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Biomedicine & Pharmacotherapy

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The effects of Radix Angelica Sinensis and Radix Hedysari ultrafiltrationextract on X-irradiation-induced myocardial fibrosis in rats

Chengxu Maa, Zhaoyuan Fub, Huan Guoc, Huiping Weib, Xinke Zhaob, Yingdong Lia,⁎

a College of Integrated Traditional Chinese and Western Medicine, Gansu University of Chinese Medicine, Lanzhou 730000, ChinabAffiliated Hospital of Gansu University of Chinese Medicine, Lanzhou 730000, Chinac School of Basic Medical Sciences, Lan Zhou University, Lanzhou 730000, China

A R T I C L E I N F O

Keywords:Radix Angelica Sinensis and Radix Hedysariultrafiltration extractRadiation-induced myocardial fibrosisMicroRNA-21OsteopontinTranscription factor AP-1

A B S T R A C T

Radix Angelica Sinensis and Radix Hedysari are traditional Chinese medicines that are used for preventing andtreating various diseases. This study aimed to investigate the effect and possible underlying mechanisms of RadixAngelica Sinensis and Radix Hedysari ultrafiltration extract (RAS-RH) on X-irradiation-induced cardiac fibrosis inrats. Our data demonstrated that (a) a single dose of total body irradiation (TBI) at 8 Gy resulted in cardiacfibrosis, whereas the control hearts exhibited less collagen and fibrosis. RAS-RH mitigated these morphologicalinjuries. (b) TBI resulted in an increase in the serum levels of transforming growth factor β1 (TGF-β1) andtroponin-I (TnI). RAS-RH inhibited the release of TBI-induced serum TGF-β1 and the TnI levels. (c) TBI inhibitedthe apoptosis of primary rat cardiac fibroblasts, whereas RAS-RH induced the apoptosis of primary rat cardiacfibroblasts after X- irradiation. (d) TBI resulted in an increase in the expression of osteopontin (OPN), c-fos, c-jun, miRNA-21 and collagen1α (COL1α) in primary rat cardiac fibroblasts, and RAS-RH mitigated the TBI-induced increased expression of OPN, c-jun, miRNA-21 and COL1α. In conclusion, these results demonstrate thatRAS-RH exerts antifibrotic effects possibly through inducing the apoptosis of fibroblasts, inhibiting the release ofserum TGF-β1, reducing the levels of serum TnI and reducing the expression of OPN, c-jun, miRNA-21 andCOL1α. Therefore, RAS-RH may potentially be developed as a medical countermeasure for the mitigation ofradiation-induced myocardial fibrosis.

1. Introduction

Although radiotherapy has been used in the management of thor-acic malignancy and has led to a significant improvement in patientsurvival, this treatment has brought about a new range of cardiovas-cular disorders that are induced by radiation injury [1–3]. Radiation-induced heart disease (RIHD) is the leading cause of benign death inpatients who undergo thoracic radiation therapy, and this disease mayoccur until several years after the patient receives radiation. RIHDprimarily manifests as asymptomatic myocardial ischemia, pericarditis(both acute and chronic forms), coronary artery disease (acceleratedatherosclerosis), conduction abnormalities, valvulitis, myocarditis, andheart failure [4,5]. Radiation-induced myocardial fibrosis (RIMF) re-presents both acute and chronic stage RIHD based on pathology, andthis condition is thought to be a major risk factor for adverse myo-cardial remodeling and vascular changes. With the use of more con-formal regimens, heart toxicity associated with radiation therapy has

declined. However, certain risks must be carefully considered to reducemortality and morbidity of patient with thoracic malignancies. Thus,the development of a safe and effective drug for the prevention andtreatment of RIMF is essential.

RIMF is characterized by the proliferation of cardiac fibroblasts andthe excessive deposition of collagen and other extracellular matrix(ECM) components [6]. Moreover, RIMF is associated with a variety ofgrowth factors, proteolytic enzymes, angiogenic factors and fibrogeniccytokines [7]. Osteopontin (OPN) is a multifunctional cytokine that isinvolved in myofibroblast activation in cardiac fibrosis. Johan et al.showed that OPN is essential in the activation of AP-1(transcriptionfactor) and the subsequent transcription of miRNA-21 in cardiac fi-brosis [8]. Robert et al. reported that fibroblasts are statically non-conductive cell populations that secrete structural proteins, biochem-ical molecules and extracellular matrix proteins which form collagennetworks to maintain normal myocardial morphology [9]. In addition,Thum et al. reported that miRNA-21 regulates fibroblast survival and

https://doi.org/10.1016/j.biopha.2019.01.057Received 23 November 2018; Received in revised form 15 January 2019; Accepted 16 January 2019

⁎ Corresponding author at: College of Integrated Traditional Chinese and Western Medicine, Gansu University of Chinese Medicine, 35 Dingxi East Road, Lanzhou730000, China.

