Ai-Hua Li, Qing-Qing Wang, Ying-Chun Qin , Yi-Lin Xie , Zhi-Qiang Yan

1. College of Medicine, Anhui University of Science and Technology, Huainan 232001, China

2. Zhongshan Hospital of Traditional Chinese Medicine, Zhongshan 528401, China

3. Shanghai Jiao Tong University-Minhang Campus, School of Life Science and Biotechnology, Shanghai Key Laboratory for Reproductive Medicine,Shanghai 200240, China

4. Anhui University of Science and Technology Affiliated Fengxian Central Hospital, Shanghai 201499, China

Keywords:Canagliflozin Cardiac hypertrophy Cardiac fibrosis SHR

ABSTRACT Objective: To investigate the effects of cagliazin, a sodium-glucose cotransporter 2 inhibitor(SGLT-2I), on ventricular remodeling in spontaneously hypertensive rats (SHR) through renin angiotensin system (RAS) and transforming growth factor -β1(TGF-β1). Methods: The experiment was divided into 4 groups: normal blood pressure control group, SHR group, cagliet net low-dose group (30mg/kg), cagliet net high-dose group (60mg/kg), once a day for 8 weeks.Normal blood pressure rats (WKY) were used as the control group to measure blood pressure with tail sleeve sphygmomanometer (BP) and blood glucose level was measured with glucose meter Cardiac function was evaluated by echocardiography, cell area of left ventricle was evaluated by histomorphology, real-time quantitative polymerase chain reaction and protein imprinting hybridization were used to detect TGF-β1 Smad4 renin from type I collagen (Col1a)type III collagen (Col3a) matrix metalloproteinase 2(MMP-2) Expression results of angiotensin II1 type receptor 1(AGTR1) and Angiotensin II2 type receptor 2 (AGTR2). Results:After 8 weeks of administration, the cardiac weight/body weight ratio (HW/BW) of left ventricular weight/heart weight ratio (LVW/HW) of kaglinet low-dose group and high-dose group was statistically significant compared with that of spontaneous hypertensive rats (P< Compared with SHRs, the expression of Col1a, Col3a, MMP2, TGF-β1, Smad4, Renin AGTR1 was significantly down-regulated and the expression of AGTR2 was up-regulated in cagliet net lowdose and high-dose groups Conclusions: Cagliazin can improve hypertension-induced cardiac remodeling by regulating RAS and TGF-β1/Smad signaling pathways. Conclusion: From the results, canaglifozin was found to ameliorate pressure overload-induced cardiac remodeling by regulating the RAS and TGF-β1/Smad signaling pathway.

1. Introduction

Cardiovascular diseases are the major cause of morbidity and mortality worldewide[1]. hypertension is an independent risk factor for cardiovascular disease, in the pathogenesis of cardiovascular disease and pathological ventricular remodeling play an important role [2]. As one of multiple organs affected by high blood pressure, blood pressure rise for a long time to be able to cause to myocardial hypertrophy and pathological characteristic of muscle fibers into ventricular remodeling Hypertension promotes hypertrophic growth of cardiomyocytes and increases the size of cardiomyocytes [3]. Cardiomyocyte hypertrophy is considered to be an adaptive response to overload stimulation. However, continuous stimulation can lead to cardiac decompensation and heart failure[4]. Hypertension can promote fibroblast proliferation and induce overexpression of type I collagen (Col1a) and type III collagen(Col3a), which accumulate in the interstitium [4]. These two types of collagen can lead to myocardial fibrosis and ultimately lead to left ventricular dysfunction[3, 4]. Ventricular hypertrophy is an independent risk factor for cardiovascular events in hypertensive patients, and reversing cardiac hypertrophy can help reduce the risk of cardiovascular complications in patients.

