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Abstract

BACKGROUND:

Hypertension is a well-recognized risk factor for cardiovascular, which is also a critical factor in causing myocardial fibrosis (MF).

OBJECTIVE:

The study aimed to explore the effect of Atractylenolide II (ATL-II) on MF and oxidative stress in spontaneous hypertension rats (SHR).

METHODS:

The body weight of rats after injection of ATL-II was quantitatively analyzed. The left ventricular function of SHR was evaluated by Echocardiographic. HE staining, Masson trichrome staining, left ventricular mass index (LVMI) and immunofluorescence was applied to investigate the effects of ATL-II on MF. RT qPCR was used to detect the Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin mRNA expression levels in myocardial slices. The effect ATL-II on cardiomyocyte apoptosis was detected by TUNEL staining and western blot. An immunohistochemistry assay was conducted to detect $\alpha$ -SMA protein and TGF- $\beta$ 1 protein. The contents of H ${}_{2}$ O ${}_{2}$ , GSH-PX, SOD, and MDA were measured by colorimetry.

RESULTS:

ATL-II could dose-dependently improve the BW of SHRs ( $P<$ 0.05) and enhance myocardial function. Moreover, ATL-II effectively reduced cardiomyocyte apoptosis in SHRs. Alternatively, ATL-II could inhibit the Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin mRNA and protein expression levels in SHRs. ATL-II could ameliorate oxidative stress by improving the activities of SOD and GSH-PX and lowering the contents of H ${}_{2}$ O ${}_{2}$ and MDA in ATL-II-treated SHRs, which reach about 80%.

CONCLUSION:

ATL-II could exert an inhibiting effect on MF and oxidative stress in SHRs. Hence, ATL-II may hold promise for the treatment of MF and oxidative stress in Spontaneous Hypertension.

Keywords

Atractylenolide II myocardial fibrosis oxidative stress spontaneous hypertension rats apoptosis

1. Introduction

Hypertension is a well-recognized risk factor for cardiovascular, which is also a critical factor in causing MF [1]. MF, a serious adverse effect of hypertension on the heart, is a common pathological damage caused by various cardiovascular diseases [2, 3]. Especially, MF is irreversible, which becomes a difficult problem in clinical treatment [4].

MF is the result of excessive deposition of Collagen fibers in the extracellular matrix, which causes heart failure and ventricular hypertrophy [5, 6, 7]. MF markers include collagen I, fibronectin, $\alpha$ -SMA, and vimentin. These markers are usually used to assess the severity of the disease [8]. The mechanism of MF caused by hypertension is relatively complex. Some studies showed that oxidative stress (OS) was closely related to MF pathogenesis [9, 10].

Oxidative stress is the result of the imbalance between reactive oxygen species (ROS) production and reduced antioxidant capacity in the organism [11]. Oxidative stress plays a critical role in the pathophysiology of cardiovascular diseases, such as hypertension, MF, and cardiomyocyte apoptosis [12]. Oxidative stress causes excessive production of reactive oxygen species (ROS), which is an important event in the development of cardiovascular diseases. Meanwhile, previous studies have found hypertension had a relationship with oxidative stress, oxidative stress could raise blood pressure [13]. Besides, some studies confirmed MF induced by hypertension may be closely related to the decrease of antioxidant enzymes [14, 15].

Atractylodes macrocephala (AM) is an important traditional herbal medicine in Asia, which is widely used to treat gastrointestinal, cardiovascular, and immune system diseases [16]. Many natural compounds were extracted from Atractylodes macrocephala, such as polysaccharides, sesquiterpenoids, and volatile oils. These extracts have various pharmacological effects, such as anti-tumor, anti-inflammatory, and regulating the digestive system [17]. Existing researches indicate that AT-II which is a major sesquiterpenoid extracted from AM exhibits anti-hyperglycemia anti-cancer and anti-oxidant activities [18, 19, 20]. However, few studies paid much attention to MF caused by hypertension.

Hence, the purpose of the research is to explore the efficiency of ATL-II on MF and oxidative stress in SHRs, which would provide valid data for the application of ATL-II to treat myocardial diseases associated with MF and oxidative stress in the future.

