Effects of Paeonol Combined with Panax Notoginseng Saponins on Nrf2/ARE Pathway in Rats with Diabetic Cardiomyopathy

Yanfang FAN, Xiaoli YAN, Zhuoya JIA, Kaihang SHI, Kai CAO

Chengde Medical University, Chengde 067000, China

Abstract [Objectives] To investigate whether paeonol (Pae) combined with panax notoginseng saponins (PNS) can protect the myocardium of rats with diabetic cardiomyopathy (DCM) through improving the antioxidant capacity of the rats by activating the Nrf2/ARE pathway. [Methods] The rats were fed with high-fat and high-sugar diet for 6 weeks, and combined with intraperitoneal injection of small-dose STZ to build a type II diabetes model; the model rats were randomly divided into a model group, a Pae group of 80 mg/kg, and a PNS group of 100 mg/kg, a Pae 80 mg/kg+PNS 100 mg/kg, and a metformin group 157.5 mg/kg; rats of normal group and model group were injected with an equal volume of sodium carboxymethyl cellulose (1%), 10 rats in each group. After 8 weeks, 3 rats in the model group were taken for histopathological examination. Changes in myocardial fibrosis and myocardial collagen formation indicated that the building of DCM model was successful. qRT-PCR and Western blot were used to detect the expression levels of Nrf2, HO-1, Col-I, and Col-III in myocardial tissue of each group of rats. [Results] Compared with the normal group, the mRNA and protein expression levels of Nrf2 and HO-1 in myocardial tissue of the DCM group rats were significantly reduced, and the mRNA and protein expression levels of Col-I and Col-III were significantly increased; compared with the DCM group, the mRNA and protein expression levels of Nrf2 and HO-1 in the myocardial tissue of each drug group increased to varying degrees, and the combined drug group increased more significantly than that of the single drug group; the mRNA and protein expression levels of Col-I and Col-III were reduced to varying degrees, and the combined drug group declined more significantly than that of the single drug group. [Conclusions] Paeonol combined with panax notoginseng saponins can up-regulate the expression of Nrf2 and HO-1 in myocardial tissue, inhibit the expression of type I and type III collagen. The mechanism may be related to improving the DCM myocardial fibrosis through activating the Nrf2/ARE pathway.

Key words Paeonol, Panax notoginseng saponins, Diabetic cardiomyopathy, Myocardial fibrosis, Nrf2/ARE

1 Introduction

Diabetic cardiomyopathy (DCM) is a main complication of diabetes. Myocardial fibrosis is an essential pathological feature of DCM. In addition, diabetic myocardial fibrosis is one of the main causes of the onset and death of heart failure[1]. DCM has many pathological mechanisms, including microvascular changes, oxidative stress, inflammatory response, and fibrosis. Among them, oxidative stress is a key factor[2]. The Nrf2/ARE signaling pathway has been found to be the most important endogenous antioxidant stress pathway. At present, the treatment of DCM mainly depends on biochemical hypoglycemic agents such as insulin, tolbutamide and the like. However, these drugs are often not satisfactory due to considerable side effects. At present, there is no exact drug for the treatment of DCM, which may be related to the single target of the drug. Traditional Chinese medicine has the advantage of multiple targets, so it is necessary to develop new, better and safe traditional Chinese medicine to control and treat DCM.

Paeonol (Pae) is the main active component of the root bark ofPaeoniasuffruticosa(family ranunculus) and the plant ofPycanostelmapaniculatum(Bunge) K. Schum (family Rumaceae). Paeonol is antitumor and has the function of lowering blood sugar and insulin resistance in type II diabetic rats[3].Panaxnotoginsengsaponins (PNS) is the main active component of Panax notoginseng (Burk.) F.H.Chen, mainly used to prevent cardiovascular and cerebrovascular diseases. Its pharmacological effects include anticancer, neuroprotection, hypoglycemia and prevention of diabetes complications[4].