E-mail address: lydj412@163.com (Y. Li).

Biomedicine & Pharmacotherapy 112 (2019) 108596

0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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growth factor secretion, thereby promoting cardiac fibrosis [10].Traditional Chinese medicinal herbs have attracted increasing at-

tention for the prevention and treatment of RIMF. Radix AngelicaSinensis is one of the most commonly used traditional Chinese medicinalherbs and is reported to have multiple therapeutic effects, including theamelioration of inflammation, diabetes, and cardiovascular disorders[11]. It was also reported that Radix Angelica sinensis protects hema-topoietic stem/progenitor cells against X-ray-irradiation-induced aging[12]. Radix Hedysari is another frequently prescribed herb in traditionalChinese medicine and has been shown to play a vital role in anti-he-patic and anti-pulmonary fibrosis [13–15]. In addition, Zheng et al.demonstrated that the combination of Angelica sinensis and Radix As-tragali can achieve the maximal therapeutic effect in danggui buxuetang [16]. However, to the best of our knowledge, the effect of RadixAngelica Sinensis and Radix Hedysari ultrafiltration extract (RAS-RH) onRIMF has not been reported. In this study, we investigated the effectand possible underlying mechanisms of RAS-RH on X-irradiation-in-duced myocardial fibrosis in rats. Given that TBI may also occur outsideof the clinical setting, such as in a radiation accident, we used a ra-diation protocol that mimicked a nuclear accident [17–20].

2. Materials and methods

2.1. Reagents

Eagle’s Minimum Essential Medium (MEM) and fetal bovine serum(FBS) were obtained from Solarbio (Solarbio, Beijing, China). Trypsinwas obtained from Biotopped Life Sciences (Biotopped Life Sciences,Beijing, China). Collagenase II was obtained from Gibco (Gibco, Beijing,China). The Annexin V-FITC /PI Apoptosis Detection Kit was obtainedfrom Multisciences (Multisciences, Zhejiang, China). TRIzol reagentswere purchased from Ambion (Ambion, Carlsbad, CA). ThePrimeScript™ RT reagent kit and mRNA RT-PCR Detection Kit wereobtained from Promega (Promega, Beijing, China). The miRNA First-Strand cDNA Synthesis Kit and miRNA qRT-PCR Detection Kit wereobtained from Fulen Gene (Fulen Gene, Guangzhou, China). Rabbitanti-Col1α, rabbit anti-OPN, rabbit anti phospho-c-fos and rabbit anti c-fos were obtained from Abcam (Abcam, Cambridge, UK). Rabbit antiphospho-c-jun and rabbit anti c-jun were obtained from AffinityBiosciences (cell signaling technology, Beijing, China).

2.2. Plant materials

RAS-RH was used in the form of an ultrafiltration extract of thefollowing raw materials: Radix Angelica Sinensis (Umbelliferae, driedroot of Angelica sinensis (Oliv.) Diels, 400 g), Radix Hedysari(Leguminosae, dried root of Hedysarum polybotrys Hand.-Mazz,2000 g). Radix Angelica Sinensis and Radix Hedysari were obtained fromTasly Zhong Tian Pharmaceutical Company (Dingxi, China). Each plantmaterial was authenticated by Xicang Yang, Affiliated Hospital ofGansu University of Chinese Medicine (Lanzhou, China) according tothe methods of the Chinese Pharmacopoeia. RAS-RH was prepared bythe Gansu Academy of Membrane and Technology (Lanzhou, China);this institution has obtained the patenting rights (Patent No:CN200910021504.0, CN200910021505.5). The separation of the crudeextract was performed by members of our team using chromatographicmethods. The major active constituents therein were determined to beRadix Hedysari polysaccharides, Angelica ferulic acid, formononetin,astragaloside and astragalus polysaccharides [21]. The membrane usedwas a hollow fiber-type polyacrylonitrile (PNA) ultrafiltration mem-brane (Lanzhou, China). According to the manufacturer, the molecularweight cutoff (MWCO) was 100 kDa. RAS-RH was refined with waterdecoction at a pressure of 0.04 kPa/m3 at 25 °C and a flow rate of 72 L/h/m2. Approximately 4000mL of filtrate was then condensed to800mL, which was equivalent to 1 g of RAS-RH /mL of liquid medicine.