There is increasing evidence that renin angiotensin system (RAS)is involved in pathological ventricular remodeling [5]. Angiotensin gen is synthesized in the liver and lysed by renin into the effector molecule angiotensin II(AngII)[6]. As an important member of RAS, Ang II plays a role by interacting with angiotensin II receptor (AGTR1) [7]. RAS components can also be produced by intracellular and local tissues and play an important role in tissue remodeling [8]. Studies have found that cardiomyocytes and fibroblasts simultaneously express AGTR1 and angiotensin II type 2 receptor (AGTR2)[9]. Ang Binding of II to AGTR1 activates several intracellular signaling pathways, including the pro-fibrotic transforming growth factor -β(TGF-β) pathway [5]. TGF-β1 pathway plays a key role in stimulating fibroblast proliferation and migration and promoting the deposition of extracellular matrix [10].Angiotensin converting enzyme (ACE) inhibitors and Ang Receptor II blockers are widely used in the treatment of cardiovascular disease[11]. However, the morbidity and mortality of heart failure remain high and therefore a new strategy for the treatment of pathological ventricular remodeling and heart failure is needed.

Cagliazin is a member of a class of sodium-glucose cotransporter-2 inhibitors (SGLT2i) that reduce blood glucose by blocking the glomerular reabsorption of glucose [12]. Cagliazin has been used in the treatment of patients with type 2 diabetes. In addition to lowering blood glucose, there is increasing evidence that cagliazin has cardiovascular protective effects Meta-analysis showed that treatment with cagliazin significantly reduced heart failure events[13]. It has been reported that cagliazin can improve cardiometabolic and diastolic functions, reduce vascular hardness, and lower blood pressure[13, 14]. Kagligin has antioxidant and anti-inflammatory effects in vivo and in vitro studies [15]. Kagligin significantly improves cardiac function and inflammatory response in diabetic mice by inhibiting mTOR/HIF-1α signaling pathway[15]. Cagliazin alleviates isoproterenol-induced cardiac oxidative stress by stimulating a variety of antioxidant and anti-inflammatory signaling pathways [16]. Cagliazin alleviates cardiac oxidative stress, improves cardiac ischemia-reperfusion injury, and improves cardiac function by stimulating antioxidant and anti-inflammatory signaling [17].Similarly, other members of SGLT2i, such as Empagliflozin, have been reported to reduce myocardial fibrosis in diabetic mice and maintain myocardial function in a mouse model of stress-induced heart failure [18, 19]. Although the beneficial effects of cagliazin on cardiovascular disease have been studied, the effect of cagliazin on cardiac remodeling induced by hypertension has not been reported.Smad pathways and can prevent hypertensive myocardial fibrosis and pathological myocardial hypertrophy.

2. Materials and methods

2.1 Experimental animals and drug therapy

Male spontaneously hypertensive rats (SHR) and normal blood pressure rats (WKY) were purchased from Beijing Weitonglihua Experimental Animal Co., LTD. {Lot No. :SCXK (Beijing) 2016-0006} and fed with ordinary feed and tap water. The rats were kept at standard temperature and light condition and divided into WKY group (n=8). SHR group (n=8) Low-dose canagliflozin group (30mg/kg/day) (n=8) and high-dose Canagliflozin group(60mg/kg/day) (n=8) Canagliflozin was dissolved in 0.8% sodium carboxymethyl cellulose by gavage once a day. The WKY group and THE SHR group were treated with sodium carboxymethyl cellulose only. Systolic blood pressure (SBP) and diastolic blood pressure(DBP) were measured by tail-sleeve method. Beijing, China) Blood pressure, blood glucose and body weight of rats were continuously monitored once a week before and during the 1st to 8th week of administration, and fasting 8-hour blood glucose was measured weekly with a glucose meter (Fish Leap, China) during the same time period All animal experiments were conducted in accordance with the Animal Management Regulations of the Ministry of Health of China (Document no. 55, 2001) and approved by the Animal Ethics Committee of Southern Medical University.

2.2 Echocardiography

After 8 weeks of treatment, ultrasound system was used (Vevo 2100, VisualSonic, Inc,Toronto, Canada) and 15mhz high-frequency probe echocardiography in laboratory animals rats anesthetized with isoflurane (1.5%) were evaluated for: end diastolic left ventricular posterior wall thickness end systolic left ventricular volume end diastolic end systolic ventricular septal thickness ejection fraction.

