Korean J Physiol Pharmacol 2021; 25(2): 131-137
Published online March 1, 2021 https://doi.org/10.4196/kjpp.2021.25.2.131
Copyright © Korean J Physiol Pharmacol.
Jing Cheng1, Chaoyang Ren2, Renli Cheng3, Yunning Li4, Ping Liu1, Wei Wang1,*, and Li Liu1,*
1Department of Pharmacy, Wuhan Fourth Hospital, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Dong Xi Hu Municipal Healthcare Security Administration, 3Department of Orthopedics, Wuhan Fourth Hospital, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430033, Hubei, 4Department of Traditional Chinese Medicine, 986 Hospital of Air Force, Xian 710054, Shanxi, China
Correspondence to:Li Liu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aging is the process spontaneously occurred in living organisms. Cardiac fibrosis is a pathophysiological process of cardiac aging. Mangiferin is a wellknown C-glucoside xanthone in mango leaves with lots of beneficial properties. In this study, rat model of cardiac fibrosis was induced by injected with 150 mg/kg/d Dgalactose for 8 weeks. The age-related cardiac decline was estimated by detecting the relative weight of heart, the serum levels of cardiac injury indicators and the expression of hypertrophic biomakers. Cardiac oxidative stress and local inflammation were measured by detecting the levels of malondialdehyde, enzymatic antioxidant status and proinflammatory cytokines. Cardiac fibrosis was evaluated by observing collagen deposition via masson and sirius red staining, as well as by examining the expression of extracellular matrix proteins via Western blot analysis. The cardiac activity of profibrotic TGF-β1/p38/MK2 signaling pathway was assessed by measuring the expression of TGF-β1 and the phosphorylation levels of p38 and MK2. It was observed that mangiferin ameliorated D-galactose-induced cardiac aging, attenuated cardiac oxidative stress, inflammation and fibrosis, as well as inhibited the activation of TGF-β1/p38/MK2 signaling pathway. These results showed that mangiferin could ameliorate cardiac fibrosis in D-galactose-induced aging rats possibly via inhibiting TGF-β/p38/MK2 signaling pathway.
Keywords: Aging, Heart fibrosis, Inflammation, Mangiferin, Oxidative stress
Population aging is a global public health problem . Aging is the deteriorative process spontaneously occurred in living organisms and characterized by progressive decline in multiple organ functions and systemic immunity . As a result, aging is suggested to be a major risk factor for varieties of diseases. It is reported that cardiovascular diseases (CVDs) (including hypertension, cardiac hypertrophy and heart failure) contribute to about 31% of all deaths worldwide . In fact, CVDs have already been the major health problems in both developed countries and developing countries . Aging affects the heart at functional, structural and molecular levels [5,6]. These aging-related CVDs have become the leading cause of death in elderly people . CVDs, especially cardiac hypertrophy, are the common pathological changes in aging process . Cardiac hypertrophy is a compensatory change of the heart in order to adapt to hypertension, vascular heart diseases and so on . Cardiac hypertrophy and its accompanied myocardial fibrosis are usually responsible for heart failure . Extracellular matrix (ECM) remodeling and collagen deposition are the characteristics of organ fibrosis. Aging promotes cardiac fibrosis . However, there is no effective treatment for attenuating the aging-associated cardiac hypertrophy and fibrosis .
Mangiferin is a well-known C-glucoside xanthone and widespread in herbal medicines and fruits including mango leaves (
Mangiferin (purity > 95%) and D-gal (purity > 99%) were purchased from Aladdin Co, Ltd. (Shanghai, China). MMI-0100 (sequence: YARAAARQARAKALARQLGVAA) was synthesized by Genscript Biotechnology, Co. (Jiangsu, China). The commercial kits for the analysis of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The commercial kits for the analysis of interleukin (IL)-1β IL-6 and tumor necrosis factor (TNF)-α were purchased from ELISA Lab, Co. Ltd. (Hubei, China). The antibodies of phosphorylation-mitogen activated protein kinase activated protein kinase II (p-MK2) and β-myosin heavy chain (β-MHC) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The antibodies of p-p38, α-smooth muscle actin (α-SMA), collagen (Col)-I, Col-III, natriuretic peptides A (ANP) and BNP were purchased from from Abcam, Co. (Cambridge, UK). The antibody of transforming growth factor-β1 (TGF-β1) was purchased from Proteintech Group, Inc. (Chicago, IL, USA).
