Dr Tao Ding, Pharmacodynamic and Toxicological Evaluation Center, Jilin Academy of Traditional Chinese Medicine, 155 Chuangju Street, Changchun, Jilin 130000, P.R. China
*Contributed equally
Chronic cardiac inflammation and fibrosis can progress into severe forms of cardiomyopathy.
Myocarditis is cardiac inflammation that can be caused by viruses, bacteria or autoimmune diseases (
Supportive therapies and immunosuppression remain to be the main treatment methods for myocarditis (
Alkaloids and saponins are the main pharmacologically active components of
A total of 96 male BALB/c mice aged 6-8 weeks (weight, 18-20 g) were purchased from Changchun Changsheng Gene Pharmaceutical Co., Ltd. [license number: SCXK (Liao)-2020-0001; eligibility nos. 2107262101007676 and 21072621030386012]. Mice were housed in a barrier system at a temperature of 23±2˚C, humidity of 60±10% and a 12/12-h light/dark cycle. All animals had free access to food and water. The present study was approved by the animal Ethics Committee of the Jilin Academy of TCM (approval no. JLSZKYDWLL2021-003; Changchun, China).
The
Optimization of the compound formula was performed using the uniform design model (a mathematical method) to reduce the experiment times, the uniform design and data analysis were performed using DPS v. 6.55 software. (
After 1 week of adaptive feeding, BALB/c mice were randomly divided into the following groups based on body weight (24 animals per group): Control; EAM; KX-High (KX dose, 275 mg/kg); and KX-Low (KX dose, 138 mg/kg). All mice, except for those in the control group, received subcutaneous (back, inguinal on both sides) injections of 0.2 ml emulsion I on days 0, 7, 21 and 42 at the beginning of the test. Porcine cardiac myosin (MilliporeSigma) was dissolved in potassium phosphate buffer (pH=6.8) and mixed thoroughly 1:1 with complete Freund's adjuvant (MilliporeSigma), yielding a solution with 200 µg porcine cardiac myosin in 0.2 ml of the emulsion (
In the control group, each animal was injected with 0.2 ml emulsion II (potassium phosphate buffer with complete Freund's adjuvant at a 1:1 ratio) following the same protocol. High and Low KX solutions were prepared by dissolving 275 or 138 mg KX, respectively, in 10 ml distilled water. Mice in the KX-high and KX-low groups were intragastrically fed by oral gavage (10 ml/kg) with the experimental drug daily from day 0 until sacrifice (day 21 or 60). Mice in the control and EAM groups received an equivalent volume of distilled water. All animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then euthanized through cervical dislocation. In addition, mice were anesthetized and sacrificed before the experimental endpoints in case of persistent self-injurious behavior, non-healing wounds and loss of appetite.
Mice were sacrificed after 21 or 60 days, before their bodyweight and heart weight were measured. The heart tissues were fixed in 15% formalin at a temperature of 23±2˚C for not less than 48 h, embedded in paraffin and sectioned into transverse sections (5-µm thickness) for H&E staining (at a temperature of 23±2˚C for 5-20 min). For the H&E staining, myocardial tissue was examined under a light microscope at a magnification of x400, the macroscopic score was determined using a five-point scale: 0, no inflammation; 1, limited discoloration; 2, numerous small lesions; 3, diffused discoloration not exceeding one-third of the heart surface; and 4, diffused discoloration exceeding one-third of the heart surface (
The serum levels of CK-myocardial band (CK-MB), LDH and the cardiac tissue levels of cardiac troponin I (cTn-I), collagen type I (Col I), collagen type III (Col III) and the inflammatory markers IL-1β, IL-6, TNF-α and TGF-β1 were detected using mouse ELISA kits (R&D Systems China Co., Ltd.: CK-MB, cat. no. 202109; LDH, cat. no. 202108; cTn-I, cat. no. 202108; Col I, cat. no. 202110; IL-1β, cat. no. 202109; IL-6, cat. no. 202108; TNF-α cat. no. 202107 and TGF-β1 cat. no. 202109). Cardiac homogenate (10%) was prepared by grinding 100 mg of heart tissue in 1 ml of saline. In microplates, standards (50 µl) were added into predefined wells, samples (10 µl serum or cardiac homogenate) and sample diluent (40 µl) were added into testing sample wells, while blank wells were left empty. In the wells for standards and samples, horseradish peroxidase-labelled conjugates (100 µl) were added before sealing the plates for incubation at 37˚C for 60 min. After washing the plates 5 times, substrates A (50 µl) and B (50 µl) were added into each well. After incubation at 37˚C for 15 min, stop solution (50 µl) was added to each well, and the absorbance of each well was measured at 450 nm within 15 min (cat. no. ELx800; BioTek Instruments, Inc.).
