The Protective Effect of Sheng Mai Yin on Diabetic Cardiomyopathy via NLRP3/Caspase-1 Pathway

Sheng Mai Yin (SMY) has therapeutic effects on myocardial infarction (MI), heart failure (HF), diabetic cardiomyopathy (DCM), and myocarditis. To study whether SMY can relieve pyroptosis and play a protective role in diabetic cardiomyopathy, a molecular docking technique was used to predict the possible mechanism of SMY against DCM. Then, a DCM rat model was induced by intraperitoneal injection of streptozotocin (STZ), divided into 5 groups: the DM group (model), SMY-L group (2.7 mL/kg SMY), SMY-M group (5.4 mL/kg SMY), SMY-H group (10.8 mL/kg SMY), and Met group (120 mg/kg metformin). Rats in the CTL group (control) and DM group were given normal saline. After 8 weeks, the levels of blood glucose, lipids, and myocardial enzymes were detected according to the kit instructions. Cardiac function was detected by echocardiography. HE and Masson were used to observing the pathological changes, collagen deposition, and collagen volume fraction (CVF). The apoptosis rate of cardiomyocytes was determined by Tunel. The IL-1β level was determined by ELISA and RT-PCR. The expressions of NLRP3, caspase-1, and GSDMD were measured using RT-PCR and Western blotting. The docking results suggested that SMY may act on NLRP3 and its downstream signal pathway. The in vivo results showed that SMY could reduce blood glucose and lipid levels, improve heart function, improve histopathological changes and myocardial enzymes, and alleviate cardiomyocyte apoptosis and myocardial fibrosis. SMY inhibited the mRNA and protein expressions of NLRP3, ASC, Caspase-1, and GSDMD and IL-1β production. SMY can reduce DCM by regulating the NLRP3/caspase-1 signaling pathway, providing a new research direction for the treatment of DCM.


Introduction
Diabetic cardiomyopathy (DCM) is a specifc cardiac manifestation of diabetes patients and is the main cause of morbidity and mortality of diabetes patients around the world. In China, 33.9% of patients with type 2 diabetes have cardiovascular disease [1]. Te pathogenesis of DCM involves cardiac infammation and changes in metabolic characteristics, characterized by early left ventricular hypertrophy and diastolic dysfunction, manifested in myocardial cell hypertrophy, apoptosis, and myocardial interstitial fbrosis. With the progression of the disease, DCM gradually evolved into systolic dysfunction with reduced ejection fraction and eventually developed into heart failure [2,3]. DCM is characterized by a variety of pathophysiological variables, including oxidative stress, apoptosis, cardiac fbrosis, impaired angiogenesis, and altered glycolysis metabolism, although its pathogenesis is unknown. Presently, there is no particular medicine available for the treatment of DCM, and the majority of patients sufer from heart failure. Identifying possible treatment targets is crucial for reducing the morbidity and mortality associated with DCM.
Numerous studies have demonstrated that the nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain-containing receptor 3 (NLRP3) infammasome play a signifcant role in the pathophysiology and research advancement of DCM and is a new pharmacological target for treating DCM and related complications [4,5]. In individuals with DCM, activation of the NLRP3 infammasome has been found to generate or exacerbate cardiac infammation, myocardial cell necrosis or apoptosis, myocardial fbrosis, and cardiac failure [6,7].
Sheng Mai Yin (SMY) is composed of ginseng, radix ophiopogonis, and Schisandra chinensis. Some studies have shown that the Sheng Mai compound preparation composed of three Chinese herbs has therapeutic efects on heart diseases such as myocardial ischemia-reperfusion, coronary heart disease, and heart failure [8][9][10]. By activating AMPKα and decreasing oxidative stress injury mediated by NADPH oxidase, Sheng Mai San (SMS) has been reported to protect the myocardium against diabetes [11]. Trough stimulation of the Nrf2/Keap1 signaling pathway, Sheng Mai injection (SMI) inhibits apoptosis, decreases CK, LDH, and MDA levels, and increases SOD activity, therefore reducing DOXinduced cardiotoxicity [12]. However, the mechanism of action of SMY on DCM is yet unknown, and it has to be determined whether SMY may play a protective function in DCM by acting on the NLRP3/Caspase-1 pathway.

