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Mechanism of cAMP-PKA Signaling Pathway Mediated by Shaoyao Gancao Decoction (芍药甘草汤) on Regulation of Aquaporin 5 and Muscarinic Receptor 3 Levels in Sjögren’s Syndrome

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Abstract

Objective

To investigate the mechanism of cAMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction (芍药甘草汤, SGD) on the regulation of aquaporin 5 (AQP5) and muscarinic receptor 3 (M3R) levels in Sjögren’s syndrome (SS).

Methods

Of the 30 mice, 5 were randomly selected as control, and others were used for creating SS model. After successful modeling, mice were randomly divided into 5 groups (n=5 per group) and intragastrically administered with saline (8 mL/kg), pilocarpine (1.4 mg/kg), or low, medium and high doses SGD (0.14, 0.21, 0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae, respectively) for 6 weeks. Human labial gland acinar cells were treated with pilocarpine or varying doses of SGD with saline as the placebo. Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice. The serum levels of anti-SS antigen A (SS-A), anti-SS antigen B (SS-B), M3R, and α-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3R in acinar cells. Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA, cAMP, Epac1, AQP5, M3R, nuclear factor kappa-B (NF-κB), and tumor necrosis factor (TNF)-α in submandibular gland tissues and cells of each group.

Results

Compared to normal mice, body weight, 5-min salivary secretion, 30-min secretion of tears and breakup time of tear film of model mice decreased at 1–6 weeks after immunization (all P<0.05), whereas water intake increased (all P<0.05). In the model group, glands of the submandibular glands showed atrophy, accompanied by acini of different sizes, decreased numbers and loose arrangement, with catheter dilatation and different degrees of lymphocyte infiltration. Conditions of mice in SGD groups were improved. The positive expression of AQP5 and M3R were higher in the acinar cells treated with all doses SGD compared to the normal group; serum levels of SS-A, SS-B, and α-fodrin were lower, and that of M3R was higher in all doses SGD treated animals than the model or pilocarpine treated ones (all P<0.05). Compared to the model and pilocarpine groups, the mRNA and protein levels of NF-κB and TNF-α were lower in mice or cells treated with medium or high-dose SGD (all P<0.05), while those of PKA, Epac1, AQP5 and M3R were higher (all P<0.05).

Conclusion

SGD can improve symptoms of SS by regulating the cAMP-PKA signaling pathway and increasing AQP5 and M3R levels.

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Authors and Affiliations

  1. Department of Rheumatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, China

    Dan Wang, Ben Li, Yu Sun & Dong-hui Wei

  2. Department of Emergency, Shanghai Traditional Chinese and Western Medicine Integrated Hospital, Shanghai University of Traditional Chinese and Western Medicine, Shanghai, 200082, China

    Hao Zhao

Authors
  1. Dan Wang
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  2. Hao Zhao
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  3. Ben Li
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  4. Yu Sun
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  5. Dong-hui Wei
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Contributions

Zhao H contributed to the study concepts, data analysis, statistical analysis and manuscript review; Wang D contributed to the study design, definition of intellectual content and manuscript preparation; Sun Y contributed to the literature research; Wang D, Wei DH and Li B contributed to the experimental studies; Zhao H and Sun Y performed the data acquisition; Li B edited the manuscript. All authors have read and approved the submitted version.

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Correspondence to Hao Zhao.

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The authors report no conflicts of interest in this work.

Additional information

Supported by the National Nature Science Foundation of China (No. 81503380)

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Wang, D., Zhao, H., Li, B. et al. Mechanism of cAMP-PKA Signaling Pathway Mediated by Shaoyao Gancao Decoction (芍药甘草汤) on Regulation of Aquaporin 5 and Muscarinic Receptor 3 Levels in Sjögren’s Syndrome. Chin. J. Integr. Med. 26, 502–509 (2020). https://doi.org/10.1007/s11655-020-3205-5

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