NXK consists of two herbs, Radix ginseng Rubra and llex pubescens. The herbs were mixed according to the ratio of 23:77. The concentrated NXK powders were produced by Sci-tech Industrial Park, Guangzhou University of Chinese Medicine, under internationally certified good manufacturing practice guidelines (Serial Number: TA2017014).

Four-week-old C57BL/6 male mice were obtained from the Guangdong Animal Experimental Center. The mice were housed in an environment with a temperature of 22 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. The animal experiment protocols conformed to the Guidelines for the Care and Use of Laboratory Animals published by the National Academy Press (National Institutes of Health Publication No. 85-23, revised 1996) and were approved by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (permit number: 20200608003).

The mice were divided into two groups: the TAC group and the sham group. Heart failure was achieved via transverse aortic constriction (TAC) on eight-week-old mice. The animals were intubated and ventilated with room air using a small-animal respirator (Harvard Inspira ASVv, Massachusetts, United States) and anesthetized with 1.0% isoflurane inhalation. The left side of the chest was then opened at the second intercostal area. A 7-0 silk string was passed under the transverse thoracic aorta between the innominate and left common carotid arteries and snared around the aorta with a 28-gauge needle. After ligation, the needle was removed, and the skin was sutured. The sham group underwent the same surgery but without constriction.

The mice were then divided into six groups (10 mice/group): Sham, TAC, TAC + low dose NXK (NXK-L), TAC + medium-dose NXK (NXK-M), TAC + high-dose NXK (NXK-H), and TAC + Perindopril. Intramuscular injection of penicillin was administered to all groups for 3 days.

To determine the optimal dose of NXK, we used 0.42 g/kg/d as the low dose, 0.83 g/kg/d as the medium dose, and 1.65 g/kg/d as the high dose. Three days after surgery, the TAC + NXK-L, TAC + NXK-M, and TAC + NXK-H groups were intragastrically administered 0.42 g/kg/d, 0.83 g/kg/d, and 1.65 g/kg/d NXK, while the TAC + Perindopril group was given 3.6 g/kg for 28 days.

Mice in the lipopolysaccharide (LPS) and TAK-242 groups were intraperitoneal injected with saline or TLR4 inhibitor (TAK-242; 2 mg/kg), respectively, followed by LPS (12 mg/kg; intraperitoneal injection) 1 h after the surgery. The sham and TAC groups were given sterile water. The mice were used for experiments 8 weeks after the surgery.

Echocardiography studies were performed using a Vevo 2,100 imaging system (FUJIFILM Visual Sonics Inc., Canada). The mice were anesthetized with 2% isoflurane inhalation. The M-mode image of the middle layer of the nipple muscle was used to measure the left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), end-systolic volume (ESV), end-diastolic volume (EDV), left ventricular posterior wall end-diastole (LVPWD) and end-systole (LVPWS). The left ventricular ejection fraction (LVEF) was calculated as (LVEDV—LVESV)/LVEDV; the left ventricular fraction shortening (LVFS) was calculated as (LVEDD—LVESD)/LVEDD; the stroke volume (SV) was calculated as (EDV − ESV). Blind observers used Vevo 2,100 workstation 1.7.1 to analyze the digital images offline.

The heart tissue was fixed in 4% paraformaldehyde and then immersed in graded series of alcohol: 50% ethanol for 30 min, 70% ethanol for 60 min, 80% ethanol for 60 min, 90% ethanol twice for 30 min each, and absolute ethanol for 30 min, followed by 60 min in isobutanol + carbonic acid. Subsequently, the specimens were embedded in wax for 90 min and cooled. Masson’s Trichrome staining was performed on the paraffin-embedded heart sections to assess myocardial fibrosis. Sections were also blocked and then incubated with antibodies against CD11b and Ly6C. Nuclei were stained with DAPI. Nikon Eclipse microscope with NIS Elements software was used to capture the images. ImageJ software was used to measure the area of cardiac fibrosis (the ratio of fibrotic area to total myocardial area). At least 20 fields (200X) were analyzed in each section.

The heart samples were snap-frozen in liquid nitrogen and placed on ice for 30 min before extracting total RNA using TRIzol reagent. cDNA was synthesized using TIANScript RT kit^® (TIANGEN BIOTECH, China) according to the manufacturer’s instructions. The RT-PCR amplification reaction was 20 μl, containing 10 μl LightCycler FastStart DNA MasterMix, 1 μl cDNA, 7 μl RNase/DNase-free water, and 500 nM of each primer. The primer sequences are listed in Supplementary Table S1. The expression of the target gene was quantified by the standard curve and the 2-ΔΔCt method. β-actin was used as an internal control to normalize the levels of the target genes.

Total protein was extracted from the heart tissue according to the instructions provided in the total protein extraction kit, and the protein concentration was determined (Ca. P0013B, Beyotime, Shanghai, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% and 12%). Then, the protein was transferred to polyvinylidene fluoride (PVDF) membrane (Ca. IPVH00010, EMD Millipore, Billerica, MA, United States), blocked with 5% skimmed milk in Tris-buffered saline-Tween 20 (TBST) for 90 min at 37°C, and probed with the following primary antibodies: TLR4 at 1:500 dilution; MyD88, NF-κB p65, P-IκBα, IκBα, TLR2, TLR7, TLR9 and beta-tubulin at 1:1,000 dilution. Subsequently, the membranes were incubated with the corresponding secondary antibody. The immunoreactive bands were developed using ECL, and the ProteinSimple imaging system was utilized to scan the target protein. ImageJ software was used to analyze the image.