2.3 Tissue section

The left ventricle was fixed overnight with 4% paraformaldehyde at 4 times and then the tissue was dehydrated and embedded in paraffin. The sections were 4μm thick and stained with hematoxylin and eosin (HE) to detect morphology and assess myocardial fibrosis with trichromatic massone staining (Solarbio, Beijing, China)section images were obtained using a microscope (BX53; Olympus Corporation, Tokyo, Japan) analyzed the results using ImageJ software.

In this study, we found that cagliazin regulates RAS and TGF-β1/

2.4 Real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted from myocardial tissue samples using TRIzol reagent (Invitrogen, USA) and PCR was performed using RT Master Mix (MedChemExpress, New Jersey, USA) reverse transcription of total RNA into cDNA using a 2 SYBR premixed Ex Taq kit with ROX (Kangwei, China) rT-PCR reaction at 95 under 2 min,95 5 s,60 30 s, followed by 40 cycles of signal detection on ABI Q6 machine (Applied Biosystems, USA) With GAPDH as internal reference and 2-δδ Ct method, PCR primer sequences used in this study were calculated as follows:

2.5 Western blotting

Left ventricular tissue was lysed with RIPA buffer containing protease inhibitors (MedChemExpress, New Jersey, 100 After boiling for 10 min, the proteins were separated by 10% SDSpolyacrylamide gel and transferred to nitrocellulose membrane(Thermofish, USA). The membrane was sealed with 5%skim milk powder Tris buffer at room temperature for 1 h,then with type I collagen (Col1a)(1:1000,Proteintech, USA)Type III collagen (Col3a)(1:1000,Proteintech, USA) TGFβ1(1:1000,CST, USA) Smad4(1:1000,CST, USA) Antibodies to GAPDH(1:1000,Proteintech, USA) and β -tubulin(1:1000,Proteintech, USA) were incubated overnight at 4 ℃ and then the protein bands were exposed using enhanced chemiluminescence reagent (Shenger, China) after incubation with secondary antibody Images were obtained and analyzed using Tanon Image Analyzer(Tanon Science, Shanghai, China).

2.6 Statistical Analysis

The GraphPad 7 software was used for statistical analysis. The results were expressed as mean standard deviation of comparison between groups. One-way ANOVA was used for analysis (as blood pressure, weight and blood sugar at time points). 0.05 was considered statistically significant.

3. Results

3.1 Effects of cagligin on blood pressure in SHR rats

To observe the effect of cagliazin on blood pressure, the systolic and diastolic blood pressure of rats were measured by tail-sleeve method for 8 weeks. Compared with the WKY group, the systolic and diastolic blood pressure of the SHR group was significantly higher than that of the WKY group, and the difference was statistically significant (P< 0.01) after 2-8 weeks of administration, systolic blood pressure in the sr-cana (30mg/kg) group was lower than that in the SHR group (60mg/kg), and the difference was statistically significant (P< 0.01) after 2 weeks of administration, the diastolic pressure of sr-cana (60mg/kg) group was lower than that of SHR group; After 3-8 weeks of administration, the diastolic blood pressure of the sr-cana (30mg/kg) group was lower than that of the SHR group (60mg/kg), and the difference was statistically significant(P< 0.01) see Table 1 and Figure 1.

Table1 Effect of canagliflozin on blood pressure in SHRs(±s)

Table1 Effect of canagliflozin on blood pressure in SHRs(±s)

