling pathway. Taken together, our studies reinforce the notion that the anti-fibrotic effect of geniposide is beneficial in skeletal muscle, thus, constitutes a promising approach to combat skeletal muscle fibrosis. Methods Preparation of crude extract of Gardenia fruit pomace Gardenia fruit pomace was kindly provided by Professor Youzuo Zhang of Agricultural and Food Science College of Zhejiang A&F University, which is original from Lin’an, ZhengJiang province, China. We milled the pomace and sieved it through a 60 mesh screen before use. Soaked powders using water bath at 60°C for three times. All filtrate was combined, cooled and centrifuged at 4000 rpm for 10 min. Supernatant was collected and isolated using HPD-100A macroporous resin column chromatography. Successive elutions used water, 5% ethanol, 10% ethanol with 2 BV (BV=bed volume) and 20% ethanol with 5 BV (elution velocity=1 BV/h). 20% ethanol eluent was collected and then concentrated by a rotary evaporator at 50°C 6


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until ethanol evaporating totally. The concentrated solution was freeze-dried into a powder which was stored at -20°C for further use, then the aqueous extract had been prepared. Then we soaked powders of Gardenia fruit pomace in 60% ethanol with stirring at room temperature for 8 h twice to prepare the alcohol extract. The following procedures of purification were the same with the preparation of aqueous extract. After freeze-drying, the alcohol extract was prepared and stored at -20°C. HPLC analysis Geniposide standard solution was prepared with concentration of 246 µg/mL, and was dissolved in methanol. The aqueous extract and alcohol extract were weighed exactly dissolving in methanol, diluted and then we shaked the solutions up. Then centrifuged them at 4000 rpm for 10 min and collected the supernatants. The standard and sample solutions were filtrated by 0.45 µm microfiltration membrane prior to HPLC analysis. Chromatographic conditions: Octadecyl silane chemically bonded silica was used as bulking agent (4.6 mm×250 mm, 5 µm), and the column temperature was maintained at 30°C. The analysis was achieved with gradient elution using (A) 0.05% phosphoric acid in water and (B) methanol as the mobile phase. The gradient applied was as follows: 0-20 min 90% A and 10% B, 20-30 min to 50% A, 30-40 min to 80% A, and 40-50 min 90% A and 10% B (v/v). Flow rate was 1.0 mL/min and injection volume was 10 µL. The effluent was monitored with a UV detector at 238 nm used for quantification of geniposide. Under these conditions, the geniposide was separated at the baseline and identified by comparing its retention time with the corresponding peak in the standard solution. 7



Animals experiments Male mice between 2 and 3 weeks used in this study were obtained from Shanghai Experimental Animal Center, which were maintained at a temperature of 23±3°C and a humidity of 35±5% under a 12-h dark/light cycle (lights on at 06:30 a.m.) in a specific pathogen-free animal facility. This study’s procedures and protocols were approved by the Animal Care and Use Committee of the School of Food Science and Technology, Jiangnan University (No. JN 2013-4). After conforming with new environment, male mice were distributed in 7 groups randomly. One group was served as control and other groups were needed to build an acute contusion model of the gastrocnemius (GAS). The model was constructed according to the previous method.2 In brief, after anesthetization by intraperitoneal (i.p.) injection with chloral hydrate (0.7 ml/100 g), both left and right hindlimbs of the mouse were fixed on a plate respectively, GAS was exposed and a stainless steel ball (2 cm in diameter) with a weight of 15 g was released at a height of 1 m as an impactor on the target muscle twice. Mice received geniposide (Sigma Aldrich #SML0153) of 25 mg/kg/d body weight through i.p. for 3 weeks (n=3 per group) as test groups. Mice were maintained in a comprehensive lab animal monitoring system (CLAMS, Columbus Instruments, Columbus, OH) for 24 h to allow mice to adapt to this environment and motor activity and food intake were continuously recorded during the next 24 h as described previously. Mice from test and control groups were sacrificed on days 7, 14 and 21, respectively, and after operation, all mice were sacrificed 21 days after contusion, and then both left and right GAS, tibialis anterior (TA), soleus (SOL) were isolated. Lean 8