Protein extracts from cardiac tissue were prepared in Radio-Immunoprecipitation Assay (RIPA) lysis buffer (CoWin Biosciences; cat. no. 21821) containing protease and phosphatase inhibitors. Proteins were separated through 10% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking in 5% non-fat milk diluted in TBS-Tween 20 (0.1% Tween; CoWin Biosciences) in the dark at 4˚C overnight, the membranes were incubated with primary antibodies (Santa Cruz Biotechnology, Inc.) against activated kinase 1 (TAK1)-binding protein 1 (TAB1, cat. no. sc-166138; 1:100 dilution), NF-κB (cat. no. sc-8008; 1:200 dilution), phosphorylated (p)-NF-κB (cat. no. sc-136548; 1:200 dilution), IκB (cat. no. sc-74451; 1:100 dilution), p-IκB (cat. no. sc-8404; 1:200 dilution), IKKα (cat. no. sc-7606; 1:200 dilution), TGF-β1 (cat. no. sc-130348; 1:200 dilution), Smad2 (cat. no. sc-101153; 1:200 dilution), Smad4(cat. no. sc-7966; 1:200 dilution), Col Ⅰ (cat. no. sc-376350; 1:100 dilution) or GAPDH (cat. no. sc-365062; 1:100 dilution) for 2 h at 37˚C. Subsequently, the membranes were washed by TBS-Tween-20 and incubated with a secondary HRP-conjugated antibody (m-IgGκ, cat. no. sc-516102; 1:1,000 dilution or m-IgG Fc, cat. no. sc-525409; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 1.5 h at 37˚C. Next, the incubated membranes were washed again and visualized using an enhanced chemiluminescence detection kit (CoWin Biosciences). The levels of target proteins were normalized to those of GAPDH with Gel imaging system (ChemiDoc-It 510 Imager, Ultra-Violet Products Ltd).
GraphPad Prism software (version 9.0; GraphPad Software Inc.) was used for all statistical analyses. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Bonferroni post hoc tests for comparisons between different time points and groups and one-way ANOVA with Tukey's post hoc test or Kruskal-Wallis followed by Dunn's post hoc test for the comparison among multiple groups. The macroscopic scores are presented as the median + interquartile range, whilst all other values are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
After 21 days, mice in the EAM group weighed significantly less compared with those in the control group (P<0.01;
After 60 days, the mice in the EAM group weighed significantly less compared with those in the control group (P<0.01;
In the control group, no changes were observed in the body weight of mice sacrificed after 21 and 60 days. In the EAM group, the HW/BW was significantly increased (P<0.01;
After 21 days, the serum levels of CK-MB, LDH and cTn-I were significantly increased in the EAM group compared with those in the control group (P<0.01;
After 60 days, the serum levels of CK-MB, LDH and cTn-I were significantly increased in the EAM group compared with those in the control group (P<0.01;
In control mice, there were no changes observed between 21 and 60 days. However, the levels of CK-MB, LDH and cTn-I were significantly lower after 60 days compared with those after 21 days (P<0.01;
After 21 days, the levels of IL-6, IL-1β and TNF-α were significantly increased in the EAM group compared with those in the control group (P<0.01;
After 60 days, the levels of IL-6, IL-1β and TNF-α were significantly higher in the EAM group compared with those in the control group (P<0.01;
There were no changes observed in terms of the inflammatory cytokines measured in the present study in the control mice between 21 and 60 days. However, in the EAM group the levels of IL-6, IL-1β and TNF-α were significantly lower in the myocardium of mice after 60 days compared with those in mice after 21 days (P<0.01;
After 21 days, the expression levels of TAB1, IKKα, p-IκB/IκB, p-NF-κB/NF-κB and TGF-β1 were significantly increased in EAM mice compared with those in the control group (P<0.01;