Note: Compared with SHR group, #P<0.01

Systolic pressure(mmHg)Time(week) WKY SHR SHR-CANA(30mg/kg) SHR-CANA(60mg/kg)0 140.70±4.40# 201.80±21.91 196.40±10.62 207.80±9.43 1 139.50±5.43# 209.40±8.70 199.00±4.58 196.30±6.21 2 150.70±9.44# 230.30±14.49 206.80±5.80# 191.80±10.59#3 144.00±8.17# 212.30±7.88 186.70±12.78# 187.20±12.81#4 147.60±12.09# 217.50±16.20 183.70±6.13# 183.70±10.85#149.30±10.07# 226.50±13.71 178.40±15.29# 185.80±17.57#6 138.00±5.60# 208.00±7.024 185.70±12.23# 173.30±5.73#7 156.80±9.70# 235.70±5.30 205.30±16.38# 202.60±14.73#8 147.20±1.97# 234.60±15.78 183.90±10.78# 175.80±11.5#5 2.661 P＝0.001 Diastolic pressure (mmHg)Time(week) WKY SHR SHR-CANA(30mg/kg) SHR-CANA(60mg/kg)0 113.60±2.20# 165.50±11.89 169.30±8.30 166.00±18.43 1 111.90±4.90# 164.10±8.46 172.80±15.03 166.20±6.80 2 112.80±3.20# 192.10±9.96 187.00±11.26 172.30±4.57#3 114.80±3.50# 188.60±5.06 151.40±6.73# 142.30±12.06#4 112.80±5.69# 174.10±9.36 146.70±8.84# 141.20±16.40#5 119.10±3.18# 174.00±10.42 144.30±12.90# 134.70±11.28#6 114.50±3.82# 164.80±2.43 133.80±9.95# 115.20±5.61#7 108.30±1.87# 195.70±13.80 158.80±7.41# 133.00±2.94#8 104.30±4.63# 191.90±12.34 140.00±10.65# 132.80±5.13#F 7.832 P＜0.001 F

Figure 1. Effect of canagliflozin on blood pressure in SHRs.

3.2 Effects of cagligin on blood glucose and body weight in SHR rats

The effect of kagligin on blood glucose was studied, as shown in figure 2A. Compared with the SHR group, the blood glucose in the shr-cana group (30mg/kg) and the shr-cana group (60mg/kg) decreased 1-8 weeks after administration, and the difference was statistically significant (P< 0.01) However, there was no significant difference in blood glucose in the WKY group because of the effect of cagliazin on weight loss in diabetic patients and animal models [13, 20], we investigated the effect of cagliazin on SHR body weight. Compared with THE SHR group, the shR-CANA group (30mg/kg) and the SHR-CANA group (60mg/kg) showed a significant decrease in body weight (P< 0.01), however, there was no significant difference in body weight in the WKY group, as shown in Table 2 and Figure 2.

Figure 2. Effect of canagliflozin on blood glucose and body weight in SHRs.

Table 2 Effect of canagliflozin on blood glucose and body weight in SHRs (±s)

Table 2 Effect of canagliflozin on blood glucose and body weight in SHRs (±s)

Note: Compared with SHR group, #P<0.01

Blood glucose(mmol/L)Time（week) WKY SHR SHR-CANA(30mg/kg) SHR-CANA(60mg/kg)0 6.73±0.22 7.23±0.81 6.90±0.22 6.80±0.37 1 7.00±0.56 7.83±0.55 6.60±0.34# 6.55±0.58#2 7.00±0.56 7.03±0.76 5.68±0.76# 5.60±0.36#3 6.48±0.66 6.70±0.55 4.95±0.26# 4.98±0.81#4 6.30±0.32 6.20±0.53 4.40±0.32# 4.00±0.24#5 6.33±0.67 6.80±0.50 5.08±0.26# 4.15±0.17#6 6.40±0.50 6.50±0.41 4.93±0.28# 4.78±0.28#7 6.53±0.92 7.08±1.10 4.38±0.70# 4.48±0.36#8 6.50±0.59 6.63±0.46 4.68±0.33# 4.18±0.30#F 2.553 P＝0.001 Weight(g)Time(week) WKY SHR SHR-CANA(30mg/kg) SHR-CANA(60mg/kg)0 313.00±8.29 319.30±17.67 304.50±8.89 312.50±16.92 1 327.80±19.33 330.80±7.27 292.00±9.42# 296.00±9.90#2 334.80±15.44 337.80±3.10 267.80±4.19# 291.30±6.24#3 343.00±8.76 336.00±7.87 280.50±5.75# 271.30±3.86#4 335.30±6.70 338.00±4.69 258.50±10.34# 259.80±2.87#5 339.80±6.70 350.80±7.32 257.00±14.67# 255.00±8.60#6 332.50±6.25 347.00±4.76 262.80±10.50# 246.30±7.46#7 341.00±3.74 348.80±7.54 258.00±7.70# 244.00±8.60#8 342.50±10.41 339.00±6.83 250±13.76# 252.00±5.29#F 51.027 P＜0.001