Molecular
Docking. CB-Dock was used to predict the binding afnity between the main ingredients of SMY and NLRP3. Te major components of SMY were 11 chemical components found in rat serum: ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rg2, ginsenoside Rb1, ginsenoside Rd, ginsenoside Rc, ophiopogonin D, schisandrin, schisandrol B, and schisandrin B [13]. Te structures were downloaded from the PubChem website and then the hydrogens and charge were added. Te PDB format of NLRP3 (ID: 6npy) [14] was downloaded from RCSB (https://www.rcsb.org/), waters and het groups were deleted, and hydrogens were added, and then the docking procedure was submitted [15].

Establishment of the Rat Model
. After a week of adaptive feeding, 10 male SD rats were chosen at random for the CTL group and fed a standard diet. Other rats were fed a highcalorie diet (composition: lard 5%, sugar 5%, yolk powder 5%, and cholesterol 1%). Each group of rats was fed routinely for six weeks. Rats in the control group were treated intraperitoneally with citric acid-sodium citrate bufer (pH 4.2) after 6 weeks, whereas rats in the other groups were injected intraperitoneally with 1% STZ at 35 mg/kg. After 3 days, blood was taken by needling at the tail tips of rats in each group, and the diabetic model was successfully prepared when the blood glucose was ≥16.7 mmol/L [16].

Drug Treatment.
Successfully modeled SD rats were randomly assigned to fve groups: DM (model), SMY-L (2.7 mL/kg SMY), SMY-M (5.4 mL/kg SMY), SML-H (10.8 mL/kg SMY), or Met (120 mg/kg metformin). Both the CTL group and the model group received the same amount of normal saline. All groups received the same gavage intervention for eight weeks.

Detection of Blood Glucose and Lipid Levels.
After fasting for 12 h, rats in each group were anesthetized with pentobarbital sodium, and blood was taken from the abdominal aorta to the vein collection. After 30 min, the supernatant was centrifuged at 1500 r/min for 15 min and stored at −80°C. Blood glucose, GHB, TG, TC, LDL-C, and HDL-C values were measured according to the kit's instructions.

Detection of Cardiac Function by Echocardiography.
All rats were anesthetized with isofurane and administered 2D echocardiography for calculating ejection fraction (EF) and fractional shortening (FS) [16].

Detection of Pathological
Staining. Te myocardial tissue was fxed in 4% paraformaldehyde at room temperature, dehydrated, embedded in parafn, and sliced into 5 μm sections. Diferent sections were stained with HE and Masson and observed under a microscope, which was used to observe the pathological changes, collagen deposition in the myocardial interstitium, and collagen volume fraction (CVF). Evidence-Based Complementary and Alternative Medicine 2.9. Detection of Myocardial Enzymes. Te contents of CK, ANP, and BNP were detected according to the kit's instructions.

Detection of Apoptosis Rate.
In each slice, fve visual felds were randomly selected for microscopic examination, and the total number of cardiomyocytes and the apoptotic number of cardiomyocytes were counted in each feld. Apoptosis rate � apoptotic cardiomyocytes number/all cardiomyocytes number × 100%.

Detection of IL-1β
Levels. IL-1β levels in rat myocardial tissue were detected by a commercial test kit. Te optical density (OD) was selected at 450 nm and read by a microplate reader (BioTek, USA).

