Moxibustion enables protective effects on rheumatoid arthritis-induced myocardial injury via transforming growth factor beta1 signaling and metabolic reprogramming

doi:10.19852/j.cnki.jtcm.20230802.005

Abstract

Abstract:

OBJECTIVE: To examine the effects of moxibustion on myocardial injury and myocardial metabolomics in rats with rheumatoid arthritis (RA) based on the transforming growth factor beta1 (TGF-β1)/Smads signaling pathway.
METHODS: One hundred rats were treated with saline [normal control (NC) group] or complete Freund’s adjuvant (CFA) by right plantar injection for the RA model group, and the latter were randomly divided into 4 groups. Tripterygium wilfordii polyglycoside tablets (雷公藤多苷片, TPT) have anti-inflammatory and are widely used in the clinical treatment of RA, therefore serving as a positive control group. Three days post injection rats were given TPT tablet (TPT group), acupuncture therapy (APT group), and moxibustion treatment (MOX group) for 15 consecutive days, while NC group and model group were equally grasped and fixed and received normal saline. Rat joint swelling scores and arthritis index (AI) were evaluated in each group before the CFA challenge, therapy and after receiving therapy. Myocardial ultrastructure was observed by electron microscope. Enzyme-linked immunosorbent assay was used to detect cardiac troponin I (cTnI) levels in rat myocardial tissue. Quantitative reverse transcription polymerase chain reaction and Western blotting analysis were used to measure the mRNA and protein levels of TGF-β signaling molecules including TGF-β1, Smad2, Smad3, Smad4, and Smad7. Myocardial metabolomics was analyzed using gas chromatography-mass spectrometer.
RESULTS: Compared with model group, RA model rats receiving TPT, acupuncture, or moxibustion therapy all showed reduced joint swelling scores and AI (all P < 0.01) and improved myocardial damage, whereas rats treated with moxibustion were found to be more marked. Consistently, the expressions of cTnI, TGF-β1, Smad2, Smad3, and Smad4 were found to be elevated in model rat group in contrast to NC rats and were significantly downregulated in TPT, APT and MOX group when compared with model group, while the levels of Smad7 showed the opposite result (all P < 0.01). Moreover, the dissection of metabolomics suggested a novel metabolite biomarker panel including D-Xylulose 5-phosphate, dihydroxyacetone phosphate, arachidonic acid, etc was defined and implicated in amino acid, glucose, and fatty acid metabolic processes as revealed by principal component analysis and partial least squares discriminant analysis.
CONCLUSION: Moxibustion prevents RA-induced inflammatory response and offers potent therapeutic effects on myocardial dysfunctions. The protective effects might be associated with its role in TGF-β1 inactivation and metabolic reprogramming.

Key words: moxibustion, arthritis, rheumatoid, transforming growth factor beta1, smad proteins, signal transduction, myocardial injury, metabolomics

WANG Miao, ZHU Yan, ZHAO Hui, ZHAO Hongfang. Moxibustion enables protective effects on rheumatoid arthritis-induced myocardial injury via transforming growth factor beta1 signaling and metabolic reprogramming[J]. Journal of Traditional Chinese Medicine, 2023, 43(6): 1190-1199.

Figures/Tables 4

Table 1 Evaluation of rat joint swelling scores and AI in each group ($\bar{x}$ ± s)

Group	n	Joint swelling scores (%)			AI (points)
Group	n	Before modeling	Before therapy	After therapy	Before modeling	Before therapy	After therapy
NC	20	1.75±0.23	1.83±0.11	1.85±0.24	0	0	0
Model	20	1.76±0.17	2.95±0.14^a	2.68±0.11^a	0	9.34±0.67^a	7.02±1.08^a
MOX	20	1.79±0.10	2.93±0.17	2.35±0.09^b	0	9.33±0.09	3.62±0.91^b
APT	20	1.77±0.13	2.93±0.14	2.36±0.12^b	0	9.29±1.05	3.68±1.02^b
TPT	20	1.78±0.11	2.91±0.15	2.41±0.09^b	0	9.31±0.15	4.11±0.97^bc

Figure 1 Protecting effects on the myocardial ultrastructure with or without therapy (×15000) A-E: electron microscopic ultrastructural basis of rat heart muscles (n = 7). A: NC group treated only with normal saline; B: model group treated with CFA and normal saline; C: TPT group treated with CFA with TPT solution by gavage (dose 8 mg·kg-1·d-1); D: APT group treated with CFA and acupuncture; E: MOX group treated with CFA and suspension moxibustion. In the figures, red arrows indicate mitochondria and yellow arrows indicate transverse striations. NC: normal control; TPT: tripterygium wilfordii polyglycoside tablets; CFA: complete Freund’s adjuvant; MOX: moxibustion; APT: acupuncture.

Figure 2 Expression of TGF-β signaling molecules upon therapy A: the heart mRNA expression of TGF-β1, Smad2, Smad3, Smad4, and Smad7 in rats receiving indicated treatment. Approximately 100 mg were used for Total RNA of heart tissues were extracted, reverse-transcribed and subjected to real-time PCR using indicated primers. All PCR reactions were done in triplicates. Showed is data from 3 independent experiments. B: total proteins were isolated, quantified, and separated by SDS-PAGE, followed by Western blotting analysis using indicated antibodies. 1: NC group treated only with normal saline; 2: model group treated with CFA and normal saline; 3: TPT group treated with CFA with TPT solution by gavage (dose 8 mg·kg-1·d-1); 4: APT group treated with CFA and acupuncture; 5: MOX group treated with CFA and suspension moxibustion. NC: normal control; TPT: tripterygium wilfordii polyglycoside tablets; CFA: complete Freund’s adjuvant; MOX: moxibustion; APT: acupuncture; TGF-β1: transforming growth factor beta1. Data were expressed as mean ± standard deviation (n = 7). aP < 0.01, compared with NC group; bP < 0.01, compared with model group; cP < 0.05, dP < 0.01, compared with MOX group.

Figure 3 TIC of myocardial samples Representative TIC diagrams for each group are shown above (n = 5). A: NC group treated only with normal saline; B: MOX group treated with CFA and suspension moxibustion; C: model group treated with CFA and normal saline; D: TPT group treated with CFA with TPT solution by gavage (dose 8 mg·kg-1·d-1); E: APT group treated with CFA and acupuncture. Visual inspection of the TIC chromatograms of all samples revealed that the instrumental analysis of all samples had a strong signal, high peak capacity, and good retention time reproducibility. TIC: total ion flow chromatogram; NC: normal control; CFA: complete Freund’s adjuvant; MOX: moxibustion; TPT: tripterygium wilfordii polyglycoside tablets; APT: acupuncture; GC/MS: Gas Chromatography-Mass Spectrometer. GC/MS detected a large number of peaks from each sample, such as the raw instrumental data files of the samples were analyzed by deconvolution using the default parameters of the AMDIS software (National Institute of Standards and Technology). Software algorithms were used to pre-process and analyze the acquired mass spectrometry data to convert the complex mass spectrometry signals into useful qualitative and quantitative data.

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