2.3. Animal groups and treatment

Male Wistar rats weighing 200 to 230 g were obtained from theAnimal Breeding Center of Gansu University of Traditional ChineseMedicine (Lanzhou, China). The following three groups of rats (n=7)were used for the study: 1) The control group, in which sham-irradiatedrats served as controls. 2) The X-ray group, in which the rats received asingle dose of total body irradiation (TBI) at 8 Gy that was administeredusing a PXi-225 (North Branford, USA). These rats were fed in thebarrier facility for 30 days and served as positive controls. 3) The RAS-RH+X-ray group, in which, 24 h after TBI, the rats were given RAS-RH(25 or 50 or 100mg/kg/day) for 30 days. All animal experiments wereperformed in accordance with the relevant guidelines and regulationsof the responsible authorities.

2.4. Cardiac histology

To evaluate tissue damage at 30 days after irradiation, the entirehearts of rats from the control, X-ray, and RAS-RH+X-ray groups werefixed in 10% formalin (v/v) and embedded in paraffin. Then, 4-μmsections were processed with hematoxylin and eosin (H&E) or Masson’strichrome staining, according to standard methods.

2.5. Enzyme linked immunosorbent assay (ELISA)

To investigate the effects of RAS-RH on X-irradiation-inducedmyocardial fibrosis in vivo, serum from the control, X-ray and RAS-RH+X-ray rats was collected for a determination of the concentrationsof TGF-β1, TnI and brain natriuretic peptide (BNP) using a TGF-β1ELISA kit (Abcam, Cambridge, UK), a Rat TnI ELISA kit and a Rat BNPELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China), re-spectively.

2.6. Isolation and culture of primary cardiac fibroblasts

Cardiac fibroblasts were isolated from the control, X-ray and RAS-RH+X-ray groups by enzymatic digestion as described previously,with modifications [8,22]. Briefly, the hearts were obtained aseptically,cleaned, rinsed with sterile phosphate-buffered saline (PBS, pH 7.4) andminced into small pieces. The heart pieces were digested at 37 °C with0.5 mg/mL trypsin (twice, 8 min each) and then 1mg/mL collagenase II(3 times, 15 min each). The released cells from the three collagenasedigestions were pooled and filtered through a 200-μm sieve to removethe heart debris. Then, the obtained cells were separated by a 30-minperiod of preplating in MEM supplemented with 5% BCS at 37 °C in 5%CO2. The unattached cells were removed, and the plates were washedwith MEM several times. Under these conditions, 95% of the resultingattached cells are cardiac fibroblasts [22].

2.7. Apoptosis assay

Apoptotic cardiac fibroblasts were stained using FITC-conjugatedannexin V and propidium iodide (PI). The stained cells were analyzedwith a flow cytometer (ACEA Biosciences, Hangzhou, China) using theCellquest software (ACEA Biosciences, Hangzhou, China).

2.8. miRNA/RNA isolation and quantification analysis

RNA extraction was performed using TRIzol reagent according tothe manufacturer’s instructions. The expression of the indicatedmiRNAs was analyzed after the RNA was reverse transcribed usingspecific RT primers according to the manufacturer's guidelines. Theexpression level of miRNA-21 was calculated using the cycle threshold(Ct) value, and expression was normalized to that of snRNA U6. ThemRNA expression levels of col1α, OPN, c-fos and c-jun and the internalcontrol GAPDH were analyzed by quantitative real-time-polymerase

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chain reaction (qRT-PCR) on an iCycler (Thermo Fisher Scientific,Shanghai, China). All reactions were performed in triplicate, and theexpression data (after being normalized to GAPDH levels) were ana-lyzed using the 2−ΔΔCt method [23]. The primers are shown in Table 1.