Eight weeks adult male Wistar rats (180–220 g) were obtained from the Centers of Disease Control and Prevention of Hubei Province, China. Rats were maintained under standard environment (temperature of 22°C ± 3°C, humidity of 50% ± 10% and 12 h light:12 h dark cycle). After acclimatization to the laboratory conditions for one week, the rats were randomly assigned into 5 groups (n = 8): the vehicle control group (Veh), the D-gal-induced aging group (aging), the aging-MMI-0100 group (MMI-0100), the aging-100 mg/kg mangiferin group (100 mangiferin) and aging-50 mg/kg mangiferin group (50 mangiferin). The rats from aging, MMI-0100, 100 mangiferin and 50 mangiferin groups were injected with 150 mg/kg/d D-gal for 8 weeks . Animals from 100 mangiferin and 50 mangiferin groups were daily intragastric administration of 100 and 50 mg/kg mangiferin for 8 weeks, respectively. The low dose of mangiferin was set according to previous report . The high dose of mangiferin was set as 2 times of low dose. The rats from MMI-0100 were daily intraperitoneal injected with 50 µg/kg MMI-0100 . At the end of the 8 weeks experimental period, the animals were anesthetized with pentobarbital Na (i.p. 100 mg/kg). Heart tissue samples were collected, weighted and excised. One part fresh tissue was used for subsequent Western blot analysis. One part tissue was fixed in 4% paraformaldehyde, embedded in paraffin and cut into thin sections. Then, the sections were stained by masson and sirius red. One part tissue was made to the 10% tissue homogenate using ice cold 0.9% sodium chloride for the subsequent assay of cardiac oxidative stress and inflammation. All the experiments were performed in accordance with the Chinese legislation and the ethical rules of NIH Guidelines for the Care and Use of Laboratory Animal, and were approved by the ethics committee of affiliated Puai Hospital of Tongji Medical College, Huazhong University of Science and Technology (approval No. PAH2018-12).
The age-related cardiac decline was estimated by detecting the relative weight of heart, the levels of serum cardiac markers (CK and LDH) and the expression of hypertrophic makers (ANP, BNP, and β-MHC) . The relative weight of heart was showed as heart index (HI). HI (mg/g) = the weight of heart (mg) / the weight of body (g). The levels of serum CK and LDH were detected using commercial kits according to instructions. The expression of cardiac ANP, BNP, and β-MHC was detected by Western blot analysis. Rat heart tissue was ground in liquid nitrogen. The protein samples were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis. Then, the samples were transferred to a PVDF membrane
Cardiac fibrosis was evaluated by observing collagen deposition
Rat cardiac oxidative stress was assessed by detecting the activities of enzymatic antioxidants SOD and CAT, as well as the level of lipid peroxidation marker MDA. Cardiac inflammation was evaluated by detecting the levels of IL-1β, IL-6, and TNF-α. The performance was carried out in accordance with the instructions of commercial kits.
The cardiac activity of profibrotic TGF-β1/p38/MK2 signaling pathway was assessed by measuring the expression of TGF-β1 and the phosphorylation levels of p38 and MK2
The values were presented as mean ± standard deviation. The bar graphs were made using Origin 6.0. Results were analyzed statistically by one-way ANOVA followed by Tukey's multiple comparison using SPSS 17.0.1 (SPSS Inc., Chicago, IL, USA). Differences were considered as significant at p < 0.05.
It can be seen from Table 1 that D-gal injection significantly enhanced the heart weight (wt) and HI compared to the Veh. When compared to the aging group, MMI-0100 and mangiferin (50 and 100 mg/kg/d) significantly reduced the heart wt and HI. As showed in Fig. 1, when compared to the Veh, the serum levels (Fig. 1A) of CK and LDH, as well as the cardiac expression (Fig. 1B) of hypertrophic makers ANP, BNP, and β-MHC were significantly increased in the rats from Aging group. When compared to the Aging group, MMI-0100 and mangiferin (50 and 100 mg/kg/d) significantly decreased the levels of CK and LDH, and the expression of ANP and BNP. MMI-0100 and 100 mg/kg/d mangiferin significantly decreased the expression of β-MHC.
Table 1 . Effects of mangiferin on body weight (wt), heart wt and heart index (HI) in D-gal-induced aging rats.
|Body wt (g)||Heart wt (g)||HI (mg/g)|
|Veh||449.1 ± 12.5||1.35 ± 0.07**||3.00 ± 0.07**|
|Aging||454.4 ± 15.6||1.64 ± 0.07##||3.61 ± 0.04##|
|MMI-0100||452.1 ± 12.0||1.40 ± 0.06**||3.09 ± 0.06**,##|
|50 mangiferin||451.8 ± 13.8||1.54 ± 0.07*,##||3.40 ± 0.07**,##|
|100 mangiferin||449.9 ± 13.1||1.47 ± 0.08**,#||3.27 ± 0.08**,##|
D-gal, D-galactose; Veh group, vehicle control group; Aging group, D-gal-induced aging group. Compared to aging group, *p < 0.05, **p < 0.01. Compared to Veh group, #p < 0.05, ##p < 0.01.
Fig. 2 describes the cardiac collagen deposition. When compared to the Veh, D-gal injection significantly enhanced both the proportion of masson-positive and sirius red-positive area. When compared to the Aging group, MMI-0100 and mangiferin (50 and 100 mg/kg/d) significantly reduced the proportion of masson-positive and sirius red-positive area.