2. Methods

2.1 Animals and treatments

Spontaneous hypertension rats (SHRs) and Wistar Kyoto rats (WKYs) (male, 200–250 g, 6 weeks old) were purchased from the Model Animal Research Center of Nanjing University, China (SCXK2012-0005). The rats were randomly divided into 5 groups ( $n=$ 6 rats per group): control group, C group (WKY); model group, M group (SHR; model $+$ low group, ML group (SHR $+$ ATL-II 10 mg ${}^{-1}$ $\cdot$ kg ${}^{-1}$ $\cdot$ d ${}^{-1}$ ; model $+$ medium group, MM group (SHR $+$ ATL-II 30 mg ${}^{-1}$ $\cdot$ kg ${}^{-1}$ $\cdot$ d ${}^{-1})$ and model $+$ high group, MH group (SHR $+$ ATL-II 60 mg ${}^{-1}$ $\cdot$ kg ${}^{-1}$ $\cdot$ d ${}^{-1}$ ) [21]. The rats of the ML group, MM group, and MH group were intraperitoneally injected with ATL-II weekly for 8 weeks, and the other groups were treated with saline. Moreover, 6 weeks after ATL-II injection, the rats were sacrificed under anesthetization. Additionally, primary cardiomyocytes from rats were isolated and cryopreservation according to previously described protocols [22]. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine (Jiangsu, China).

2.2 LVMI assay

The fed rats should be weighed in body weight (BW) every week. After 6 week’s treatment, the rats were sacrificed and the hearts were collected. Hearts and left ventricles were weighed, respectively. LVMI was obtained by calculating the ratio of BW to left ventricular mass (LVM).

2.3 Echocardiography assessment

After ATL-II injection for 6 weeks, the rats’ cardiac function was measured by an echocardiography system (EPIQ 7C, Jiangsu, China). The rats were anesthetized with 2% sodium pentobarbital, and their chest hairs were removed. In the M-mode curve, the relevant indexes were recorded: left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVSV), left ventricular end-diastolic volume (LVDV), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). LVEF and LVFS were used as parameters to indicate cardiac function [23].

2.4 HE and Masson staining

Myocardial tissue samples were fixed with 4% formaldehyde solution (Aladdin, Shanghai, China), embedded in paraffin, and then cut down into slices. After deparaffinized, HE and Masson’s staining were performed to evaluate cardiac fibrosis following the manufacturer’s guidance of the HE and Masson staining kit (Jiancheng Bioengineering Institute, Nanjing, China). Lastly, the slices were inspected under a light microscope (SteREO Discovery.V20, OPTON, Germany).

2.5 Immunofluorescence assay

Cardiomyocytes from rats were fixed in 4% paraformaldehyde (Aladdin, Shanghai, China) at 25 ${}^{\circ}$ C for 30 min, and then permeabilized with 0.5% Triton X-100 (Aladdin, Shanghai, China) for 20 minutes at room temperature. Subsequently, the fixed cells were incubated with 5% bovine serum albumin (Aladdin, Shanghai, China) for 20 minutes at 25 ${}^{\circ}$ C. Moreover, the cells were treated with rabbit polyclonal antibodies against $\alpha$ -SMA (Invitrogen, Hudson, CA, USA, 1:500 dilution) and TGF- $\beta$ 1 (Invitrogen, Hudson, CA, USA, 1:500 dilution) at 4 ${}^{\circ}$ C overnight. After cells were washed, cells were incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibodies (Invitrogen, Hudson, CA, USA) for 50 minutes at 37 ${}^{\circ}$ C. Besides, the cells were incubated with 4’,6-diamidino-2-phenylindole (DAPI) (Aladdin, Shanghai, China) for 10 minutes. Finally, the cells were observed by confocal laser scanning microscopy (Leica, Wetzlar, Germany).

2.6 Immunohistochemistry assay

The samples were digested with enzymes and dehydrated with alcohol. Then the samples were treated with rabbit polyclonal antibodies against $\alpha$ -SMA (Invitrogen, Hudson, CA, USA, 1:500 dilution) and TGF- $\beta$ 1 (Invitrogen, Hudson, CA, USA, 1:500 dilution) at 4 ${}^{\circ}$ C overnight. Next, the slides were washed with PBS and then incubated with the corresponding secondary antibody at 30 ${}^{\circ}$ C for 30 min. Afterward, the slides were developed by diaminobenzidine (DAB) (Solarbio, Beijing, China). The positive and negative indicators of immune response showed brown and blue in the nucleus, respectively.