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mass was measured using a NMR Analyzer (Bruker, Milton, ON, Canada). The right GAS was fixed in neutralized 10% formalin for histological study, other tissues were frozen in lipid nitrogen and stored at -80°C until future analysis. Cell culture and transfection The mouse C2C12 myoblast cells were maintained at 37°C and 5% CO2 in DMEM (Gibco, Thermo Fisher Scientific) containing 10% FBS (Gibco, Thermo Fisher Scientific) and 1% P/S (Gibco, Thermo Fisher Scientific). When C2C12 myoblasts reached 80-90% confluence, cells were differentiated into myotubes in differentiation medium, consisting of DMEM containing 2% Horse serum (Gibco, Thermo Fisher Scientific), and 1% P/S. To determine the effects of geniposide in C2C12 cells which have been differentiated for 4 d, several ways of stimulating C2C12 cells plated in 6-well plates were as follows. i) The cells were treated with the aqueous extract (112.4 mg/mL) and alcohol extract (154.0 mg/mL) at a certain volume for 12h. ii) Aqueous extract, alcohol extract, geniposide stimulated C2C12 cells separately at the geniposide’s concentration of 0.2, 0.4, 0.8 mg/mL for 12 h. iii) Cells were treated with geniposide at the concentration gradient of 0, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL for 12 h and for time gradient of 0, 1, 2, 4, 8, 12 h at 0.4 mg/mL. iv) 20 ng/ml TGF-β (Santa cruz) stimulated the cells individually and costimulated cells with 0.4 mg/mL geniposide for 12 h. v) Upon cells reaching 50-60% confluence, plasmid pcDNA3.1 and Smad4 transfection in C2C12 cells was performed using Lipofectamine LTX and Plus Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instruction.30 After cell transfection for 6 h, C2C12 cells were moved 9



to differentiation medium. then cells were treated with geniposide (0.4 mg/mL) at 24 h and harvested at 48 h after transfection. Hoechst 33258 (Sigma Aldrich) (0.5 µg/mL) was used to analyze cytotoxicity of geniposide in C2C12 cells at the concentration gradient of 0, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL for 12 h. DMSO was used as a control. Western blot Western blot analysis was performed as previously described.31 In brief, the culture cells were harvested, lysed with lysis buffer and the frozen tissue samples were lysed in RIPA lysis buffer (Beyotime Biotechnology). Equal amounts of protein were separated by SDS-PAGE and subsequently transferred on to PVDF membranes according to standard protocol. Antibodies included Anti-Col I (Abcam), anti-Vim (Abcam), anti-α-SMA (Abcam), anti-Smad4 (Cell Signaling), and anti-GAPDH (Kang Chen, Shanghai, China) were used as primary antibodies. RNA isolation and quantitative real-time PCR analysis Trizol reagent (Invitrogen) was used to extract total RNA from C2C12 cells and derived tissue samples and the RNAs were quantified by Nanodrop (Thermo Scientific, Waltham, USA). First strand cDNA was synthesized using the Prime Script RT system (Takara). Quantitative real-time PCR (RT-qPCR) was performed on an ABI 7900 RT-PCR system (Applied Biosystem). Each sample was run in triplicate. The specific primer sequences were shown in Table 1 and the expression of 18s was used as an internal control. Histological analysis The tissues of right GAS were fixed in neutralized 10% formalin for 72 h and 10


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were subsequently dehydrated through an alcohol gradient (30%–100%), cleaned, and finally embedded in paraffin wax, and then sections were made perpendicular to the direction of the muscle fiber with 5 µm in thickness using a microtome (SM2500; Leica Microsystems, Wetzlar, Germany). The samples harvested immediately and harvested at 7, 14, and 21 days after contusion were observed using hematoxylin and eosin (HE) and Sirius Red staining. Sample sections collected 4 days after electroporation transfection were observed by immunohistochemical stain using Col I antibody (Sigma). These sections were visualized by inverted light microscopy (IX71SBF2; Olympus Corporation, Tokyo, Japan) with DP72 Manager (Olympus Corporation) to capture digital images.30 Statistical analysis Data were shown as the mean ± standard error of the mean (SEM) from samples collected in triplicate. To detect the difference between two groups, Student’s t-test was used. To analyze the difference between three groups, one-way ANOVA was used. The significance was presented as *, P < 0.05, **, P < 0.01, and ***, P < 0.005. An insignificant difference was expressed as NS. GraphPad Prism 5.0 (GraphPad Software, La Jolla, USA) was used for all statistical analysis. Results The extracts from Gardenia fruit pomace play an inhibitory role in the fibrotic process in vitro