2.9. Protein analysis

Rat cardiac fibroblasts were lysed using RIPA lysis buffer. Totalprotein was isolated and then transferred onto polyvinylidene di-fluoride (PVDF) membranes. The membranes were incubated overnightat 4 °C with rabbit anti-Col1α, rabbit anti-OPN, rabbit anti phospho-c-fos, rabbit anti phospho-c-jun, rabbit anti c-fos or rabbit anti c-jun, andthen the membranes were washed for 30min in TBST. Antibodybinding was visualized by chemiluminescence of the HRP-conjugatedgoat anti-rabbit secondary antibodies.

2.10. Statistical analysis

Data are presented as the mean ± standard deviation (SD) unlessotherwise stated. Groups were compared by one-way ANOVA followed

by the LSD or Tamhane tests using SPSS 17.0 (Chicago, IL). P-va-lues< 0.05 were considered to be statistically significant for all sta-tistical analyses.

3. Results

3.1. RAS-RH mitigates cardiac morphological changes after X-irradiation

H&E staining revealed morphological changes in the hearts 30 daysafter TBI. There was no histological evidence of necrosis or inflamma-tion in any treatment group, compared to the control group (Fig. 1A).Masson’s trichrome staining revealed myocardial fibrosis and irregularcollagen deposition in the hearts. TBI resulted in an increase in myo-cardial fibrosis and collagen deposition in the hearts of the irradiatedrats compared with control group. The group subjected to 50 and100mg/kg/day of RAS-RH+X-ray exhibited less collagen and fibrosiscompared to the X-ray group (Fig. 1B). These results indicated that 50and 100mg/kg/day of RAS-RH partially mitigated the appearance ofTBI-induced fibrosis of the myocardium.

3.2. RAS-RH inhibits the release of TGF-β1 in the serum after X-irradiation

As shown in Fig. 1C, TBI increased the TGF-β1 content up to3930.89 ± 98.05 pg/mL, which was significantly higher than that ofthe control group, 244.80 ± 17.74 pg/mL. However, after treatmentwith 25, 50 or 100mg/kg/day RAS-RH, the TGF-β1 content were re-duced to 3756.04 ± 144.61, 2533.09 ± 156.77 and2206.40 ± 68.35 pg/mL, respectively. Treatment with 50 and 100mg/kg/day RAS-RH significantly reduced the content of TGF-β1 whencompared with the X-ray group. However, 50 and 100mg/kg/day RAS-RH had no significant effect on the content of TGF-β1; therefore,50mg/kg/day RAS-RH was used for the following studies.

Table 1Primer sequences used for real-time RT-PCR analyses.

Gene Corporation Catalog#

miRNA-21-3P FulenGen RmiRQP0929miRNA-21-5P FulenGen RmiRQP0316U6 FulenGen RmiRQP9003Col1α FulenGen RQP054226Spp1 FulenGen RQP049204c-jun FulenGen RQP050276c-FOS FulenGen RQP050333GADPH FulenGen RQP049537

Fig. 1. RAS-RH mitigates cardiacmorphological changes and inhibitsthe release of TGF-β1 after X-irradia-tion in vivo. A, Representative imagesof rat hearts following hematoxylinand eosin (H&E) staining. Nuclei wasstained purple blue and cytoplasmstained red. B, Representative resultsof Masson’s trichrome staining of therat hearts. Myocardium was stainedpurple and collagen stained blue(400× magnification). C, Serum levelsof TGF-β1 from the control, X-ray andRAS-RH+X-ray groups (n= 3,*P < 0.05, **P < 0.01 vs. the controlgroup; #P < 0.05, ##P < 0.01 vs.the X-ray group; &P < 0.05, &&P <0.01 vs. the 25 mg/kg/day of RAS-RH+ X-ray group) (For interpretation ofthe references to colour in this figurelegend, the reader is referred to theweb version of this article).

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3.3. RAS-RH inhibits serum TnI levels after X-irradiation

To assess the effect of RAS-RH on cardiac fibrosis, we measured thelevels of the cardiac blood biomarkers TnI and BNP. As shown inFig. 2A and B, TBI increased the TnI level to 4530.59 ± 143.80 pg/mL,which was significantly higher than the TNI level of the control group,500.39 ± 8.33 pg/mL. After treatment with 25, 50 or 100mg/kg/dayRAS-RH, the TnI level was reduced to 4230.68 ± 56.00,1290.45 ± 114.15 and 1320.71 ± 210.30 pg/mL, respectively.Treatment with 50 and 100mg/kg/day RAS-RH significantly reducedthe level of TnI when compared with the X-ray group. However, X-irradiation did not significantly elevate the serum BNP levels. More-over, after treatment with RAS-RH, the BNP levels did not change sig-nificantly.