3.3 Kaagligin can improve myocardial hypertrophy induced by hypertension

After 8 weeks of treatment with cagliazin, transthoracic echocardiography and histology were used to assess cardiac structure and function in all animals compared with the SHR group. There were no statistically significant differences in end-diastolic ejection fraction and left ventricular volume between the SHR group and THE WKY group. However, ventricular septal thickness and left ventricular posterior wall thickness were observed in the SHR group The volume at the end of contraction period increased compared with WKY group, and the difference was statistically significant(P< 0.05) compared with the SHR group, the ventricular septal thickness at the end of contraction volume of the left ventricular posterior wall thickness in the shr-cana group (30mg/kg) and the shrcana group (60mg/kg) were all decreased, and the difference was statistically significant (P< 0.05), as shown in Figure 3 and Table 3, the ratio of heart weight/body weight (HW/BW) and left heart weight/heart weight (LHW/HW) was detected. Compared with the WKY group, HW/BW and LHW/HW were increased in the SHR group, and the difference was statistically significant (P< 0.05),compared with the SHR group, the HW/BW and LHW/HW of the SHR-CANA(30mg/kg) group and the SHR-CANA(60mg/kg) group were decreased, and the differences were statistically significant(P< 0.05) HE staining results showed that the cross-sectional area of cardiomyocytes in THE SHR group was significantly increased compared with that in the WKY group, and the difference was statistically significant (P< 0.01) compared with the SHR group,the cross-sectional area of myocardial cells in the shr-cana (30mg/kg) group was significantly reduced in the shr-cana (60mg/kg)group, and the difference was statistically significant (P< 0.01),see Figure 4 and Table 4.

Figure 5 Effects of canagliflozin on cardiac fibrosis in SHRs.

Table 4 Effects of canagliflozin on cardiac hypertrophy in SHRs.(±s)

Table 4 Effects of canagliflozin on cardiac hypertrophy in SHRs.(±s)

Note: Compared with SHR group, #P<0.01

Group n Heart/body weight(g/g) Left ventricle/heart weight(g/g) HE staining (area of cardiomyocyte, μm2)WKY 8 0.0033±0.00015# 0.69±0.0056# 6565.00±375.50#SHR 8 0.0040±6.923e-005 0.80±0.023 14030.00±885.40 SHR-CANA(30mg/kg) 8 0.0037±1.431e-005# 0.72±0.032# 7067.00±371.60#SHR-CANA(60mg/kg) 8 0.0035±0.00013# 0.73±0.047# 5770.00±557.60#F 32.95 8.45 169.8 P 0.0001 0.0027 0.0001

Figure 3. Effects of canagliflozin on left ventricle cardiac hypertrophy and function in SHRs.

Figure 4 Effects of canagliflozin on cardiac hypertrophy in SHRs.

Table 3 Effects of canagliflozin on left ventricle cardiac hypertrophy and function in SHRs. (±s)

Table 3 Effects of canagliflozin on left ventricle cardiac hypertrophy and function in SHRs. (±s)

Note: Compared with SHR group, #P<0.05

Group n Lvpw(mm) Ivst(mm) End-systolic volume(μL)WKY 8 1.68±0.20# 2.68±0.14# 85.45±21.19#SHR 8 2.09±0.25 3.32±0.27 125.4±23.44 SHR-CANA(30mg/kg) 8 1.67±0.20# 2.80±0.39# 91.09±9.76#SHR-CANA(60mg/kg) 8 1.63±0.17# 2.47±0.22# 84.97±14.15#F 5.52 8.95 5.88 P 0.0085 0.0010 0.0066