To study the effect of Gardenia fruit pomace on the fibrotic process, Gardenia fruit pomace was extracted and purified with two extractive methods and the aqueous 11



extract and ethanol extract were prepared. Then we determined the content of geniposide in non processed Gardenia fruit pomace, aqueous extract and ethanol extract from Gardenia fruit pomace. Figure 1A showed the retention time and peak area of standard material geniposide, and as shown in Figure 1B-D, the retention time of the main peak of the solution was consistent with Figure 1A. The content of geniposide (its structure was shown in Figure 2) from aqueous and ethanol extracts was 674 and 421 mg/g, respectively, while the content of geniposide in unprocessed Gardenia fruit pomace was 68.2 mg/g. After treatment of the two extracts in C2C12 myotubes, a significant decrease of Col I, Vim and α-SMA at both protein (Figure 3A) and mRNA (Figure 3B-D) levels were noticed compared with control. These data exhibited an inhibitory role of the extracts from Gardenia fruit pomace in fibrosis process in C2C12 myotubes. As the content of geniposide in the extracts was high, we wondered whether geniposide mainly played an anti-fibrotic role in C2C12 myotubes. As shown in Figure 4A, Col I, α-SMA and Vim were determined to compare the anti-fibrotic effect of aqueous, ethanol extracts and single chemical geniposide by stimulating C2C12 myotubes (the concentration of geniposide was 0.4 mg/mL). Data showed that geniposide also obviously decreased the pro-fibrotic genes (particularly Col I) compared with two extracts. And in RT-qPCR analysis (Figure 3B-C), expressions of Col Ⅰ and Vim were lowest with the treatment of geniposide. What’s more, inflammatory factors TNF-α and IL-6 were also determined at mRNA level (Figure 3D-E). With the treatment of aqueous, ethanol extracts and geniposide, the expression of IL-6 was significantly decreased but TNF-α had no obvious change 12


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compared with control, which couldn’t fully explain the anti-inflammatory property of extracts and geniposide during fibrotic process in C2C12 myotubes. Thus, we speculated that geniposide could suppress fibrotic process. Geniposide plays an inhibitory role in the fibrotic process in vitro Firstly, cytotoxicity of geniposide in C2C12 myotubes was carried out by Hoechst. As shown in Figure S1, the amount of C2C12 myotubes treated with 0.4 mg/mL geniposide had no significant difference compared with control. Then we focused on exploring whether geniposide of Gardenia fruit pomace worked on reducing skeletal muscle fibrosis. Then we used geniposide stimulating C2C12 myotubes to verify the effect. C2C12 myotubes were treated with different concentrations of geniposide for different time. As expected, some changes of the expressions of α-SMA, Vim, and Col I at both mRNA and protein levels in response to geniposide treatment were found. In concentration gradients, with the treatment of geniposide, the mRNA expressions of α-SMA, Vim and Col I were significantly decreased in a dose-dependent manner compared with control (Figure 5A-C). Besides, the protein levels of α-SMA, Vim and Col I presented alike downtrend (Figure 5D). In time gradients (Figure 6A-D), with the treatment of 0.4 mg/mL geniposide for 12 h, the expressions of α-SMA, Vim and Col I reached the lowest at both mRNA and protein levels. These data suggested that geniposide reduced the degree of fibrosis in muscle cells. Geniposide mitigates skeletal muscle fibrosis in vivo We further verified the anti-fibrosis effect of geniposide in vivo. Acute contusion 13



induced the accompanying occurrence of skeletal muscle fibrosis, we analyzed mice’s motor activity, food intake, lean mass, muscle weight and histological results. As shown in Figure S2A-D, there was no significant difference of animal state and muscle weight with and without geniposide. The morphology of normal skeletal muscle cell is complete and nucleus is close to the edge of the cell. After skeletal muscle injury, HE staining (Figure 7A) showed that cell morphology was destroyed seriously. Sirius Red staining and immunohistochemical (Col I) staining (Figure 7B-C) displayed that there was no formation of collagen fibers and Col I before injury but collagen fibers and Col I deposition were highly increased after injury. With time prolonged (after 21 d), condition of skeletal muscle fibrosis was improved including recovered cell morphology, significantly reduced amounts of collagen fibers and Col I. As shown in Figure 7D-F, compared with normal skeletal muscle, the treatment of geniposide exhibited a positive effect on injured skeletal muscle in which cell morphology was much better and contents of collagen fibers and Col I were lower at same point. Moreover, the protein and mRNA levels of α-SMA, Vim and Col I were found to be obviously decreased by geniposide injection compared with control (Figure 7G-H). Taken together, these results proved that geniposide mediated anti-fibrosis effect on injured skeletal muscle. Geniposide attenuates TGF-β-induced fibrotic process To test whether the effect of geniposide on fibrotic process was dependent on TGF-β, TGF-β was used to stimulate C2C12 myotubes, which activated fibrotic process. The expressions of α-SMA, Vim, and Col I at both mRNA and protein levels 14