Previous research of our research team[5]found that the combined application of Pae and PNS can inhibit the gene expression of type I and type III collagen of cardiac fibroblasts, and can also improve rat myocardial fibrosis caused by ventricular remodeling after myocardial infarction. However, there is still no report about whether the combination of Pae and PNS has function of resisting the myocardial fibrosis of DCM. In accordance with the modern medicine’s understanding of the occurrence and development of diseases, we used an animal model of diabetic cardiomyopathy to investigate whether there is a synergistic effect and mechanism of the combined application of Pae and PNS in improving the myocardial fibrosis of DCM.

2 Data and methods

2.1GeneraldataHealthy male Sprague-Dawley (SD) rats, body mass of (200±10) g (Beijing Vital River Laboratory Animal Technology Co., Technology Co., Ltd., license No.SCXK (Beijing) 2012-0001). Paeonol (Xi’an Xiaocao Botanical Development Co., Ltd., purity 99%, batch No.xc20160617), Panax notoginseng saponins (Xi’an Xiaocao Botanical Development Co., Ltd., purity 80%, batch No.xc20151102), Metformin Tablet (Shanghai Sine Pharmaceutical Laboratories Co., Ltd., National Medicine Permit No.H31020246), blood glucose paper (Beijing Yicheng Bioelectronics Technology Co., Ltd.), high-fat and high-sugar feed (60.4% base feed, 20% sucrose, 10% lard, 1.5% cholesterol, 8% egg yolk powder, 0.1% sodium cholate), rat basic feed (Beijing Keao Xieli Feed Co., Ltd.). PCR reverse transcription kit (Takara, batch No.RR420A), amplification kit (Takara, batch No.RR047A), Collagen I (Col-I), Collagen III (Col-III), Nrf2, HO-1, GADPH primers (Sangon Biotech (Shanghai) Co., Ltd.), Collagen I, Collagen III, Nrf2, HO-1, GADPH primary antibody (Abcam, USA), Horseradish labeled goat anti-rabbit IgG (H+L) (KPL, USA), transfer electrophoresis instrument (DYY-BC Beijing Liuyi Biotechnology Co., Ltd.), PCR instrument (iCycle7 Thermal Cycler Genetimes Technology Inc.), fluorescence quantitative PCR (Mx3000P, Bio-Rad, USA).

2.2Preparationofratmodelofdiabeticcardiomyopathy

Selected 80 SPF-grade male SD rats with weight of (200±10) g, adaptive fed for one week. Randomly selected 10 rats as the normal group, and the remaining rats were fed with high-fat and high-sugar feeds. After 6 weeks, they were fasted for 16 h, and intraperitoneally injected STZ 25 mg/kg for 2 consecutive days (dissolved in 0.1% citrate buffer, pH 4.3). The rats of normal group were injected with an equal volume of citric acid buffer. On the third day and seventh day after STZ injection, blood was collected from the tail vein to measure fasting blood glucose in rats. Blood glucose≥16.7 mmol/L was considered successful in building the type II diabetes model. the model rats were randomly divided into a model group, a Pae group of 80 mg/kg, and a PNS group of 100 mg/kg, a Pae 80 mg/kg+PNS 100 mg/kg, and a metformin group 157.5 mg/kg; rats of normal group and model group were injected with an equal volume of sodium carboxymethyl cellulose (1%), 10 rats in each group. After 8 weeks, 3 rats in the model group were taken for histopathological examination. Changes in myocardial fibrosis and myocardial collagen formation indicated that the building of DCM model was successful[6-7]. Myocardial tissue was taken for molecular biology testing.