Detection of mRNA Expression by RT-PCR.
Myocardial RNA was extracted from the left ventricular myocardium using the Trizol reagent. Using a reverse transcription kit, an equal quantity of RNA from each sample was reversely transcribed into cDNA. For PCR amplifcation of the same number of reverse transcription products, SYBR Green was used. RT-PCR was conducted with the LightCycler ® 96 PCR apparatus (Roche, Switzerland). Te results were analyzed using 2 −ΔΔCq method to evaluate the mRNA levels of NLRP3, ASC, Caspase-1, and GSDMD. Te primers used in the research are shown in Table 1.

Detection of Protein Expression by Western Blotting.
Te heart tissue was lysed in lysate, and total protein was extracted. On a 10% to 15% polyacrylamide gel, the lysates were separated and transferred to an NC membrane. After blocking the NC membrane with 5% skim milk powder, the following antibodies were incubated at 4°C overnight: anti-NLRP3 (1 : 1000), anticleaved caspase-1 (1 : 1000), anti-ASC (1 : 1000), anti-GSDMD (1 : 1000), anti-GAPDH (1 : 5000), and anti-tubulin (1 : 5000). Te primary antibody was incubated at 4°C for an overnight before being incubated at room temperature with the secondary antibody for 2 h. Te density of protein bands was detected using the ECL chemical substrate luminescence kit, and the protein bands were imaged in Tanon5200 imaging system (Tanon, China).
2.14. Statistical Analysis. Te data were given as mean-± standard deviation (SD), and statistical analysis was performed using SPSS 23.0. A one-way ANOVA was utilized to determine the signifcance between the groups. P < 0.05 was regarded as statistically signifcant.

Docking Results.
Te primary components of SMY exhibited binding afnity with NLRP3. Te superior binding afnities of Ophiopogonin D, Schisandrol B, and Ginsenoside Rf to NLRP3 may provide active monomer compounds for future investigation ( Table 2 and Figure 1). Te docking data revealed that SMY may operate on NLRP3 and its downstream signaling pathway, which was confrmed by in vivo tests.

SMY Reduced Blood Glucose and Lipid Levels in Model
Rats. Te abnormal blood glucose and blood lipid levels suggested that the DCM model had been developed successfully [17]. After 8 weeks, the levels of blood glucose, GHB, TG, TC, and LDL-C were substantially higher in the DM group than in the CTL group (P < 0.01), although the level of HDL-C did not change signifcantly. After therapy with SMY-M and SMY-H, blood glucose, GHB, TG, TC, and LDL-C levels were reduced (P < 0.05, P < 0.01) ( Figure 2). Rat models demonstrated that SMY may successfully lower blood glucose and blood lipid levels.

SMY Improved the Heart Function in Model Rats.
Echocardiography was performed to determine the heart function of each group of rats, with EF and FS serving as the primary indices. Te EF and FS of the DM group were considerably lower than those of the CTL group (P < 0.01), while SMY and Met reversed the FS and LVEF (P < 0.05, P < 0.01) ( Figure 3). Te results suggested that SMY has a certain ameliorative efect on diabetic cardiac function injury.

SMY Improved the Histopathological Changes and
Myocardial Enzymes in Model Rats. Te fndings of the HE staining demonstrated that the CTL group possessed normal morphology, a full myocardial structure, and an organized arrangement of muscle fbers. Te DM group had myocardial fracture, myocardial fber organization abnormality, and infammatory cell infltration. Te myocardial fbers of the Met group and the SMY group were arranged in a relatively orderly manner with a small amount of infammatory infltration. Creatine kinase (CK) is mainly used in the diagnosis of myocardial infarction, which is widely in skeletal muscle, cardiac muscle, and brain tissue. Serum atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) activities are used to diagnose and monitor the course and efcacy of heart failure [18]. CK, ANP, and BNP levels were considerably greater in the DM group than in the CTL group (P < 0.01); SMY and Met lowered CK, ANP, and BNP levels (P < 0.05, P < 0.01) (Figure 4). Te results showed that SMY could signifcantly reduce the changes in cardiac pathology and myocardial enzymes in model rats.