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were significantly higher in C2C12 myotubes treated with TGF-β in the absence of geniposide. While concomitant geniposide treatment obviously lowered both levels of α-SMA, Vim, and Col I in C2C12 myotubes exposed to TGF-β (Figure 8A-D), which indicated that the anti-fibrotic role of geniposide might be based on the interference with the TGF-β-related pathway. Down-regulated Smad4 is required for the anti-fibrotic effect of geniposide We continued to detect mRNA and protein levels of Smad4, obtained similar results as mentioned above (Figure 8E-F) and hypothesized that targeting TGF-β mediated Smad4 Signaling in the process of skeletal muscle fibrosis. Whether the mechanism performed through TGF-β/Smad4 signaling pathway was investigated. As shown in Figure 3F-G, we observed that aqueous, ethanol extracts and geniposide decreased the expression of Smad4 in C2C12 myotubes and among these treatments, geniposide made Smad4 reach a lowest point. Then it was found that the mRNA and protein levels of Smad4 significantly decreased in C2C12 myotubes treated with geniposide in a dose- and time-dependent manner, respectively (Figure 5&6E-F). In addition, similar in vivo results were obtained from GAS muscle of mice with injured skeletal muscle at protein levels (Figure 7I). As geniposide modulated the expression of TGF-β/Smad4 axis, we hypothesized that whether the effect of geniposide on skeletal muscle fibrosis was altered by Smad4 overexpression. As expected, Smad4 overexpression increased the protein levels of Smad4, Col I, α-SMA and Vim, while concomitant geniposide treatment decreased the protein levels of Smad4, Col I, α-SMA and Vim in C2C12 myotubes, suggesting that 15



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the altered expression of these pro-fibrotic genes was as a direct effect of de-repression via Smad4 (Figure. 9A). Besides, RT-qPCR analysis revealed that Smad4 overexpression reversed the anti-fibrotic phenotype of geniposide (Figure. 9B). In agreement with our hypothesis, these results collectively implied that geniposide inhibited skeletal muscle cell fibrosis through TGF-β/Smad4 signaling pathway. Discussion Fibrosis is involved in many organs, such as liver, heart, and the occurrence of fibrosis causes excessive production and deposition of ECM.32, 33 Skeletal muscle fibrosis compromises myofibre contractility, and the accumulation of myofibroblasts results in scar tissue formation and ultimately impairs issue, organ function and predisposes to further injury, and the treatment options for the condition remain severely limited.34 Recent years, studies have shown that some natural products alleviate fibrosis. For example, Ginkgo biloba extract mitigates liver fibrosis, Gualou Xiebai Decoction ameliorates myocardial fibrosis and so on.35,

36

As for skeletal

muscle, Sitziat et al. have found that dietary natural polyphenols could reduce muscle fibrosis deposition and myofiber necrosis in Duchenne muscular dystrophy model, and in Toshiyuki’s study immobilization-induced skeletal muscle fibrosis was attenuated by astaxanthin supplementation.37, 38 However, natural products for treating contusion induced muscle injury have not been reported yet. In the present study, we investigated the expressions of α-SMA, Vim, and Col I at both protein and mRNA levels in C2C12 myotubes treated with crude extracts from Gardenia fruit pomace and the results demonstrated that G. jasminoides has anti-fibrosis effect on muscle cells. 16


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Whether the main bioactive component of Gardenia fruit pomace played anti-fibrosis role and underlying mechanism needed to be further explored. The Gardenia fruit, which is widely used as a traditional Chinese, has been observed much efficacy such as antioxidant, anti-inflammatory, anti-depressant, neuroprotective activities. The constituents of Gardenia fruit include iridoids, iridoid glucosides, crocins, organic acids, flavonoids, volatile oil, and among them geniposide, crocin are major bioactive compounds.19 Nowadays, Gardenia fruit is applied to making tea, natural food colorant, food additive, squeezing into edible oil and so on.39, 40 After squeezing oil, the rest of gardenia fruit is Gardenia fruit pomace which is used for extracting geniposide, gardenia yellow and crocin yet but it has not been fully utilized.41-43 In the study, due to our improved purification method, the quantitative analysis of crude extracts by HPLC revealed the main presence of geniposide. Our results showed that the content of geniposide in aqueous extract and ethanol extract from Gardenia fruit pomace was 674 and 421 mg/g, respectively, which was much higher than other studies’ content of geniposide and Gardenia fruit pomace could be utilized fully. 44, 45 Geniposide is one of iridoids and is the dominant active component in Gardenia fruit, which was considered to be responsible for various biological effects of the herb. However, the inhibitory effect of geniposide on skeletal muscle fibrosis has never been investigated. We found that crude extracts from Gardenia fruit pomace exhibited anti-fibrotic role in vitro. Therefore, due to the high content of geniposide in crude extracts, we continued to investigate the expressions of α-SMA, Vim, and Col I at 17