All values were reported as mean ± standard error of the mean (SEM). The normality of data distribution was evaluated using the Shapiro-Wilk test. Data with skewed distribution were transformed by calculating their natural logarithm to approximate normal distribution. One-way analysis of variance (ANOVA) followed by Dunnett’s test or the Student Newman-Keuls (SNK) test were used to analyze the differences between the groups. Differences were considered statistically significant at p < 0.05.

Cardiac function was evaluated via echocardiography (Figure 1A; Table 1). Compared to the sham group, LVDS was increased in the TAC group (p < 0.05), while LVEF and LVFS were decreased (all p < 0.01). Compared to the TAC group, the three NXK groups showed significant increases in LVEF and LVFS (all p < 0.01, Figure 1B; Table 1), thus suggesting a protective role of NXK in cardiac hypertrophy. However, there were no differences in LVPWD and LVPWS between the groups (all p > 0.05).

RT-PCR was used to detect the RNA expression levels of BNP and ANP in all groups. The expression levels of BNP and ANP were significantly increased in the TAC group compared to the sham group (p < 0.001, Figure 1C, Supplementary Table S2). Furthermore, the expression levels of BNP and ANP in the Perindopril and the NXK low-, medium-, and high-dose groups were significantly lower than that of the TAC group (all p < 0.001, Figure 1C, Supplementary Table S2). Taken together, these results demonstrated the therapeutic efficacy of NXK in the heart failure model.

To further investigate the cardio-protective role of NXK, we examined the effect of NXK on myocardial fibrosis. HE staining showed that the hearts in the TAC group were significantly larger than that of the sham and drug groups, resulting in increased pressure load and myocardial hypertrophy. Next, we used Masson’s Trichrome staining to measure myocardial fibrosis. The degree of fibrosis was higher in the TAC group than in the sham and the drug groups (Figure 2A). Moreover, a larger fibrotic area was detected in the myocardium of the TAC group, where the greatest pressure load was observed compared to the sham group. In contrast, there was significantly smaller fibrotic area in the NXK group (Figures 2A,B, and Supplementary Table S3).

The mRNA expression levels of α-SMA, TGF-β, collagen I (Col1), collagen III (Col3), and matrix metalloprotein 9 (MMP9) were increased in the TAC group compared with the sham group (Figures 2C,D), and the mRNA expression levels of TGF-β in the NXK low, middle, high dose groups were significantly decreased vs. the TAC group (p < 0.05 vs. TAC, Figures 2C,D, Supplementary Table S4). The relative expression levels of Col1, Col3, α-SMA and MMP9 were significantly decreased in the NXK low, middle, high dose groups (p < 0.001 vs. TAC, Figures 2C,D, Supplementary Table S4). These results indicated that NXK treatment remarkably decreased myocardial fibrosis in the CHF model.

Immunofluorescence demonstrated that CD11b + Ly6C + monocytes/macrophages were significantly increased in the model group. In contrast, infiltration of CD11b + Ly6C + monocytes/macrophages were inhibited in the Perindopril and NXK groups (Figure 3A).

Next, we detected the RNA expression levels of TNF-α, IL-6, IL-1, VCAM-1, and ICAM-1 among the groups. Compared to the sham group, the expression levels of IL-1, IL-6, TNF-α, VCAM-1, and ICAM-1 were significantly increased in the TAC groups (p < 0.001, Figure 3B; Table 2). Moreover, the expression levels of IL-1, IL-6, and TNF-α were decreased in the Perindopril group and the NXK high-, middle-, and low-dose groups (p < 0.001 vs. TAC). The results also showed decreased expression levels of VCAM-1 and ICAM-1 in the drug intervention group compared with the TAC group (p < 0.05, Figure 3B; Table 2).

NF-κB is a pivotal transcription factor that regulates inflammation, immune responses, and cell survival. In this study, the levels of the target proteins, TLR4, MyD88, and NF-κB p65 ( Figure 4 ), were significantly increased in the TAC group compared to the sham group. Moreover, the relative expression levels of TLR4 and MyD88 proteins in the high-, medium-, and low-dose groups of NXK and the Perindopril group were significantly lower compared to the TAC group (p < 0.01 vs. TAC, Figure 4 and Supplementary Table S5). The relative expression of NF-κB p65 was significantly lower in the high-, medium-, and low-dose groups compared to the TAC group (p < 0.05, Figure 4 and Supplementary Table S5). Besides, the expression of total IκBα and the phosphorylation of IκBα (P-IκBα) were further measured. The P-IκBα protein level was increased after TAC treatment and decreased after NXK treatment. Moreover, total IkBα was increased with decrease in p-IkBα after NXK treatment (p < 0.01 vs. TAC, Figure 5 and Supplementary Table S6).

Next, we used the TLR4 inhibitor TAK242 and agonist LPS to evaluate the effect of NXK. As shown in Figure 6A, the relative protein expression levels of NF-κB p65 were significantly decreased in the TAK242, NXK (M), LPS + NXK(M) and the TAK242 + NXK (M) groups compared with the LPS groups (p < 0.05, Figure 6A, Supplementary Table S7). Treatment with TAK242, NXK (M), LPS + NXK(M) and the TAK242 + NXK (M) significantly reduced the protein levels of P-IκBα (p < 0.01, Figure 6B, Supplementary Table S7).

The relative expression levels of TLR2, TLR7 and TLR9 were also evaluated. As shown in Figure 7A, the relative protein expression levels of TLR2, TLR7 and TLR9 were significantly increased in the TAC group compared with the sham group, and the protein levels of TLR2 and TLR9 in the NXK medium-dose group were significantly decreased compared with the TAC group (p < 0.01, Figure 7B). In addition, the expression of TLR7 in the NXK group showed no significant difference compared with the TAC group (p > 0.05, Figure 7B, Supplementary Table S8).