SMY Alleviates Cardiomyocyte Apoptosis and Myocardial
Fibrosis in Model Rats. Te DM group's nuclei were discovered to be pale brown, and their apoptosis rate was signifcantly greater than that of the CTL group (P < 0.01).
Compared to the DM group, SMY and Met treatment signifcantly decreased the amount of pale brown nuclei and the apoptosis rate (P < 0.05, P < 0.01) (Figure 5(a)). Masson staining revealed that the muscle fbers of the myocardium were red and the collagen fbers were blue. In the CTL group, the myocardial fbers were organized and only a modest Evidence-Based Complementary and Alternative Medicine   number of collagen fbers were formed, whereas the DM group had abundant collagen fber depositions. Pretreatment with SMY and Met resulted in a signifcant decrease in the collagen content of cardiac tissue. Collagen volume fraction (CVF) can be utilized to examine morphological alterations of the left ventricular myocardium. CVF levels were considerably greater in the DM group than in the CTL group (P < 0.01), but SMY and Met lowered CVF levels (P < 0.01) ( Figure 5(b)).

SMY Decreased IL-1β Level in Rat Myocardial Tissue.
As shown in Figure 6, IL-1β level in rat myocardial tissue increased signifcantly in the DM group compared with the CTL group (P < 0.01). IL-1β level was decreased signifcantly in the Met and SMY groups (P < 0.05). SMY inhibited the mRNA expressions of NLRP3, ASC, caspase-1, GSDMD, and IL-1β.
Compared to the CTL group, the mRNA expressions of NLRP3, ASC, caspase-1, GSDMD, and IL-1β increased considerably in the DM group (P < 0.01). In contrast to the DM group, SMY and Met were able to reverse the mRNA overexpressions (P < 0.05, P < 0.01) (Figure 7).