protein and mRNA levels in C2C12 myotubes after the treatment of aqueous and ethanol extracts and single chemical geniposide. Results indicated that geniposide similarly decreased expressions of pro-fibrotic genes. We speculated that geniposide was responsive for main anti-fibrotic activity of Gardenia fruit. With the treatment of geniposide in C2C12 myotubes at concentration and time gradients and through testing the expressions of α-SMA, Vim, and Col I at both protein and mRNA levels, we observed that the anti-fibrotic effect of geniposde reached maximum benefit synthetically when the concentration of geniposide was 0.4 mg/mL and the time is 12 h. Besides, the result was further verified in vivo. We established acute contusion of skeletal muscle in mouse model and still tested the expressions of pro-fibrotic genes (α-SMA, Vim and Col I) at both protein and mRNA levels and conducted histological analysis. The results of treatment groups injected geniposide compared with control showed that geniposide had a positive effect on injured skeletal muscle. In a word, it was proved that geniposide indeed alleviated skeletal muscle fibrosis in vitro and in vivo. Next, we continued to explore the underlying mechanism of anti-fibrosis of geniposide. TGF-β, the ubiquitous pro-fibrotic cytokine, plays a key role in the pathogenesis of organ fibrosis, which promotes activation and differentiation of fibroblasts into myofibroblasts and enhances ECM proteins such as collagens and fibronectins (include Vim) synthesis via inducing Smad activation.2, 38 Smad4, the only identified Co-Smad in mammals, plays a very central role in the TGF-β/Smads signaling pathway.46, 47 Therefore, we aim to test whether the anti-fibrotic effect of geniposide is 18


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related to the TGF-β/Smad4 signaling pathway. In the current study, we found that when C2C12 myotubes exposed to TGF-β with the treatment of geniposide, the expressions of α-SMA, Vim, Col I, Smad4 at both protein and mRNA levels and TGF-β at mRNA level were obviously lowered compared with TGF-β-treated cells in the absence of geniposide. Besides, similar results in vivo were obtained from GAS muscle of mice with injured skeletal muscle. What’s more, we found that compared with control, the expressions of Col I, α-SMA and Smad4 at levels of protein and mRNA were obviously increased after transfected with Smad4. While C2C12 myotubes with the treatment of geniposide, these expressions clearly decreased, indicating that geniposide alleviated Smad4-stimulated fibrotic process. Accordingly, geniposide might function in skeletal muscle fibroblasts by inhibiting TGF-β/Smad4 signaling pathway. However, our study didn’t not discuss and explore the possibility of other mechanisms underlying the skeletal muscle fibrosis process and the geniposide might be related to other signaling pathways. Another possibility was that not only the geniposide had an anti-fibrotic effect, other components of iridoids might have a synergistic effect. What’s more, though in our study anti-inflammation of aqueous and ethanol extracts and geniposide was not obvious in C2C12 myotubes, we could focus on injured skeletal muscle to explore their anti-inflammation, which might contribute to healing process. Thus, we can pay more attention to continue thorough research. In summary, we demonstrated that the major bioactive component from G. jasminoides, geniposide, was able to reduce the fibrosis of injured skeletal muscle and 19



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the underlying mechanism might be involved an induction of the TGF-β/Smad4 signaling pathway. This study suggests that geniposide and Gardenia fruit may be promising strategies in the treatment of skeletal muscle fibrosis progression. Author information Corresponding Author Li Wang: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Lihu Avenue 1800, Wuxi 214122, China.

Phone:

86-510-85329099;

Fax:

86-510-85329099,

Email:

[email protected] Hao Ying: Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Rd., Shanghai 200031, China. Phone: 86-21-54920247; Fax: 86-21-54920291, E-mail: [email protected]. Funding This work was financially supported by the National Natural Science Foundation of China (grant 31471617, 31471679，31671890 and 31525012), the Fundamental Research Funds for the Central Universities (No. JUSRP51708A and No. JUSRP11842)

and

the

Ministry

of

Science

and

(2016YFA0500102 and 2016YFC1304905). Notes The authors declare no competing ﬁnancial interest. Abbreviations used DMEM dulbecco’s modified eagle’s medium 20


Techonology

of

China

Page 21 of 39


FBS fetal bovine serum P/S penicillin/streptomycin HS horse serum DMSO Dimethyl sulfoxide RIPA radio immunoprecipitation assay SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis PVDF polyvinylidene fluoride GAPDH glyceraldehyde-3-phosphate dehydrogenase