2.7 RT-qPCR assay

The TRIzol reagent (Invitrogen, Hudson, CA, USA) was applied to extract the total RNA of myocardial tissues. Then cDNA was synthesized using the RT-PCR kit (Invitrogen, Hudson, CA, USA). Moreover, RT-qPCR was performed with a CellsDirect ${}^{\text{TM}}$ RT-qPCR kit (Invitrogen, Hudson, CA, USA). Finally, mRNA expression levels of Collagen I, $\alpha$ -SMA, Fibronectin, Vimentin, and Nox4 were viewed and calculated with the 2 ${}^{\Delta\Delta\text{Ct}}$ method. $\beta$ -actin was used as endogenous control.

2.8 Western blot assay

The total protein of myocardial tissues was extracted with RIPA lysate (Beyotime, Shanghai, China), and total protein concentration was measured using the BCA protein assay kit (Beyotime, Shanghai, China). Protein samples were subjected to SDS/PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Subsequently, the membranes were incubated for 1 hour in a blocking buffer with 5% bovine serum albumin. Moreover, the membranes were incubated with primary antibodies against Vimentin (Invitrogen, Hudson, CA, USA, 1:1000 dilution), Fibronectin (Invitrogen, Hudson, CA, USA, 1:100 dilution), $\alpha$ -SMA(Invitrogen, Hudson, CA, USA, 1:1000 dilution), Collagen I (Invitrogen, Hudson, CA, USA, 1:1000 dilution), Nox4 (Invitrogen, Hudson, CA, USA, 1:2000 dilution) and $\beta$ -actin (Invitrogen, Hudson, CA, USA, 1:500 dilution) at 4 ${}^{\circ}$ C overnight, and the corresponding HRP labeled secondary antibody (Invitrogen, Hudson, CA, USA, 1:1500 dilution) at room temperature for 1.5 hours. Finally, protein expression levels were determined by ECL (Beyotime, Shanghai, China) method. $\beta$ -actin was used as the control group.

2.9 TUNEL staining

A one-step TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) was applied to measure cardiomyocyte apoptosis according to the manufacturer’s instructions. Cardiomyocytes were fixed in 4% paraformaldehyde at room temperature for 15 minutes and then permeabilized with 0.01% Triton X-100 for 10 minutes at room temperature. Then Cardiomyocytes were incubated with TUNEL mix for 1 hour at 37 ${{}^{\circ}}$ C. Finally, cardiomyocytes were washed with PBS another 3 times and inspected via confocal laser scanning microscopy (Leica, Wetzlar, Germany). The cardiomyocyte apoptosis ratio was calculated.

2.10 H ${}_{2}$ O ${}_{2}$ , GSH-PX, SOD, and MDA determination

After 10% chloral hydrate (Aladdin, Shanghai, China) anesthesia, rats’ blood was obtained from the abdominal aorta. Then blood was centrifuged at 3000 rpm for 15 min, the serum was separated and cryopreserved at $-$ 80 ${{}^{\circ}}$ C. Assay Kits (Jiancheng, Nanjing, China) were respectively applied to detect the levels of H2O2, GSH-PX, SOD, and MDA according to the manufacturer’s instructions by colorimetry.

2.11 Data analysis

Statistical analysis was performed by SPSS 17.0 software (SPSS Inc., Chicago, USA). All experiments were repeated three times, and the data were presented as mean $\pm$ standard deviation (SD) with means of one-way ANOVA and LSD test. The level of significance was considered significant when $P<$ 0.05.

3. Results

3.1 The effect of ATL-II on cardiac function in SHRs

The rats BW were weighed to verify whether ATL-II affected the BW of SHRs. As Fig. 1A shows that rats BW of the ML group was the lowest ( $P<$ 0.05), which showed high concentration ATL-II could improve the BW of SHRs.

Figure 1.

ATL-II enhances cardiac function in SHRs. (A) Quantitative analysis of rats BW after ATL-II injection. (B) Cardiac function was assessed by echocardiography in rats after ATL-II injection for 6 weeks. All data were presented by means $\pm$ SD ( $n=$ 3). ${}^{**}P<$ 0.01 vs. C group; ${}^{\#}P<$ 0.05, ${}^{\#\#}P<$ 0.01 vs. M group.

Moreover, cardiac function was assessed by echocardiography in rats after different dosages of ATL-II injection for 6 weeks. The results indicated that LVEDD, LVESD, LVEDV, and LVESV were significantly increased in the M group, and EF and FS were significantly decreased in the M group ( $P<$ 0.05), which suggested heart failure was observed. LVEDD, LVESD, LVEDV, and LVESV were significantly reduced after ATL-II treatment compared with the M group. Specifically, EF and FS were significantly improved after ATL-II treatment ( $P<$ 0.05) (Fig. 1B). Hence, Echocardiography analysis indicated that ATL-II could enhance cardiac function in SHRs, which was shown via a dose-dependent manner.