The expression of NF-κB and TGF-β1 in the myocardium was next examined using immunohistochemistry. The results showed that their levels were significantly increased (P<0.01) in the EAM group compared with those in the control group. However, these were significantly reversed (P<0.01 or P<0.05) after intervention with KX (
After 21 days, the levels of TGF-β1 were significantly higher in the myocardium of mice in the EAM group compared with those in the control group (P<0.01). By contrast, the levels of TGF-β1 was significantly lower in the KX-high group compared with those in the EAM group, in a dose-dependent manner (
After 60 days, the levels of TGF-β1, Col I and Col III were significantly increased in the myocardium of mice in the EAM group compared with those in the control group (P<0.01). Compared with those in the EAM group, these levels were significantly lower in mice after intervention with KX (P<0.01 or P<0.05;
After 60 days, the levels of TGF-β1, Smad2, Smad4, Col I and NF-κB in the myocardium were significantly increased in EAM mice compared with those in the control group (P<0.01). These increases were significantly reversed in mice after treatment with KX (P<0.01 or P<0.05;
NF-κB and TGF-β1 expression in mouse myocardium was subsequently assessed using immunohistochemistry. These results showed that the expression of NF-κB and TGF-β1 was significantly increased in EAM mice compared with that in the control group (P<0.01), which was in turn significantly reversed after treatment with KX (P<0.01;
Myocarditis can manifest with a variety of symptoms, such as palpitations, chest pain and arrhythmia (
An animal model of EAM was previously produced through the injection of cardiac myosin into susceptible rodents, which lack the immune tolerance mechanisms, to recreate the inflammatory and fibrotic stages of myocarditis (
In the present study, histopathological analysis of the myocardium revealed marked inflammatory cell infiltration and the presence of edematous and necrotic tissues in EAM mice sacrificed after 21 days. These observations may be attributed to the activation of the TAB1/NF-κB pathway and the promotion of inflammatory factor expression (IL-6, IL-1β and TNF-α). There were no changes observed in the control group between 21 and 60 days. However, the EAM mice showed reduced inflammatory cell infiltration after 60 days. In addition, increased myocardial tissue damage and fibrosis was observed after 60 days compared with those after 21 days. This may be due to reduced secretion of inflammatory factors in the myocardium, increased activation of the TGF-β1/Smad2 pathway and an increase in the expression of the profibrotic factors TGF-β1, Col I and Col III. These findings suggest that damage may have changed from excessive inflammation at 21 days to myocardial fibrotic damage at 60 days. These are consistent with the results reported in previous studies (
TNF-α is mainly secreted by mononuclear macrophages and is an important inflammatory and immunomodulatory factor. It is responsible for the immunopathological damage of myocardial cells (
The present study showed that treatment of EAM mice with KX ameliorated myocardial injury in a dose-dependent manner, in addition to inhibiting the secretion of TNF-α, IL-6, IL-1β and NF-κB signaling proteins. Therefore, KX may reduce inflammation through the TAB1/NF-κB pathway to reduce myocardial injury. Numerous modern pharmacological studies have previously shown that the
TGF-β1 is an anti-inflammatory cytokine secreted by macrophages that are activated by regulatory T-cells (
Based on the present findings, KX may be efficacious for the clinical treatment of myocarditis by attenuating the inflammatory response whilst alleviating the clinical symptoms in acute myocarditis. KX may be effective in delaying progression even in patients in which the treatment of myocarditis is delayed. This provides an important reference value for the clinical application of KX.
However, the present study remain associated with a number of limitations. There was lack of a positive control group.