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Figure legends Figure 1. HPLC chromatogram of geniposide (A) HPLC chromatogram of geniposide in standard solution (246 µg/mL). (B) HPLC chromatogram of geniposide in non processed Gardenia fruit pomace. (C) HPLC chromatogram of aqueous extract of Gardenia fruit pomace. (D) HPLC chromatogram of ethanol extract of Gardenia fruit pomace. Figure 2. The structure of geniposide Figure 3. Inhibitory effects of extracts from Gardenia fruit pomace on fibrosis process in vitro (A) C2C12 myotubes were treated with the aqueous extract and the alcohol extract from Gardenia fruit pomace (the concentration of geniposide is 0.4 mg/mL) and 24


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DMSO was served as a control for 12 h. Protein levels of α-SMA, Col I and Vim were determined by western blot analysis. GAPDH was served as a loading control. (B-D) The effect of the aqueous extract and the alcohol extract from Gardenia fruit pomace (the concentration of geniposide is 0.4 mg/mL) on the mRNA expression of Col I, Vim and α-SMA was evaluated by RT-qPCR analysis in C2C12 myotubes. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). NS, not significant. Figure 4. Geniposide, main component of aqueous and alcohol extracts, might be responsible for mian anti-fibrotic activity (A, F) C2C12 myotubes were treated with aqueous, alcohol extracts and single chemical geniposide (the concentration of geniposide is all 0.4 mg/mL) and DMSO was served as a control for 12 h. Protein levels of Col I, α-SMA, Vim and Smad4 were determined by western blot analysis. GAPDH was served as a loading control. (B-E, G) The effect of aqueous, alcohol extracts and single chemical geniposide (the concentration of geniposide is all 0.4 mg/mL) on the mRNA expression of Col I, Vim, TNF-α, IL-6 and Smad4 was evaluated by RT-qPCR analysis in C2C12 myotubes. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). Geni, geniposide; NS, not significant.

Figure 5. Inhibitory effects of geniposide on fibrosis process in vitro in 25



concentration gradients (A-C, E) The relative mRNA levels of α-SMA, Vim, Col I and Smad4 were quantified in C2C12 myotubes with geniposide (0, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL) treatment for 12 h by RT-qPCR analysis. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). (D, F) C2C12 myotubes were treated with the indicated amounts of geniposide (0, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL) followed by western blotting of the protein expression of α-SMA, Col I, Vim and Smad4 for 12 h. GAPDH was served as a loading control. Figure 6. Inhibitory effects of geniposide on fibrosis process in vitro in time gradients (A-C, E) The relative mRNA levels of α-SMA, Vim, Col I and Smad4 were quantified in C2C12 myotubes with geniposide (0.4 mg/mL) treatment at different time points (0, 1, 2, 4, 8, 12 h) by RT-qPCR analysis. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). (D, F) C2C12 myotubes were treated with geniposide (0.4 mg/mL) for 0, 1, 2, 4, 8, 12 h and protein levels of α-SMA, Col I, Vim and Smad4 were determined by western blot analysis. GAPDH was served as a loading control. Figure 7. Geniposide could attenuate injured skeletal muscle fibrosis in vivo (A) Hematoxylin and eosin(HE) staining of GAS muscle of mice was performed at the indicated time points (0 d, 7 d, 14 d and 21 d) following acute contusion (n=3, 26


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20X). (B) Sirius Red staining of GAS muscle of mice was performed at the indicated time points (0 d, 7 d, 14 d and 21 d) following acute contusion (n=3, 20X). (C) immunohistochemical (Col I antibody) staining of GAS muscle of mice was performed at the indicated time points (0 d, 7 d, 14 d and 21 d) following acute contusion (n=3, 20X). (D) Hematoxylin and eosin(HE) staining of GAS muscle of mice was performed at the indicated time points (7 d, 14 d and 21 d) following acute contusion with or without geniposide (25 mg/kg/d body weight ) treatment (n=3, 20X). (E) Sirius Red staining of GAS muscle of mice was performed at the indicated time points (7 d, 14 d and 21 d) following acute contusion with or without geniposide (25 mg/kg/d body weight ) treatment (n=3, 20X). (F) immunohistochemical (Col I antibody) staining of GAS muscle of mice was performed at the indicated time points (7 d, 14 d and 21 d) following acute contusion with or without geniposide (25 mg/kg/d body weight ) treatment (n=3, 20X). (G, I) Western blot analysis of α-SMA, Col I, Vim and Smad4 was performed on protein lysates from TA muscle of mice following acute contusion with or without geniposide (25 mg/kg/d body weight) treatment at 14d (n=3). GAPDH was served as a loading control. (H) qPCR analysis of α-SMA, Vim and Col I was performed from GAS muscle of mice following acute contusion with or without geniposide (25 mg/kg/d body weight) treatment at 14 d (n=3). Con, control; Geni, Geniposide. Figure 8. Geniposide attenuates TGF-β-induced fibrotic process (A-C, E) RT-qPCR analysis of α-SMA, Col I, Vim and Smad4 mRNA expression in C2C12 myotubes treated with TGF-β (20 ng/ml) in the presence or absence of 27