3.4. RAS-RH induces apoptosis of primary rat cardiac fibroblasts after X-irradiation

Next, we evaluated the effect and possible underlying mechanismsof RAS-RH on X-irradiation-induced myocardial fibrosis at the cellularlevel (cells from the control, X-ray and RAS-RH+X-ray groups). Asshown in Fig. 3A and B, compared to the control group, the percentageof apoptotic fibroblasts was strongly decreased in the X-ray group(P < 0.01). However, after treatment with 50 mg/kg/day RAS-RH, therate of apoptosis increased to 17.81%±1.57% compared with8.73%±1.70% in the X-ray group. These results demonstrated thatRAS-RH induced apoptosis of primary rat cardiac fibroblasts, whichmay be the mechanism through which RAS-RH alleviates X-irradiation-induced fibrosis.

Fig. 2. The effect of RAS-RH on TnI and BNP after X-irradiation in vivo. A, TnI levels of the control, X-ray and RAS-RH+X-ray groups. B, BNP levels (n= 3,*P < 0.05, **P < 0.01 vs. the control group, #P < 0.05, ##P < 0.01 vs. the X-ray group).

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3.5. RAS-RH decreases the gene expression of miRNA-21, col1α, OPN andc-jun after X-irradiation

To elucidate the molecular mechanism underlying the effect of RAS-RH on X-irradiation-induced fibrosis, we examined the expression le-vels of miRNA-21, col1α, OPN, c-fos and c-jun in primary rat cardiacfibroblasts. Our data revealed that TBI markedly increased the levels ofmiRNA-21-3 P and miRNA-21-5 P (P < 0.01). After treatment withRAS-RH, the levels of miRNA-21-3P and miRNA-21-5P were reduced3.95-fold and 8.52-fold, respectively, in comparison with the X-raygroup (Fig. 4A, P < 0.01). In addition, the gene expression levels ofcol1α and OPN were dramatically attenuated in the RAS-RH + X-raygroup compared with the X-ray group (Fig. 4B and D). Interestingly, TBIremarkably increased the gene expression level of c-fos and c-jun.However, after treatment with RAS-RH, the expression level of c-jungene was reduced 2.71-fold in comparison with the X-ray group, andthere was no change in the expression level of c-fos (Fig. 4C).

3.6. RAS-RH decreases the protein levels of p-c-jun, OPN and col1α after X-irradiation

To further investigate the molecular mechanisms underlying theeffect of RAS-RH on X-irradiation-induced fibrosis, the protein expres-sion levels of p-c-jun, p-c-fos, OPN and col1α were examined byWestern blotting. As shown in Fig. 5A and B, p-c-fos, p- c-jun, OPN and

col1α expression were markedly increased in the X-ray group comparedwith the control group. However, after treatment with RAS-RH, theprotein expression levels of p-c-jun, OPN and col1α were slightly de-creased when compared with the X-ray group. In contrast, we failed todetect a change in the protein expression level of p-c-fos between the X-ray group and the RAS-RH+X-ray group, which indicates that c-fosmay not be involved in the RAS-RH-induced alleviation of X-irradia-tion-induced fibrosis. In conclusion, the results of the Western blotscorroborated the results of the mRNA expression levels of c-fos, c-jun,OPN and col1α. Furthermore, these results support the finding that c-jun, OPN, miRNA-21 and col1α may be involved in the RAS-RH-in-duced alleviation of X-irradiation-induced fibrosis.

4. Discussion

RAS-RH was used in the form of an ultrafiltration extract of RadixAngelica Sinensis and Radix Hedysari. Radix Angelica Sinensis and RadixHedysari have been shown to have beneficial effects in multiple disease,including hepatic fibrosis [13] and pulmonary fibrosis [24]. This studydemonstrated that RAS-RH ameliorates X-irradiation-induced cardiacfibrosis at the morphological level based on the following findings: (a)RAS-RH mitigated irregular collagen deposition in the rat hearts afterX-irradiation, and collagen deposition is a typical phenotype of fibrosis.(b) RAS-RH inhibited the release of serum TGF-β1 and the level of TnIafter X-irradiation. TGF-β1 is one of the most studied pro-fibrotic

Fig. 3. RAS-RH induces apoptosis of primary rat cardiac fibroblasts. A, Cells were stained with annexin V-FITC and propidium iodide. The percentages of cellapoptosis were assessed by flow cytometry. B, Histogram that represents a statistical analysis of image A (n= 3, *P < 0.05, **P < 0.01 vs. the control group,#P < 0.05, ##P < 0.01 vs. the RAS-RH + X-ray group).