Figure 2.

ATL-II attenuates MF in SHRs. (A) Representative images of HE Staining in myocardial sections. (B) Representative image of Masson staining in myocardial sections. (C) Representative images of $\alpha$ -SMA and TGF- $\beta$ 1 staining by immunofluorescence in Cardiomyocytes. (D) RTqPCR for Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin mRNA expression levels in myocardial sections. (E) Representative western blot image and quantitative analysis of Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin in myocardial tissues. (F) Quantitative analysis of LVMI for 4 and 8 weeks. All data were presented by means $\pm$ SD ( $n=$ 3). ${}^{**}P<$ 0.01, ${}^{***}P<$ 0.001 vs. C group; ${}^{\#}P<$ 0.05, ${}^{\#\#}P<$ 0.01 vs. M group.

3.2 The effect of ATL-II on MF in SHRs

To clarify the effect of ATL-II on MF in the SHRs, relevant indexes were measured. As shown in Fig. 2A, the results of HE staining revealed that obvious myocardial tissue damage was observed in the M group compared to the C group ( $P<$ 0.05), which was illustrated by increased blue-purple stained areas. The damage was significantly reduced in the ML group, MM group, and MH group ( $P<$ 0.05), and a few myocardial tissue damages were inspected in the MH group.

Then, Masson staining was applied to observe the myocardial sections, which showed that large amounts of collagen deposition and disorder arrangement of myocardial fibers were observed in the M group compared to the C group ( $P<$ 0.05). However, after a different dose of ATL-II treatment, myocardial fibers were arranged in order, and myocardial collagen fibers rarely appeared collagen deposition in the ML group, MM group, and MH group (Fig. 2B). The myocardial collagen fibers were stained blue, and the myocardial fibers were stained red.

Moreover, immunofluorescence assays for $\alpha$ -SMA and TGF- $\beta$ 1 showed that the protein expression levels of $\alpha$ -SMA and TGF- $\beta$ 1 of the M group were significantly higher than C group, which suggested cardiac fibrosis was increased. But after ATL-II treatment, $\alpha$ -SMA and TGF- $\beta$ 1 protein expression levels were significantly reduced, thereby suggesting decreased myocardial fibrosis ( $P<$ 0.05). $\alpha$ -SMA and TGF- $\beta$ 1 were stained bright green fluorescence (Fig. 2C).

Besides, RTqPCR and western blot assays showed that expression levels of Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin of the M group were significantly higher than C group ( $P<$ 0.05), whereas ATL-II could significantly reduce the effects (Fig. 2D and E).

In addition, LVMI was calculated when ATL-II treatment was for 4 and 8 weeks. The results showed that compared with the C group, the LVMI of the M group was significantly improved ( $P<$ 0.05). However, LVMI was dramatically decreased after ATL-II treatment ( $P<$ 0.05) (Fig. 2F). The above data demonstrated that ATL-II had a positive function in attenuating MF in SHRs, which showed a dose-dependent manner.

3.3 The effects of ATL-II on cardiomyocyte apoptosis in SHRs

The results of TUNEL staining indicated that cardiomyocyte apoptosis was obviously inspected in the M group. The apoptosis rate of the M group significantly exceeded the C group, which showed significant differences ( $P<$ 0.001). Meanwhile, ATL-II could significantly inhibit cardiomyocyte apoptosis ( $P<$ 0.05) (Fig. 3A).

Moreover, the results of RTqPCR presented that expression levels of Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin of the M group were significantly higher than the C group ( $P<$ 0.05). The effects were significantly reduced after ATL-II treatment ( $P<$ 0.05) (Fig. 3B).

As Fig. 3C shows that few $\alpha$ -SMA and TGF- $\beta$ 1 staining positive cells were expressed in the C group. Meanwhile, the number of $\alpha$ -SMA and TGF- $\beta$ 1 positive cells in the M group was significantly higher than in the C group. ATL-II significantly reduced the number of $\alpha$ -SMA and TGF- $\beta$ 1 staining positive cells in the peripheral region of the MH group. So, ATL-II dose-dependently exerted an inhibitory effect on cardiomyocyte apoptosis in SHRs.