2.3DetectionoftheexpressionofCol-I,Col-III,Nrf2andHO-1mRNAinratmyocardialtissuebyqRT-PCRThe total RNA was extracted by Trizol from myocardial tissue, and reversely transcribed RNA into cDNA according to the kit steps. Primer sequence Col-I: upstream primer: 5′-ACCGTGGTGAGACTGGTCCT-3 ′, downstream primer: 5′-TGTCACCTTGTTCGCCTGTC -3′, amplified fragment: 120 bp; Col-III: upstream primer: 5′-ACCAGGGCTGCAAGATGGAT -3, downstream primer: 5′-CTCAGCACCAGCATCTGTCC-3 ′, Amplified fragment: 122 bp; Nrf2: upstream primer: 5′-GCCTTCCTCTGCTGCCATTA-3 ′, downstream primer: 5′-AACTCCACCGTGCCTTCAGT-3′, amplified fragment: 120 bp; HO-1: upstream primer: 5′-CGTGCTCGCATGAACACTCT -3 ′, downstream primer: 5′-AGCAGGAAGGCGGTCTTAGC-3′, amplified fragment: 80 bp; GAPDH: upstream primer: 5′-GACATGCCGCCTGGAGAAAC-3 ′, downstream primer: 5′-AGCCCAGGATGCCCTTTAGT-3′, amplified fragment: 92 bp. Added to the reaction system at 95 ℃, pre-denaturated for 3 min at 95 ℃, and denatured for 5 min. The target gene was amplified at 59 ℃ annealing temperature, and extended at 72 ℃ for 30 s. The amplification curve and dissolution curve of the expression of each indicator in the myocardium of each group were obtained. The data were analyzed and calculated using the 2-ΔΔCtmethod. TheCtvalue denotes the number of cycles that the fluorescence intensity reaches the set threshold during the reaction. ΔΔCt= (Mean value ofCtof target genes in the test group-Mean value ofCtof reference genes in the test group)-(Mean value ofCtof target genes in the control group-Mean value ofCtof reference genes in the control group).

2.4DetectionofCol-I,Col-III,Nrf2andHO-1proteincontentinmyocardialtissueofratsbyWesternBlottingTotal protein was extracted from myocardial tissue, and the protein was quantified by BCA method. Take 50 μg of total protein sample loading amount, 12% SDS-PAGE gel constant-voltage electrophoresis, 130 mA stable transfer membrane on ice, sealed with 5% skimmed milk powder overnight at 4 ℃, primary antibody (dilution ratio: Nrf2 and Col-I was 1∶ 2 000 and HO-1 was 1∶4 000, Col-III was 1∶3 000, and GAPDH was 1∶10 000). Incubated for 2 h at room temperature with a shaker, washed the membrane 3 times at 15, 10, and 10 min with TBST. The secondary antibody (1∶5 000) was incubated at room temperature on a shaker for 2 h. The membrane was washed 3 times with TBST for 5 min. Dripped the ECL luminescent liquid and developed with the Tanon 6 100 chemiluminescence imaging system. The gray value of the target protein and GAPDH bands were analyzed with the aid of Quantity One software, and the relative expression level of the target protein was expressed by its ratio.

3 Results and analysis

3.1ResultsofrealtimePCRCompared with the normal control (NC) group, the mRNA expression levels of Nrf2 and HO-1 in myocardial tissue of the DCM group were significantly reduced (P<0.05), and the mRNA expression levels of Col-I and Col-III were significantly increased (P<0.05); compared with the DCM group, the mRNA expression levels of Nrf2 and HO-1 in the myocardial tissue of each drug group were increased to varying degrees (P<0.05); Col-I and Col-III mRNA expression levels were reduced to varying degrees (P<0.05); compared with the single drug group of Pae and PNS, the combined drug group had a significantly higher increase in Nrf2 and HO-1 (P<0.05), and a significantly higher decline in Col-I and Col-III (P<0.05), as shown in Fig.1.

3.2WesternblottingdetectionresultsCompared with the NC group, the protein expression levels of Nrf2 and HO-1 in the myocardial tissue of the DCM group were significantly reduced (P<0.05), and the protein expression levels of Col-I and Col-III were significantly increased (P<0.05); compared with the DCM group, the expression levels of Nrf2 and HO-1 protein in the myocardial tissue of each drug group were increased to varying degrees (P<0.05); Col-I and Col-III expression levels were reduced to varying degrees (P<0.05); compared with the single drug group of paeonol and panax notoginseng saponins, the combined drug group had a significantly higher increase in Nrf2 and HO-1 (P<0.05), and a significantly higher decline in Col-I and Col-III (P<0.05), as shown in Fig.2 and Table 1.

Note: compared with NC group:1)P<0.05; compared with DCM group:2)P<0.05; compared with Pae group:3)P<0.05; compared with PNS group:4)P<0.05; compared with MH group:5)P<0.05.