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cytokines. TnI has been extensively studied as a biomarker of cardiacfunction among patients who receive high-dose conformal radiationtherapy for thoracic malignancies [25]. We also showed that X-irra-diation markedly increased the TGF-β1 content and TnI levels and thatRAS-RH inhibited the X-irradiation-induced release of serum TGF-β1and the level of TnI, thereby mitigating X-irradiation-induced cardiacfibrosis.

Myocardial fibrosis is a complex biological process involving nu-merous cellular activities and molecules. The activity of cardiac fibro-blasts is critical during cardiac fibrosis, as these cells are associatedwith tissue remodeling during fibrosis [9]. Moreover, cardiac fibrosis ischaracterized by the excessive proliferation of cardiac fibroblasts. Ourexperimental results showed that RAS-RH is effective in inducingapoptosis of primary rat cardiac fibroblasts after X-irradiation. In-triguingly, a previous study reported that miRNA-21 overexpressioninhibits the apoptosis of cardiac fibroblasts [10]. More importantly, wedemonstrated that TBI clearly increases the expression of miRNA-21,and RAS-RH attenuates this TBI-induced increase in miRNA-21 ex-pression. We speculated that RAS-RH induces apoptosis of primary ratcardiac fibroblasts after X-irradiation, possibly by inhibiting miRNA-21expression. In addition, Hara Kang reported that miRNA-21 exerts apro-fibrotic function in radiation-induced lung fibrosis [26]. Consistentwith this finding, our data also showed an obvious increase in miRNA-

21 expression after X-irradiation. Moreover, miRNA-21-5p expressionwas increased to a greater degree than miRNA-21-3p expression after X-irradiation, which indicates that miRNA-21-5p may be more involved inX-irradiation-induced cardiac fibrosis. Likewise, we determined thatmiRNA-21-5p expression was reduced to a greater extent than miRNA-21-3p expression after treatment with RAS-RH, which suggests thatmiRNA-21-5p may play a major role in the RAS-RH-induced ameli-oration of X-irradiation-induced cardiac fibrosis.

OPN is a multifunctional cytokine that is considered to be closelyrelated to the fibrosis process in humans and animal models [27,28]. Inaddition, recent research suggests that OPN is indispensable in acti-vating the transcription factor AP-1, which subsequently regulates thetranscription of miRNA-21. AP-1, which is a dimer composed of c-fosand c-jun subunits, transcriptionally activates the expression of miRNA-21. The results of our study demonstrated that OPN expression wassignificantly increased after X-irradiation. RAS-RH remarkably atte-nuated the X-irradiation-induced increase in OPN expression. Similarly,we also demonstrated that TBI remarkably increased the levels of c-fosand c-jun. After treatment with RAS-RH, the gene expression level of c-jun was dramatically reduced; however, there was no change in theexpression level of c-fos, which implies that the RAS-RH-inducedamelioration of X-irradiation-induced cardiac fibrosis was likely un-related to c-fos.

Fig. 4. The gene expression levels of miRNA-21, col1α, c-fos, c-jun and OPN were determined by quantitative real-time-polymerase chain reaction (qRT-PCR) inprimary rat cardiac fibroblasts. A, Quantification of miRNA-21-3p and miRNA-21-5p expression levels. B, Quantification of col1α expression levels. C, quantificationof c-fos and c-jun expression levels. D, Quantification of OPN.expression levels (n=3, *P < 0.05, **P < 0.01 vs. the control group, #P < 0.05, ##P<0.01 vs. the RAS-RH+X-ray group).

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Based on previous reports and our present results, we proposed thefollowing mechanism underlying the effect of RAS-RH on mitigating X-irradiation-induced myocardial fibrosis in rats as shown in Fig. 6: X-irradiation leads to the expression of OPN, which induces the AP-1-mediated transcription of miRNA-21. Mature miRNA-21 ultimatelyleads to the expression of profibrotic genes (such as COL1α and TGF-β1) and fibroblast survival. RAS-RH may inhibit the X-irradiation-in-duced expression of OPN, which subsequently inhibits the transcriptionof miRNA-21 and ultimately mitigates X-irradiation-induced myo-cardial fibrosis. Our results partially elucidate the mechanism

underlying the effect of RAS-RH on ameliorating X-irradiation-inducedcardiac fibrosis.