Fig. 3 describes that D-gal injection stimulates cardiac oxidative stress (Fig. 3A) and inflammation (Fig. 3B). When compared to the Veh, the cardiac activities of SOD and CAT in rats from Aging group were significantly decreased. Meanwhile, the levels of cardiac MDA, IL-1β, IL-6, and TNF-α were significantly increased in rats from Aging group compared to the Veh. When compared to the Aging group, MMI-0100 and mangiferin (50 and 100 mg/kg/d) significantly increased the cardiac activities of SOD and CAT, decreased the cardiac levels of MDA, IL-1β, IL-6 and TNF-α. It indicated that mangiferin attenuated D-gal-induced cardiac oxidative stress and inflammation in rats.
Fig. 4 represents D-gal injection provokes cardiac pro-fibrogenic TGF-β1/p38/MK2 signaling pathway in rats. When compared to the Veh, the cardiac expression of TGF-β1, the phosphorylation levels of p38 and MK2, as well as the expression of ECM proteins Col-I, Col-III, and α-SMA were significantly enhanced in rats from Aging group. When compared to the Aging group, MMI-0100 and mangiferin (50 and 100 mg/kg/d) significantly reduced the cardiac expression of TGF-β1, p-p38, p-MK2, Col-I, Col-III, and α-SMA. The results showed that mangiferin inhibited D-gal-induced cardiac pro-fibrogenic TGF-β1/p38/MK2 signaling pathway.
Aging is the predisposing risk factor for cardiac hypertrophy, which severely harms the public health and brings huge time and financial burden . D-gal has been long used to establish experimental aging models in animals because that it brings an abnormal D-gal accumulation
Aging is a complex physiological process. The oxidative stress is considered to be one of the possible mechanisms underlying aging-related pathological changes . Evidences indicate that aging causes the decline in mitochondrial function and antioxidant defense systems
Chronic inflammation is another pathological mechanism for CVDs besides oxidative stress . The cardiac levels of proinflammatory mediators (like IL-6 and TNF-α) are over-generated under pathological stimuli . D-gal elevates the levels of these inflammatory cytokines in heart . Furthermore, local inflammation promotes myocyte hypertrophy and interstitial fibrosis, and finally induces cardiac hypertrophy and heart failure . We found that mangiferin attenuated D-gal-induced cardiac inflammation
Fibrosis is associated with ECM accumulation and collagen deposition, and subsequently leads to progressive architectural remodeling in nearly all organs . Cardiac fibrosis is an important pathophysiological changes in cardiac aging and observed in several cardiac diseases [8,14]. Oxidative stress can up-regulate cardiac fibrotic signaling pathway [8,17]. For example, ROS can induce cardiac fibrosis
MAPKs family proteins are involved in diverse cellular processes including cell differentiation, proliferation and apoptosis . It is known that oxidative stress enhances the expression of MAPKs proteins . P38 is a member of MAPKs. P38 signaling pathway can be activated by ROS . The phosphorylation activation of p38 promotes the proliferation and differentiation of cardiac fibroblasts, as well as stimulates collagen deposition. Ultimately, cardiac fibrosis occur . MK2 is a MAPK activated protein kinase belonging to serine-threonine kinase family. MK2 can be activated through phosphorylation under inflammation conditions . MK2 is the downstream of p38. The phosphorylation level of MK2 is in direct proportion to the phosphorylation level of p38 . MK2 is involved in regulating the levels of pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) . Furthermore, MK2 participates in p38-induced heart failure . As a result, MK2 seems to provide a bridge between oxidative stress and inflammation in cardiac fibrosis. Study illustrates that TGF-β is the upstream of MK2 . Blocking MK2 can inhibit TGF-β-induced fibroblast differentiation and ECM protein a-SMA expression . MK2 inhibitor MMI-0100 treatment successfully attenuates cardiac fibrosis and inflammation . It can be concluded that the TGF-β1/p38/MK2 is an important noncanonical fibrosis-regulated signaling pathway in age-related cardiac decline . Previous study found that mangiferin alleviated cardiac ﬁbrosis
In conclusion, the whole findings suggested that mangiferin suppressed D-gal-induced cardiac aging, ameliorated cardiac oxidative stress, inflammation and fibrosis possibly
This research is supported by the Hubei Municipal Health Commission Scientific Research Project (grant numbers WJ2019H329) and by the Wuhan Municipal Health Commission Scientific Research Project (grant numbers WX19A14 and WZ15A01).
J.C., W.W., and L.L. performed the conception and design of study. J.C. wrote the manuscript and performed the Western blot analysis. C.R. and R.C. conducted the acquisition analysis and interpretation of data. Y.L. and P.L. conducted part of the experiments and revised the manuscript.
The authors declare no conflicts of interest.
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