Figure 3.

ATL-II inhibits cardiomyocyte apoptosis in SHRs. (A) Representative image and quantitative analysis of TUNEL staining in myocardial sections. (B) RTqPCR for Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin mRNA expression levels in myocardial sections. (C) Representative Immunohistochemistry image for $\alpha$ -SMA and TGF- $\beta$ 1 in myocardial samples. All data were presented by means $\pm$ SD ( $n=$ 3). ${}^{**}P<$ 0.01, ${}^{***}P<$ 0.001 vs. C group; ${}^{\#}P<$ 0.05, ${}^{\#\#}P<$ 0.01 vs. M group.

Figure 4.

ATL-II alleviates oxidative stress in SHRs. (A) Quantitative analysis of GSH-PX, H ${}_{2}$ O ${}_{2}$ , MDA, and SOD levels. (B) RTqPCR for NOX4 mRNA expression level in myocardial sections. (C) Representative western blot image and quantitative analysis of NOX4 in myocardial tissues. All data were presented by means $\pm$ SD ( $n=$ 3). ${}^{**}P<$ 0.01 vs. C group; ${}^{\#}P<$ 0.05, ${}^{\#\#}P<$ 0.01 vs. M group.

3.4 The effect of ATL-II on oxidative stress in SHRs

Activities of GSH-PX and SOD and contents of H ${}_{2}$ O ${}_{2}$ and MDA were detected to study the effect of ATL-II on oxidative stress in SHRs. The results suggested that activities of GSH-PX and SOD were obviously lowered in the M group compared to the C group, and contents of H ${}_{2}$ O ${}_{2}$ and MDA of the M group were significantly increased ( $P<$ 0.05). But ATL-II could improve SOD and GSH-PX activities, and decrease the contents of H ${}_{2}$ O ${}_{2}$ and MDA (Fig. 4A). Meanwhile, GSH-PX activity could restore to the level of the C group.

In addition, the mRNA expression level of NOX4 was up-regulated in the M group compared to the C group ( $P<$ 0.05). Whereas, after ATL-II treatment, the expression level of NOX4 was remarkably decreased (Fig. 4B). Similar results were confirmed by western blot assay (Fig. 4C). To sum up, ATL-II had the capacity in alleviating oxidative stress in SHRs in a dose-dependent manner.

4. Discussion

Numerous studies showed that approximately 30% of the population died from cardiovascular diseases around the world, and more than half of the cardiovascular disease cases were caused by hypertension. Previous studies reported that MF was one of the key pathological features of myocardial remodeling in hypertensive. Meanwhile, MF also was an important reason for diastolic dysfunction, myocardial ischemia, and sudden cardiac death induced by hypertension. Furthermore, MF induced by hypertension was closely related to the decrease of antioxidant enzymes. In addition, Oxidative stress could increase peripheral vascular resistance and blood pressure.

In our study, M-mode echocardiography was carried out to assess myocardial function. Previous studies reported that MF caused by hypertension possessed a seriously bad effect on myocardial function Our research showed that EF and FS were increased after ATL-II treatment. ATL-II could improve myocardial function in SHRs.

Moreover, myocardial apoptosis and myocardial fibrosis were detected to assess the effect of ATL-II on MF in SHRs. Cardiac Fibroblasts (CFB) could differentiate as myofibroblasts (MFB), and then MFB participated in MF formation. In addition, some factors played a key role in the proliferation and differentiation of MF [24]. Collagen I, $\alpha$ -SMA, Fibronectin, and Vimentin would be expressed in CFB of myocardial tissues, and TGF- $\beta$ 1 promoted fibrosis and proliferation of fibroblast. Hench, these indexes were monitored to explore the MF [25]. Our study indicated that the expression levels of Collagen I, $\alpha$ -SMA, TGF- $\beta$ 1, Fibronectin, and Vimentin were significantly down-regulated after ATL-II treatment in SHRs, which were similar to the predecessors’ research findings [26, 27, 28]. Besides, our study also confirmed ATL-II could effectively attenuate MF and cardiac apoptosis. Above all, ATL-II could alleviate MF by inhibiting the expression levels of factors related to the proliferation and differentiation of MF in SHRs.