3.4 Kaglidine alleviates myocardial fibrosis in SHR

To determine whether kagligin affects myocardial fibrosis in SHR, myocardial collagen content was assessed by tricolor massone staining. Histological examination showed that the volume of myocardial interstitium collagen was increased in the SHR group compared with the WKY group, whereas in the shrcana (30mg/kg) group The sr-cana (60mg/kg) group decreased(figure 5A). Next, we measured the mRNA and protein expression of myocardial fibrosis marker Col1a and the mRNA expression of Col3a. The results showed that the expression of Col1a and Col3a was significantly increased in the SHR group compared with the WKY group Compared with the SHR group, the expression of Col1a and Col3a was inhibited in the shr-cana (30mg/kg) group and the shr-cana (60mg/kg) group, and the difference was statistically significant (P< 0.05) due to matrix metalloproteinase-2, MMP2 was associated with myocardial fibrosis. We detected the expression of MMP2. Rt-pcr and Western blot results showed that the relative mRNA and protein levels of MMP2 in the SHR group increased compared with the WKY group, and the differences were statistically significant (P< 0.01) however, compared with the SHR group,the expression of MMP2 was decreased in the shr-cana (30mg/kg)group and the shr-cana (60mg/kg) group, and the difference was statistically significant (P< 0.01), see Figure 5 and Table 5.

Table 5 Effects of canagliflozin on cardiac fibrosis in SHRs.(xˉ ± s)

3.5 Effect of kagligin on expression of AGTR1, AGTR2,Renin, TGF-β1 and Smad4 in SHR myocardial tissue

Since TGF-β signaling pathway plays an important role in myocardial hypertrophy and fibrosis, we detected mRNA and protein expressions of TGF-β1 and Smad4 in the SHR group. Compared with the WKY group, the mRNA and protein levels of TGF-β1 and Smad4 in the SHR group were significantly increased, and the differences were statistically significant (P< 0.01) however,compared with SHR group, mRNA and protein levels of TGFβ1 and Smad4 in shr-cana (30mg/kg) group and shr-cana (60mg/kg) group were decreased, and the differences were statistically significant (P< Considering the important role of the RAS system in cardiac remodeling, we evaluated some RAS members. Rtpcr results showed that mRNA levels of AGTR1 and renin were increased in the SHR group compared with the WKY group, while AGTR2 was increased in the SHR group compared with the WKY group MRNA levels were decreased, and the differences were statistically significant (P< 0.01) however, compared with the SHR group, mRNA expression of AGTR1 and Renin in the SHRCANA group (30mg/kg) and the SHR-CANA group (60mg/kg) were decreased, while that in the SHR-CANA group (30mg/kg) AGTR2 mRNA expression was significantly increased in sr-cana (60mg/kg)group, and the difference was statistically significant (P< 0.01),see Figure 7 and Table 7.

Table 6 Effects of canagliflozin on TGF-β1 and Smad4 expression in cardiac tissue of SHRs(±s)

Table 6 Effects of canagliflozin on TGF-β1 and Smad4 expression in cardiac tissue of SHRs(±s)

Note: Compared with SHR group, #P<0.01

Group n Smad4/β-tubulin(western-blotting) Smad4/GAPDH(RT-PCR) TGF-β1/β-tubulin(western-blotting) TGF-β1/GAPDH(RT-PCR)WKY 8 0.35±0.13# 0.42±0.015# 0.44±0.068# 1.00±0.00#SHR 8 1.72±0.19 1.00±0.00 1.51±0.54 2.28±0.49 SHR-CANA(30mg/kg) 8 0.46±0.13# 0.51±0.015# 0.37±0.028# 1.35±0.059#SHR-CANA(60mg/kg) 8 0.59±0.18# 0.39±0.13# 0.51±0.12# 0.90±0.47#F 45.98 16.03 11.03 13.4 P 0.0001 0.001 0.0032 0.0004

Table 7 Effects of canagliflozin on expression of AGTR1,AGTR2, Renin in cardiac tissue of SHRs(±s)

Table 7 Effects of canagliflozin on expression of AGTR1,AGTR2, Renin in cardiac tissue of SHRs(±s)

Note: Compared with SHR group, #P<0.01

Group n AGTR1/GAPDH(RT-PCR) AGTR2/GAPDH(RT-PCR) Renin/GAPDH(RT-PCR)WKY 8 1.00±0.00# 1.00±0.00# 1.00±0.00#SHR 8 4.61±0.99 0.17±0.019 4.78±1.89 SHR-CANA(30mg/kg) 8 1.24±0.46# 0.84±0.36# 1.39±0.52#SHR-CANA(60mg/kg) 8 0.54±1.6# 0.62±0.25# 0.96±0.19#F 34.34 10.53 10.51 P 0.0001 0.0011 0.0038

Figure 6 Effects of canagliflozin on TGF-β1 and Smad4 expression in cardiac tissue of SHRs

Figure 7 Effects of canagliflozin on expression of AGTR1, AGTR2, Renin in cardiac tissue of SHRs.

