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Rhein alleviates advanced glycation end products (AGEs)-induced inflammatory injury of diabetic cardiomyopathy in vitro and in vivo models

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Abstract

In diabetic patients, diabetic cardiomyopathy (DCM) is one of the most common causes of death. The inflammatory response is essential in the pathogenesis of DCM. Rhein, an anthraquinone compound, is extracted from the herb rhubarb, demonstrating various biological activities. However, it is unclear whether rhein has an anti-inflammatory effect in treating DCM. In our research, we investigated the anti-inflammatory properties as well as its possible mechanism. According to the findings in vitro, rhein could to exert an anti-inflammatory effect by reducing the production of NO, TNF-α, PGE2, iNOS, and COX-2 in RAW264.7 cells that had been stimulated with advanced glycosylation end products (AGEs). In addition, rhein alleviated H9C2 cells inflammation injury stimulated by AGEs/macrophage conditioned medium (CM). In vivo have depicted that continuous gavage of rhein could improve cardiac function and pathological changes. Moreover, it could inhibit the accumulation of AGEs and infiltration of inflammatory factors inside the heart of rats having DCM. Mechanism study showed rhein could suppress IKKβ and IκB phosphorylation via down-regulating TRAF6 expression to inhibit NF-κB pathway in AGEs/CM-induced H9C2 cells. Moreover, the anti-inflammation effect of rhein was realized through down-regulation phosphorylation of JNK MAPK. Furthermore, we found JNK MAPK could crosstalk with NF-κB pathway by regulating IκB phosphorylation without affecting IKKβ activity. And hence, the protective mechanism of rhein may involve the inhibiting of the TRAF6-NF/κB pathway, the JNK MAPK pathway, and the crosstalk between the two pathways. These results suggested that rhein may be a promising drug candidate in anti-inflammation and inflammation-related DCM therapy.

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Data availability statement

All data are available from the corresponding authors upon request.

Abbreviations

AG:

Aminoguanidine

AGEs:

Advanced glycation end products

CM:

Macrophage conditioned medium

COX-2:

Cyclooxygenase 2

DCM:

Diabetic cardiomyopathy

EF:

Ejection fraction

EMPA:

Empagliflozin

ERK:

Extra cellular regulated protein kinase

FS:

Fractional shortening

IKK:

IκB kinase

iNOS:

Inducible nitric oxide synthase

IκB:

Inhibitor of NF-κB

LVIDd:

Left ventricular end-diastolic internal diameter

LVIDs:

Left ventricular end-systolic internal diameters

LVPWd:

Left ventricular posterior wall thickness during diastole

LVPWs:

Left ventricular posterior wall thickness during systole

JNK:

C-jun n-terminal kinase

MAPK:

Mitogen-activated protein kinase

MyD88:

Myeloid differentiation primary response gene 88

MMP:

Mitochondrial membrane potential

NF-κB:

Nuclear factor-κB

PGE2 :

Prostaglandin E2

STZ:

Streptozotocin

T2DM:

Type 2 diabetes mellitus

TLR4:

Toll-like receptors 4

TNF-ɑ:

Tumor necrosis factor ɑ

TRAF6:

The tumor necrosis factor receptor-associated factor 6

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Acknowledgements

The support of the experimental platform was generously provided by the Lingnan Medical Research Center of Guangzhou University of Chinese Medicine, and we are extremely thankful for this.

Funding

This study was funded by the National Natural Science Foundation of China for Young People (Nos. 82104621, 82204809) and Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515220066).

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Authors and Affiliations

  1. The First School of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou, 510405, Guangdong, China

    Shao-Yang Zhao, Huan-Huan Zhao, Bao-Hua Wang, Cui Shao, Wen-Jun Pan & Sai-Mei Li

  2. Postdoctoral Research Station, Guangzhou University of Chinese Medicine, Guangzhou, 510405, Guangdong, China

    Shao-Yang Zhao, Cui Shao & Wen-Jun Pan

  3. Nutrition Department, LinYi People’s Hospital, Linyi, 276000, Shandong, China

    Huan-Huan Zhao

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Contributions

S-ML and S-YZ conceived and designed the experiments. S-YZ carried out the experiments and wrote the manuscript. H-HZ participated in animal experiments. B-H provided the regents. CS and W-JP helped revise the article. All authors have read and agreed to the published version of the manuscript. S-YZ is the first author of this article. Correspondence should be addressed to S-YZ and S-ML.

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Correspondence to Shao-Yang Zhao or Sai-Mei Li.

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The First Affiliated Hospital of Guangzhou University of Chinese Medicine's Experimental Animal Ethics Committee approved all of the experiments performed on animals.

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Supplementary Information

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11418_2023_1741_MOESM1_ESM.tif

Supplementary file1 Fig. S1 RAW264.7 cells were treated with AGEs or rhein alone in a concentration or time-dependent manner. (A) RAW264.7 cells were treated with AGEs (from 0 to 350 μg/ml) for 8, 16, and 24 h respectively. NO production was measured. (B) RAW264.7 cells were treated with rhein (from 0 to 100 μM) for 24 h. cell viability was measured. N.S.: no significance. #P < 0.05, ###P < 0.001 compared to the control group (TIF 2847 KB)

11418_2023_1741_MOESM2_ESM.tif

Supplementary file2 Fig. S2 AGEs isn’t an ideal agent for inducing inflammatory injury in H9C2 cells. H9C2 cells were treated with AGEs (50–500 μg/ml) for 48 h, and the cell viability was then detected by MTT. Cell viabilities were similar between AGEs-treated H9C2 cells and DMEM-treated cells. (B–C) The releases of NO and LDH were detected by Griess and LDH Cytotoxicity Assay. ##P < 0.01 compared to the control group (TIF 4777 KB)

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Zhao, SY., Zhao, HH., Wang, BH. et al. Rhein alleviates advanced glycation end products (AGEs)-induced inflammatory injury of diabetic cardiomyopathy in vitro and in vivo models. J Nat Med 77, 898–915 (2023). https://doi.org/10.1007/s11418-023-01741-7

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