Oxidative stress was defined as ROS increase and antioxidant activity reduction of vascular [29, 30, 31]. ROS comprised superoxide anion (O2), hydroxyl radical (OH), and hydrogen peroxide (H ${}_{2}$ O ${}_{2}$ ) [32]. Antioxidant enzymes, including SOD and GSH-PX, could reduce ROS production. Therefore, the levels of SOD and GSH-PX were considered important elements to evaluate the antioxidant capacity. In addition, the content of MDA was a common index to reflect the situation of oxidative stress. Our study showed ATL-II dose-dependently increased SOD and GSH-PX activities and decreased the levels of MDA and H ${}_{2}$ O ${}_{2}$ , which was consistent with the previous studies [33, 34, 35]. NADPH oxides (Nox) proteins were related to many different cardiovascular diseases: atherosclerosis, hypertension, cardiac hypertrophy and remodeling, and heart failure. Nox4 was a member of the Nox family proteins that generated ROS by catalyzing the electron transfer from NADPH to molecular O ${}_{2}$ [36]. We found that the expression level of Nox4 was significantly reduced after ATL-II treatment in SHRs, which was similar to the reference [37, 38]. These results suggested that ATL-II alleviated oxidative stress by enhancing antioxidant activities and lowing the oxidation products in SHRs.

However, we investigated the effects of ATL-II on myocardial fibrosis and oxidative stress in spontaneously hypertensive rats only at the histological level in animals. This is limited and challenging for the study of ATL-II function. Further studies investigating the safety of ATL-II at the cellular level and the molecular mechanisms underlying the effects on myocardial fibrosis and oxidative stress are also imperative. In addition, it has been recently reported that the mechanism by which hydrogen-rich saline (HRS) [39] and Asiatic acid (AA) [28] inhibit spontaneous hypertension and reduce myocardial fibrosis is also significantly related to the reduction of oxidative stress, and the mechanism may be achieved by inhibiting TGF- $\beta$ signaling pathway. Our study also showed that the expression level of Tgf- $\beta$ 1 was significantly down-regulated after ATL-II treatment in SHR. Whether ATL-II alleviates oxidative stress through the TGF- $\beta$ signaling pathway remains to be further explored. Interleukin-6 (IL-6), Signal Transducer and Activator of Transcription 3 (STAT3), Endothelin-1 (ET-1) [26], PPAR [40], ErbB [41] signaling pathways have also been shown to mediate myocardial fibrosis in spontaneous hypertension in recent literature. Whether ATL-II has the role of these signaling pathways in reducing oxidative stress needs to be further explored.

5. Conclusion

In conclusion, our study proved that ATL-II could alleviate MF and myocardial apoptosis in SHRs. Furthermore, our research also found ATL-II could protect cardiomyocytes against oxidative stress by enhancing the antioxidant activities and lowering the oxidation products, which more clearly and comprehensively pointed out the significant role of oxidative stress on MF than previous literature. We speculated that ATL-II alleviated MF by reducing oxidative stress in SHRs, but the potential molecular mechanism is still unclear. Hence, ATL-II could be a promising treatment for MF and oxidative stress caused by Spontaneous Hypertension.

Ethics statement

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine, Jiangsu, China (IRB: XMLL-2021-880).

Funding

This work was supported by Jiangsu Province Traditional Chinese Medicine Technology Development Plan Project (No. YB201968).

Availability of data and material

All data generated or analyzed during this study are included in this published article.

Author contributions

XS conceived and designed the research, obtained financing, and revised the manuscript for intellect content. LW and JS acquired and analyzed the data. JK performed the statistical analysis. RP wrote the manuscript. All authors read and approved the final manuscript.

Footnotes

Acknowledgments

Not applicable.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Atractylenolide II ameliorates myocardial fibrosis and oxidative stress in spontaneous hypertension rats

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Methods

2.1 Animals and treatments

2.2 LVMI assay

2.3 Echocardiography assessment

2.4 HE and Masson staining

2.5 Immunofluorescence assay

2.6 Immunohistochemistry assay

2.7 RT-qPCR assay

2.8 Western blot assay

2.9 TUNEL staining

2.10 H 2 O 2 , GSH-PX, SOD, and MDA determination

2.11 Data analysis

3. Results

3.1 The effect of ATL-II on cardiac function in SHRs

3.3 The effects of ATL-II on cardiomyocyte apoptosis in SHRs

4. Discussion

5. Conclusion

Ethics statement

Funding

Availability of data and material

Author contributions

Footnotes

Acknowledgments

Conflict of interest

References

2.10 H ${}_{2}$ O ${}_{2}$ , GSH-PX, SOD, and MDA determination