Results of the present study suggested that KX may reduce the inflammatory response to autoimmune myocarditis and attenuate pathological injury during the first stage of EAM through the TAB1/NF-κB pathway. Subsequently, KX may also delay the progression of autoimmune myocarditis onto myocardial fibrosis through the TGF-β1/Smad2 pathway during the second stage of EAM. This reflects the multifaceted, multi-target and multi-pathway action of KX in the treatment of myocarditis, in addition to the synergy among Chinese herbal therapies. However, in the present study, there is insufficient evidence to state that
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
LL and TD made substantial contributions to the conception and design of the study. ML and YL were responsible for the experimental procedures, data acquisition, analysis and interpretation and confirm the authenticity of all the raw data. ML performed the drafting of the article and critically revised it for important intellectual content. HX was responsible for the review of data and experiments. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved.
All experiments and animal care procedures were approved by the animal ethics committee of Jilin Academy of Traditional Chinese Medicine(approval no. JLSZKYDWLL2021-003).
Not applicable.
The authors declare that they have no competing interests.
Effects of KX on the mouse cardiac indices after 21 and 60 days. (A) Body weight (n=12 per group). (B) Changes in heart weight (n=12 per group). (C) Changes in HW/BW (n=12 per group). **P<0.01 vs. Control; #P<0.05 and ##P<0.01 vs. EAM. ΔP<0.05 and ΔΔP<0.01. EAM, experimental autoimmune myocarditis; KX, combination of
Effects of KX on myocardial injury in mice after 21 and 60 days. Changes in the levels of (A) LDH and (B) CK-MB in mouse serum. (C) Changes in the levels of cTn-I in the myocardium. (D) H&E staining of the myocardium. Scale bars, 100 µm. n=5 per group. Arrows indicate inflammatory cell infiltration (mainly lymphocytes) and myocardial injury. Quantified data plots are shown on the right. **P<0.01 vs. Control; #P<0.05, ##P<0.01 vs. EAM. ΔΔP<0.01. KX, combination of
Effects of KX on the production of inflammatory cytokines in the mouse myocardium. Changes in the levels of (A) IL-6, (B) IL-1β and (C) TNF-α in the mouse myocardium (n=12 per group). **P<0.01 vs. Control; #P<0.05 and ##P<0.01 vs. EAM. ΔΔP<0.01. KX, combination of
Effects of KX on TAB1 and NF-κB signaling in the mouse myocardium. (A) Western blot analysis of TAB1, IKKα, IκB, p-IκB, NF-κB, p-NF-κB and TGF-β1 protein expression in EAM mice after 21 days. (B) Western blot quantification analysis (n=4 per group). (C) Immunohistochemistry analysis of NF-κB and TGF-β1 expression in EAM mice after 21 days. Scale bar, 100 µm. Arrows indicate positive expression of NF-κB or TGF-β1. (D) Immunohistochemical quantification of NF-κB and TGF-β1 expression in EAM mice after 21 days. n=10 per group. **P<0.01 vs. Control. #P<0.05 and ##P<0.01 vs. EAM. TAB1, TGF-β activated kinase 1-binding protein 1; EAM, experimental autoimmune myocarditis; KX, combination of
Effects of KX on cardiac fibrosis in mice. Levels of (A) TGF-β1, (B) Col III and (C) Col I after 21 and 60 days (n=12 per group). (D) Masson's trichrome staining of EAM mice after 60 days and corresponding quantification. Scale bar, 100 µm. n=5 per group. Arrows indicate the cardiac muscle fiber. **P<0.01 vs. Control; #P<0.05 and ##P<0.01 vs. EAM. ΔΔP<0.01. EAM, experimental autoimmune myocarditis; KX, combination of
Effects of KX on TGF-β1 and Smad2 expression in the mouse myocardium. (A) TGF-β1, Smad2, Smad4, Col I and NF-κB expression in EAM mice after 60 days. (B) Western blot quantification analysis (n=4 per group). (C) NF-κB and TGF-β1 expression in EAM mice after 60 days. Scale bar, 100 µm. Arrows indicate positive expression of NF-κB or TGF-β1. (D) Quantitative results of immunohistochemical analysis (n=10 per group). **P<0.01 vs. Control; #P<0.05 and ##P<0.01 vs. the EAM. EAM, experimental autoimmune myocarditis; KX, combination of