5. Conclusion

We demonstrated that RAS-RH ameliorates X-irradiation-inducedmyocardial fibrosis in rats, and we showed that the underlying me-chanism is partially related to the induction of apoptosis in primary ratcardiac fibroblasts and the inhibition of the release of serum TGF-β1,the reduction in the level of TnI and the expression of OPN, c-jun,miRNA-21 and COL1α. Taken together, these novel findings indicatethat RAS-RH may be suitable for development as a medical counter-measure for the mitigation of RIMF. Further research is warranted toclarify the detailed mechanism underlying the effect of RAS-RH on X-irradiation-induced myocardial fibrosis.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No. 81760798, 81873132).

References

[1] S.S. Tuohinen, T. Skytta, V. Virtanen, et al., Detection of radiotherapy-inducedmyocardial changes by ultrasound tissue characterisation in patients with breastcancer, Int. J. Cardiovasc. Imaging 32 (2016) 767–776.

[2] A. Kainthola, T. Haritwal, M. Tiwari, et al., Immunological aspect of radiation-in-duced pneumonitis, current treatment strategies, and future prospects, Front.

Fig. 5. Representative images of the proteinlevels of col1α, OPN, P-c-fos and P-c-jun in thethree groups. A, The protein expression levelsof col1α, OPN, P-c-fos and P-c-jun were sig-nificantly increased in the X-ray group. Aftertreatment with RAS-RH, the expression ofcol1α, OPN and P-c-jun was slightly decreased.B, Relative protein expression levels were ana-lyzed by Image-Pro Plus 6.0 (n=3, *P < 0.05,**P < 0.01 vs. the control group; #P < 0.05,##P<0.01 vs. the RAS-RH+X-ray group).

Fig. 6. Proposed mechanism underlying the effect of RAS-RH on mitigating X-irradiation-induced myocardial fibrosis in rats. (Normal arrows represent acti-vation. T-bar arrows represent inhibition.).

C. Ma, et al. Biomedicine & Pharmacotherapy 112 (2019) 108596

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Immunol. 8 (2017), https://doi.org/10.3389/fimmu.2017.00506.[3] S.W. Yusuf, B.P. Venkatesulu, L.S. Mahadevan, et al., Radiation-induced cardio-

vascular disease: a clinical perspective, Front. Cardiovasc. Med. 4 (2017), https://doi.org/10.3389/fcvm.2017.00066.

[4] M.A. Armanious, H. Mohammadi, S. Khodor, et al., Cardiovascular effects of ra-diation therapy, Curr. Probl. Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.1005.1008.

[5] J. Spetz, J. Moslehi, K. Sarosiek, Radiation-induced cardiovascular toxicity: me-chanisms, prevention, and treatment, Curr. Treat. Options Cardiovasc. Med. 20(2018), https://doi.org/10.1007/s11936-11018-10627-x.

[6] T. Thum, J.M. Lorenzen, Cardiac fibrosis revisited by microRNA therapeutics,Circulation 126 (2012) 800–802.

[7] J. Yarnold, M.C. Brotons, Pathogenetic mechanisms in radiation fibrosis, Radiother.Oncol. 97 (2010) 149–161.

[8] J.M. Lorenzen, C. Schauerte, A. Hubner, et al., Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis, Eur.Heart J. 36 (2015) 2184–2196.

[9] R.G. Gourdie, S. Dimmeler, P. Kohl, Novel therapeutic strategies targeting fibro-blasts and fibrosis in heart disease, Nat. Rev. Drug Discov. 15 (2016) 620–638.

[10] T. Thum, C. Gross, J. Fiedler, et al., MicroRNA-21 contributes to myocardial diseaseby stimulating MAP kinase signalling in fibroblasts, Nature 456 (2008) 980–984.

[11] T. Zhong, X.Y. Duan, H. Zhang, et al., Angelica sinensis suppresses body weight gainand alters expression of the FTO gene in high-fat-Diet induced obese mice, Biomed.Res. Int. 2017 (2017), https://doi.org/10.1155/2017/6280972.