geniposide (0.4 mg/mL) for 12 h. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). (D, F) C2C12 myotubes were treated with TGF-β (20 ng/ml) in the presence or absence of geniposide (0.4 mg/mL) and DMSO was served as a control for 12 h. Protein levels of α-SMA, Col I, Vim and Smad4 were assessed by western blot analysis. GAPDH was used as a loading control. Con, control; Geni, Geniposide. Figure 9. Anti-fibrosis effect of geniposide is affected by mediating Smad4 (A) Western blot analysis of Smad4, Col I, α-SMA, Vim and GAPDH protein expressions in C2C12 myotubes, which were treated with geniposide (0.4 mg/mL) after transfected with Smad4 or negative control. After Smad4 transfection, the expression of Smad4 protein was increased compared with control. (B) RT-qPCR analysis of Col I, α-SMA and Vim mRNA expression in C2C12 myotubes treated with geniposide (0.4 mg/mL) after transfection with Smad4 or not for 12 h. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). Geni, Geniposide.

Figure S1. Cytotoxicity of geniposide in C2C12 myotubes, related to Figure 3-6&8-9 (A) Differentiated state of C2C12 cells (0 d and 4 d). (B) Hoechst 33258 staining of C2C12 myotubes that were either control (DMSO) or exposed to 0.4 mg/mL geniposide for 12 h. Con, control; Geni, geniposide. 28


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Figure S2. Motor activity, food intake, lean mass and muscle weight of mice treated with geniposide, related to Figure 7 (A-B) Motor activity and food intake of mice treated with control (DMSO) or geniposide (25 mg/kg/d body weight) for 7 d following acute contusion (n=3). (C) Analysis of lean mass of mice received control (DMSO) or geniposide (25 mg/kg/d body weight) on day 21 by NMR technique (n=3). (D) The net weights of GAS in micc treated with control (DMSO) or geniposide (25 mg/kg/d body weight) on day 21 (n=3). Con, control; Geni, geniposide; NS, not significant.

29



TOC Graphic 63x47mm (96 x 96 DPI)


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Figure 1. HPLC chromatogram of geniposide (A) HPLC chromatogram of geniposide in standard solution (246 µg/mL). (B) HPLC chromatogram of geniposide in non processed Gardenia fruit pomace. (C) HPLC chromatogram of aqueous extract of Gardenia fruit pomace. (D) HPLC chromatogram of ethanol extract of Gardenia fruit pomace. 201x123mm (300 x 300 DPI)



Figure 2. The structure of geniposide 321x167mm (300 x 300 DPI)


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Figure 3. Inhibitory effects of extracts from Gardenia fruit pomace on fibrosis process in vitro (A) C2C12 myotubes were treated with the aqueous extract and the alcohol extract from Gardenia fruit pomace (the concentration of geniposide is 0.4 mg/mL) and DMSO was served as a control for 12 h. Protein levels of α-SMA, Col I and Vim were determined by western blot analysis. GAPDH was sevrvd as a loading control. (B-D) The effect of the aqueous extract and the alcohol extract from Gardenia fruit pomace (the concentration of geniposide is 0.4 mg/mL) on the mRNA expression of Col I, Vim and α-SMA was evaluated by RT-qPCR analysis in C2C12 myotubes. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). NS, not significant. 206x163mm (300 x 300 DPI)



Figure 4. Geniposide, main component of aqueous and alcohol extracts, might be responsible for mian antifibrotic activity (A, F) C2C12 myotubes were treated with aqueous, alcohol extracts and single chemical geniposide (the concentration of geniposide is all 0.4 mg/mL) and DMSO was served as a control for 12 h. Protein levels of Col I, α-SMA,Vim and Smad4 were determined by western blot analysis. GAPDH was sevrvd as a loading control. (B-E, G) The effect of aqueous, alcohol extracts and single chemical geniposide (the concentration of geniposide is all 0.4 mg/mL) on the mRNA expression of Col I, Vim, TNF-α, IL-6 and Smad4 was evaluated by RT-qPCR analysis in C2C12 myotubes. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). Geni, geniposide; NS, not significant. 192x196mm (300 x 300 DPI)