Fig. 1 Nrf2, HO-1, Col-I, Col-III mRNA detection in myocardial tissue RT- PCR

Note: 1: NC group, 2: DCM group, 3: Pae group, 4: PNS group, 5: Pae + PNS group, 6: MH group.

Fig.2Nrf2,HO-1,Col-I,Col-IIIproteinexpressionmyocardialtissuebyWesternBlotting

GroupNrf2HO-1Col-ICol-IIINC0.951 4±0.020 20.972 3±0.023 70.497 6±0.023 50.381 1±0.020 6DCM0.790 8±0.013 41)0.603 4±0.011 51)1.052 1±0.0266 21)0.768 0±0.045 11)Pae0.876 0±0.049 22)0.964 1±0.008 02)0.863 0±0.035 12)0.527 8±0.024 82)PNS0.922 8±0.009 52) 3)0.951 9±0.007 72)0.844 9±0.017 62)0.453 5±0.010 12) 3)Pae+PNS0.991 8±0.006 52) 3) 4) 5)0.989 2±0.005 12) 3) 4)0.680 0±0.021 12) 3) 4) 5)0.430 2±0.007 52) 3) 4) 5)MH0.954 7±0.007 32) 3) 4)0.984 9±0.002 62) 3)0.787 0±0.044 92) 3) 4)0.501 4±0.023 82) 3) 4)

Note: compared with NC group:1)P<0.05; compared with DCM group:2)P<0.05; compared with Pae group:3)P<0.05;compared with PNS group:4)P<0.05; compared with MH group:5)P<0.05.

4 Discussions

Under normal physiological conditions, when the production of reactive oxygen species (ROS) in the body increases, the Nrf2/ARE signaling pathway was activated, and increases the expression of Nrf2, and the expression of downstream antioxidant enzymes SOD, HO-1 and so on. And these antioxidant enzymes can scavenge ROS, to keep the body remain in a balanced state. Some study[8]indicated that, in case of DCM, the body undergoes oxidative stress reactions, and Nrf2 activation is impaired or missing, resulting in a decline in the expression of antioxidant enzyme genes downstream of Nrf2, which cannot effectively eliminate ROS, thereby aggravating the oxidative stress response and increasing the free radical oxidative stress product MDA in the body. With the increase of ROS, TGF-β1 is over-expressed, which promotes the transformation of fibroblasts into myofibroblasts and increases collagen synthesis[9]; promotes the decline of MMP-2 expression and increases the expression of TIMP-2, inhibits collagen degradation, and ultimately increases the expression of collagen I and collagen III, leading to pathological changes of myocardial fibrosis[10]. Therefore, it is particularly important to activate the anti-oxidative stress pathway in the body and scavenge ROS to delay the occurrence and development of DCM. In this experiment, a model of diabetic cardiomyopathy with high-fat and high-glucose combined with low-dose STZ was established[11]. After 8 weeks of drug intervention, the expressions of Nrf2, HO-1, Col-I, Col-III mRNA and protein in rat myocardial tissue were detected. The results showed that the expression levels of Nrf2 and HO-1 mRNA and protein in the myocardial tissue of rats in the DCM group were significantly reduced (P<0.05). When DCM occurred, endogenous antioxidant stress pathways in myocardial tissue became impaired and cannot play an antioxidant role. The expression levels of Col-I and Col-III mRNA and protein in myocardial tissues were significantly increased (P<0.05), indicating that after the occurrence of DCM,

cardiomyocytes were injured, fibroblasts were promoted, and the expression of Col-I and Col-III were up-regulated, accordingly leading to myocardial fibrosis. Besides, the expression levels of Nrf2, HO-1 mRNA and protein in the myocardial tissue of rats in each drug group were increased to varying degrees (P<0.05); Col-I and Col-III mRNA and protein expression levels were reduced to varying degrees (P<0.05), suggesting that both Pae and PNS can inhibit myocardial cell damage and delay the occurrence and development of myocardial fibrosis. Compared with the single drug, the effect of the combined drug group was more significant (P<0.05). In summary, the results of this experiment indicated that the action mechanism of Pae combined with PNS on diabetic cardiomyopathy may be achieved through activating the Nrf2/ARE oxidative stress signaling pathway and up-regulating the expression of antioxidant enzymes like HO-1.