Discussion
DCM is a diabetes-related pathophysiological disorder characterized by structural, functional, and metabolic abnormalities in the heart that can lead to HF in the absence of coronary artery disease, hypertension, and valvular heart disease [19]. Hyperglycemia, insulin resistance, hyperinsulinemia, and increased free fatty acid metabolism cause oxidative stress, infammation, formation of advanced glycation end products, abnormal calcium homeostasis, and apoptosis, resulting in myocardial cell dysfunction, injury, and death and consequently cardiac dysfunction [20]. Te underlying molecular mechanism of DCM is not yet fully understood. During the development of DCM, excessive hyperglycemia can cause an increase in reactive oxygen species, which activate NF-κB and subsequently trigger the activation of NLRP3, driving cellular infammation and apoptosis [21]. NLRP3 is an immune-related infammatory molecule, and studies have established its strong association with the development of DCM [22]. By activating p65, ROS stimulates the activation of NLRP3. Activated NLRP3 interacts with the adaptor protein apoptosis-associated specklike protein (ASC) and procaspase-1 to create the NLRP3 infammasome and innate immune system protein complexes [23]. Ten, caspase-1 is the efector protein of the NLRP3 infammasome, cleaved by the precursor molecule procaspase-1 [24]. Caspase-1 may result in the cleavage of pyroptosis executioner gasdermin D (GSDMD) along the canonical pathway. Te GSDMD-N possesses membrane pore-forming activity by attaching to phosphoinositides in the plasma membrane, resulting in pyroptosis [25].
As we all know, Met is the frst choice for a hypoglycemic drug in the treatment of type 2 diabetes. Te UK Prospective Diabetes Study (UKPDS) confrmed that Met can reduce the progress of cardiovascular disease (UK Prospective Diabetes Study [26]. Previous studies have shown that Met could improve cardiac function damage [27] and play a cardiac protective role in diabetes [28,29]. In this study, we found that SMY had the same efect as Met in improving cardiac function damage, which led us to believe that SMY can protect the heart from diabetes. SMY, a traditional Chinese medicine, is commonly used in the clinic to treat cardiac insufciency, coronary heart disease, and heart failure [8,9,30]. SMY could restore the cardiac function of CHF rats, reduce serum biochemical indexes, reduce cardiac tissue damage, and reduce the expression levels of ALOX15 and CYP1A2, which may be related to the linoleic acid metabolic pathway [10]. SMY could reduce aortic plaque area and MMP9 expression in animal models of myocardial ischemia and atherosclerosis (AS) in response to DEP exposure. In addition, SMY also could improve left ventricular structure, morphology, function, blood fow, infarct area, myocardial damage, and ROS accumulation to varying degrees in ApoE -/mice, which had a potential protective efect in DEP-aggravated AS with myocardial ischemia [31]. Hence, SMY demonstrates potential cardiac protective actions, including antilipid peroxidation and antiinfammatory characteristics, scavenging oxygen free radicals, antiischemia and hypoxia and cardiomyocyte protection, and regulation of linoleic acid metabolism [10,31,32]. Eleven components of SMY in rat serum were determined by LC-MS/MS, which were ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rg2, ginsenoside Rb1, ginsenoside Rd, ginsenoside Rc, ophiopogonin D, schisandrin, schisandrol B, and schizandrin B [13]. Several compounds have been discovered to Evidence-Based Complementary and Alternative Medicine have potent inhibitory efects on the NLRP3 signaling pathway, providing an experimental foundation for the investigation of SMY. Ginsenoside Rg1 improved cardiac function and suppressed lipopolysaccharide (LPS)-induced apoptosis and infammation in mice. Tese efects were due to the regulation of the increased expression of toll-like receptor 4 (TLR4), NF-κB, and NLRP3 [33]. Ginsenoside Re reduced the elevated NLRP3, ASC, and caspase-1 protein expression in the hippocampus of mice with chronic restraint stress (CRS) [34]. Ginsenoside Rb1 inhibited the production of TNF-α, IL-18, and IL-1β in the hippocampus, reduced the protein expression of NLRP3, and stimulated the protein expressions of Nrf2 and HO-1 [35]. Ginsenoside Rd reduced signifcantly the activation of the NLRP3 infammasome, which is reliant on the mitochondrial translocation of p62 and mitophagy [36]. In addition, we predicted the binding afnities of 11 chemicals in SMY with NLRP3 using molecular docking and discovered that they all exhibited excellent binding ability. Te superior binding afnities of ophiopogonin D, schisandrol B, and ginsenoside Rf to NLRP3 may provide active monomer compounds for future investigation. Molecular docking indicated in this work that SMY may interact with NLRP3 and its downstream signal pathway. In vivo studies demonstrated that SMY can alleviate the symptoms of DCM by lowering blood glucose and cholesterol levels, enhancing heart function, histological alterations, and myocardial enzyme and decreasing cardiomyocyte apoptosis and myocardial fbrosis. Tese pharmacodynamic fndings imply that SMY might greatly protect the myocardium against DCM. SMY might also suppress the mRNA and protein expressions of targets in the NLRP3/caspase-1 signaling pathway in DCM (Figure 9). Te results revealed that SMY can prevent and treat DCM and that its protective impact is connected to its NLRP3/caspase-1 regulatory signaling pathway.

Conclusion
Te efects of the SMY on the DCM in vivo have been verifed. SMY might greatly reduce the incidence and progression of DCM in terms of cardiac function, myocardial enzymes, histology, apoptosis, and the signaling pathway. SMY's efect on DCM is mediated through the NLRP3/caspase-1 signaling pathway. However, whether its mechanism is directly related to the NLRP3/caspase-1 signaling pathway needs further experimental verifcation. Te next research will focus on in vitro cellular mechanisms, such as adding a specifc inhibitor of NLRP3 or using NLRP3 gene silencing in the experiment, in an attempt to better clarify the mechanism of SMY in the prevention and treatment of DCM and provide a new research direction for the treatment of DCM.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.