4. Discussion

The purpose of this study was to investigate the effect of cagliazin on cardiac remodeling induced by hypertension. Cagliazin intervention can reduce myocardial hypertrophy fibrosis and cardiac dysfunction in SHR. Cagliazin down-regulated the expression of molecular markers of fibrosis such as Col1a, Col3a, MMP-2, TGFβ1 and Smad4 In addition, we demonstrated that cagliazin inhibited AGTR1 and Renin mRNA expression, but upregulated AGTR2 mRNA levels. These results suggest that cagliazin can improve ventricular remodeling in SHR, which provides a new therapeutic target for the treatment of cardiovascular diseases.

Cagliazin was originally used to control blood glucose and was also found to have cardiovascular protection. CANVAS trials showed that cagliazin reduced the risk of cardiovascular death in patients with type 2 diabetes[12, 13]. Mechanisms for reducing cardiovascular mortality include lowering blood pressure, weight loss, improvement of vascular stiffness, improvement of blood glucose, and promotion of diuretic. Our study showed that cagliazin treatment reduced blood pressure, weight, and blood glucose. Our results are consistent with those reported in other studies A recent study showed significant weight loss in patients with type 2 diabetes after 24 weeks of treatment with cagliazin[21]. Similar results were observed in obese mice induced by a high-fat diet, where both fat mass and white adipose tissue weight were reduced[22]. This reduction may be related to the induction of calorie loss[19]. Compared with other drugs, treatment with cagliazin reduced systolic and diastolic blood pressure in patients with type 2 diabetes[23]. Blood pressure was also reduced in non-diabetic rats treated with cagliazin[17]. Some studies have shown that cagliazin promotes mice The vasodilatory sensitivity of rats and rabbits to acetylcholine may help explain the potensive effect of cagliazin[17, 24, 25]. as a hypoglycemic agent,cagliazin can reduce the circulating blood glucose concentration in diabetic mice and rats, but not in non-diabetic animals[26, 27].However, it is controversial that our experiments showed a decrease in blood glucose levels in SHRs treated with kagliazin. Thisdifference may be related to different animal models and different doses of kagliazin.

Cardiac hypertrophy is characterized by an increase in the number of myocardium, ventricular thickness, myocardial cell size, and impaired cardiac function, and is an independent risk factor for cardiovascular death[3, 4, 28]. Therefore, reducing myocardial hypertrophy is critical for preventing further death from cardiovascular disease. The effectiveness of SGLT-2I in reducing myocardial hypertrophy was reported. Shi et al observed that the increased HW/BW ratio of left ventricular diameter and left ventricular posterior wall thickness and left ventricular ejection fraction in mice with transverse aortic contraction were reduced after gliazin treatment[29]. A recent study reported that dagaglipin improved myocardial hypertrophic and left ventricular function in Ang II infusion SD rats by modulating the TGF-β1/Smad pathway[30]. In addition, heart weight and left ventricular volume gain were improved in obese rats treated with igaglipin[31]. Similarly, studies have shown that the left ventricular diastolic function of type 2 diabetes patients was significantly improved after 3 months of treatment with cagliazin[14]. It has been reported that cagliazin has a protective effect on myocardial ischemia-reperfusion injury in non-diabetic male rats, and can alleviate myocardial infarction in diabetic and non-diabetic hearts [17, 26]. Consistent with previous studies, our study showed that cagliazin treatment with SHR significantly reduced HW/BW/LVW/HW/myocardial cell size compared with SHR. Left ventricular wall thickness and ventricular septum thickness and left ventricular end-systolic volume suggest that cagliazin improves ventricular remodeling.