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Figure 5. Inhibitory effects of geniposide on fibrosis process in vitro in concentration gradients (A-C,E) The relative mRNA levels of α-SMA, Vim, Col I and Smad4 were quantified in C2C12 myotubes with geniposide (0, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL) treatment for 12 h by RT-qPCR analysis. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). (D, F) C2C12 myotubes were treated with the indicated amounts of geniposide (0, 0.05, 0.1, 0.2, 0.4, 0.8 mg/mL) followed by western blotting of the protein expression of α-SMA, Col I, Vim and Smad4 for 12 h. GAPDH was sevrvd as a loading control. 188x144mm (300 x 300 DPI)



Figure 6. Inhibitory effects of geniposide on fibrosis process in vitro in time gradients (A-C,E) The relative mRNA levels of α-SMA, Vim, Col I and Smad4 were quantified in C2C12 myotubes with geniposide (0.4 mg/mL) treatment at different time points (0, 1, 2, 4, 8, 12 h) by RT-qPCR analysis. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). (D,F) C2C12 myotubes were treated with geniposide (0.4 mg/mL) for 0, 1, 2, 4, 8, 12 h and protein levels of α-SMA, Col I, Vim and Smad4 were determined by western blot analysis. GAPDH was sevrvd as a loading control. 209x149mm (300 x 300 DPI)


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Figure 7. Geniposide could attentuate injured skeletal muscle fibrosis in vivo (A) Hematoxylin and eosin(HE) staining of GAS muscle of mice was performed at the indicated time points (0d, 7d, 14d and 21d) following acute contusion (n=3, 20X). (B) Sirius Red staining of GAS muscle of mice was performed at the indicated time points (0d, 7d, 14d and 21d) following acute contusion (n=3, 20X). (C) immunohistochemical (Col I antibody) staining of GAS muscle of mice was performed at the indicated time points (0d, 7d, 14d and 21d) following acute contusion (n=3, 20X). (D) Hematoxylin and eosin(HE) staining of GAS muscle of mice was performed at the indicated time points (7d, 14d and 21d) following acute contusion with or without geniposide (25 mg/kg/d body weight ) treatment (n=3, 20X). (E) Sirius Red staining of GAS muscle of mice was performed at the indicated time points (7d, 14d and 21d) following acute contusion with or without geniposide (25 mg/kg/d body weight ) treatment (n=3, 20X). (F) immunohistochemical (Col I antibody) staining of GAS muscle of mice was performed at the indicated time points (7d, 14d and 21d) following acute contusion with or without geniposide (25 mg/kg/d body weight ) treatment (n=3, 20X). (G,I) Western blot analysis of α-SMA, Col I, Vim and Smad4 was performed on protein lysates from TA muscle of mice following acute contusion with or without geniposide (25 mg/kg/d body weight) treatment at 14d (n=3). GAPDH was sevrvd as a loading control. (H) qPCR analysis of α-SMA, Vim and Col I was performed from GAS muscle of mice following acute contusion with or without geniposide (25 mg/kg/d body weight) treatment at 14d (n=3). Con, control; Geni, Geniposide. 205x164mm (300 x 300 DPI)



Figure 8. Geniposide attenuates TGF-β-induced fibrotic process (A-C,E) qPCR analysis of α-SMA, Col I, Vim and Smad4 mRNA expression in C2C12 myotubes treated with TGF-β (20 ng/ml) in the presence or absence of geniposide (0.4 mg/mL) for 12 h. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). (D,F) C2C12 myotubes were treated with TGF-β (20 ng/ml) in the presence or absence of geniposide (0.4 mg/mL) and DMSO was served as a control for 12 h. Protein levels of α-SMA, Col I, Vim and Smad4 were assessed by western blot analysis. GAPDH was used as a loading control. Con, control; Geni, Geniposide. 196x110mm (300 x 300 DPI)


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Figure 9. Anti-fibrosis effect of geniposide is affected by mediating Smad4 (A) Western blot analysis of Smad4, Col I, α-SMA, Vim and GAPDH protein expressions in C2C12 myotubes, which were treated with geniposide (0.4 mg/mL) after transfected with Smad4 or negative control. After transfection with Smad4, the expression of Smad4 protein was increased compared with control. (B) qPCR analysis of Col I, α-SMA and Vim mRNA expression in C2C12 myotubes treated with geniposide (0.4 mg/mL) after transfection with Smad4 or not for 12 h. Data are expressed as the mean ± SEM with three independent experiments for all panels. *P < 0.05 versus control, **P < 0.01 versus control, and ***P < 0.001 versus control (Student’s t-test). Geni, Geniposide. 193x114mm (300 x 300 DPI)


Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle

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