[12] X. Mu, Y. Zhang, J. Li, Angelica sinensis polysaccharide prevents hematopoieticstem cells senescence in D-Galactose-induced aging mouse model, Stem Cells Int.2017 (2017), https://doi.org/10.1155/2017/3508907.

[13] X. Chen, X. Liu, Y. Chen, et al., Spectrum-effect relationship on anti-hepatic fibrosiseffect of Radix hedysari, Se pu 33 (2015) 413–418.

[14] S. Hosseini, M. Imenshahidi, H. Hosseinzadeh, et al., Effects of plant extracts andbioactive compounds on attenuation of bleomycin-induced pulmonary fibrosis,Biomed. Pharmacother. 107 (2018) 1454–1465.

[15] S. Bahri, R. Ben Ali, A. Abidi, et al., The efficacy of plant extract and bioactivecompounds approaches in the treatment of pulmonary fibrosis: a systematic review,Biomed. Pharmacother. 93 (2017) 666–673.

[16] K.Y. Zheng, Z.X. Zhang, C.Y. Du, et al., Ferulic acid enhances the chemical andbiological properties of astragali radix: a stimulator for danggui buxue tang, an

ancient Chinese herbal decoction, Planta Med. 80 (2014) 159–164.[17] J.P. Williams, S.L. Brown, G.E. Georges, et al., Animal models for medical coun-

termeasures to radiation exposure, Radiat. Res. 173 (2010) 557–578.[18] M. Lenarczyk, J. Su, S.T. Haworth, et al., Simvastatin mitigates increases in risk

factors for and the occurrence of cardiac disease following 10 Gy total body irra-diation, Pharmacol. Res. Perspect. 3 (2015) e00145.

[19] R.J. Debo, C.J. Lees, G.O. Dugan, et al., Late effects of total-body gamma irradiationon cardiac structure and function in male Rhesus macaques, Radiat. Res. 186 (2016)55–64.

[20] C. Rabender, E. Mezzaroma, A.G. Mauro, et al., IPW-5371 proves effective as aradiation countermeasure by mitigating radiation-induced late effects, Radiat. Res.186 (2016) 478–488.

[21] W. Kou, Y.D. Li, K. Liu, et al., Radix Angelicae Sinensis and Radix hedysari enhanceradiosensitivity of 12C6+ radiation in human liver cancer cells by modulatingapoptosis protein, Saudi Med. J. 35 (2014) 945–952.

[22] L. Piacentini, M. Gray, N.Y. Honbo, et al., Endothelin-1 stimulates cardiac fibroblastproliferation through activation of protein kinase C, J. Mol. Cell. Cardiol. 32 (2000)565–576.

[23] V. Chandrasekar, J.L. Dreyer, Regulation of MiR-124, Let-7d, and MiR-181a in theaccumbens affects the expression, extinction, and reinstatement of cocaine-inducedconditioned place preference, Neuropsychopharmacology 36 (2011) 1149–1164.

[24] L. Wang, Y. Sun, C. Ruan, et al., Angelica sinensis is effective in treating diffuseinterstitial pulmonary fibrosis in rats, Biotechnol. Biotechnol. Equip. 28 (2014)923–928.

[25] D.R. Gomez, S.W. Yusuf, M.F. Munsell, et al., Prospective exploratory analysis ofcardiac biomarkers and electrocardiogram abnormalities in patients receivingthoracic radiation therapy with high-dose heart exposure, J. Thorac. Oncol. 9(2014) 1554–1560.

[26] H. Kang, Role of MicroRNAs in TGF-beta signaling pathway-mediated pulmonaryfibrosis, Int. J. Mol. Sci. 18 (2017), https://doi.org/10.3390/ijms18122527.

[27] P. Rubis, S. Wisniowska-Smialek, E. Dziewiecka, et al., Prognostic value of fibrosis-related markers in dilated cardiomyopathy: a link between osteopontin and cardi-ovascular events, Adv. Med. Sci. 63 (2018) 160–166.

[28] H. Zhao, W. Wang, J. Zhang, et al., Inhibition of osteopontin reduce the cardiacmyofibrosis in dilated cardiomyopathy via focal adhesion kinase mediated signalingpathway, Am. J. Transl. Res. 8 (2016) 3645–3655.

C. Ma, et al. Biomedicine & Pharmacotherapy 112 (2019) 108596

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