Myocardial fibrosis is closely related to cardiac dysfunction[3,4, 10]. As is known to all, long-term hypertension can induce myocardial fibrosis[3, 4, 10]. Myocardial fibrosis is characterized by excessive accumulation of collagen and degradation of extracellular matrix, which increases diastolic strength and leads to damage of left ventricular diastolic function[10]. Collagen is synthesized by fibroblasts and is the main component of extracellular matrix. Type I collagen and type III collagen account for 80% and ～10% of cardiac collagen, respectively Chronic stress overload can promote collagen gene expression and collagen synthesis. Multiple studies have shown that the expression of type I and III collagen in SHR heart is higher than that in WKY[32, 33]. MMP2 expression is increased in salt-sensitive hypertensive and SHR rat myocardium[34]. MMP2 expression is increased in salt-sensitive hypertensive and SHR rats Studies have shown that MMP2 regulates collagen synthesis and cardiac matrix remodeling, and MMP2 knockout can reduce myocardial hypertrophy and improve myocardial fibrosis, while MMP2 overexpression can enhance myocardial hypertrophy and myocardial fibrosis[35]. In this study, we found that collagen deposition and mRNA and protein levels of type I collagen III collagen and MMP-2 were increased in SHR rat heart tissues, which were attenuated after treatment with kagligin. In summary, our study suggests that MMP2 may be involved in the protective effect of kagligin on cardiac remodeling.

TGF-β signaling pathway plays a crucial role in the development of cardiac remodeling[9]. As a major mediator of fibrogenesis,TGFβ1 has been proven to be overexpressed and activated in the heart tissue of hypertensive rats[9]. Many studies have shown the importance of TGF-β1 in cardiac remodeling For example,TGFβ1 overexpression promoted hypertrophy and fibrosis in transgenic mice, while TGF-β1-deficient heterozygous mice reduced myocardial fibrosis[37, 38]. TGF-β1 plays a role in promoting fibrosis through the typical SMAD-dependent signaling pathway.After TGF-β1 binds to specific TGF-β II receptors, receptorregulated R-Smads (SMAD2, SMAD3) is activated and R-Smads bind to common Smad4 to form a complex, which is transported into the nucleus and regulates its downstream gene expression[6, 7,11]. TGF-β1 induces Smad4 expression in myocardial fibroblasts cultured in vitro [39]. Mc950 inhibits the upregulation of TGF-β1 and Smad4 and reduces myocardial hypertrophy and fibrosis[39].Similarly, dagaglizin inhibits endothelial hypertrophy and fibrosis by decreasing TGF-β1 expression[26]. Consistent with the above results, this study showed that cagliazin reduced the expression of TGF-β1 and Smad 4 in SHR myocardium, thereby alleviating the pathologic cardiac remodeling induced by hypertension.

Studies have reported that renin angiotensin system (RAS) is closely related to myocardial remodeling induced by hypertension[3,6, 7]. RAS, including circulating RAS and tissue RAS. More and more studies have shown that tissue RAS can affect the heart and have a more direct impact on pathological heart remodeling[6,7]. There is increasing evidence that Ang II can promote cardiac remodeling through its interaction with AGTR1, which is believed to have a cardioprotective effect[6-8]. It has been reported that continuous infusion of Ang II induced reduced expression of AGTR1 and AGTR2 in rats, accompanied by cardiac hypertrophy Myocardial fibrosis and elevated blood pressure[42]. Increased expression of Ang II and AGTR1 in the heart was found in rats fed with highsalt diet[8, 43]. Up-regulation of renin AGTR1 Ang II expression and down-regulation of AGTR2 expression in SHR myocardium[44] Regulation of RAS expression can reverse hypertensie-induced pathological cardiac remodeling The expression of ACE1 and ACE2 expression prevents cardiac hypertrophy and fibrosis in SHR[45].Pseudocalcemin R568 improves hypertensive induced cardiac remodeling by regulating the expression of renin Ang II AGTR1 and AGTR2[44]. These findings suggest that the beneficial effect of cagliazin on myocardial remodeling in SHR is mediated by regulation of RAS.

Taken together, our study suggests that cagliazin therapy reduces hypertensie-induced cardiac hypertrophic and fibrosis by modulating RAS and TGF-β1/Smad signaling pathways.

Conflict of interest

Authors declare that there is no conflict of interest.Author’s contribution Li Aihua: experimental operation index detection, writing papers;Qin Yingchun xie Yilin: Assisted in experimental modeling; Wang Qingqing: Data analysis; Yan Zhiqiang: Experimental design,experimental guidance paper revision review.