Department of Rheumatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, China.
Department of Emergency, Shanghai Traditional Chinese and Western Medicine Integrated Hospital, Shanghai University of Traditional Chinese and Western Medicine, Shanghai, 200082, China.
Chin J Integr Med. 2020 Jul;26(7):502-509. doi: 10.1007/s11655-020-3205-5. Epub 2020 Jul 6.
To investigate the mechanism of cAMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction (, SGD) on the regulation of aquaporin 5 (AQP5) and muscarinic receptor 3 (M3R) levels in Sjögren's syndrome (SS).
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.
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).
SGD can improve symptoms of SS by regulating the cAMP-PKA signaling pathway and increasing AQP5 and M3R levels.
研究中药芍药甘草汤（SGD）通过环磷酸腺苷-蛋白激酶 A（cAMP-PKA）信号通路对干燥综合征（SS）中水通道蛋白 5（AQP5）和毒蕈碱受体 3（M3R）水平调节的作用机制。
30 只小鼠中，随机选取 5 只作为对照组，其余用于建立 SS 模型。建模成功后，将小鼠随机分为 5 组（每组 5 只），分别灌胃生理盐水（8 mL/kg）、毛果芸香碱（1.4 mg/kg）或低、中、高剂量 SGD（分别含 0.14、0.21、0.35 g 白芍和 0.01 g 甘草），连续 6 周。用毛果芸香碱或不同剂量 SGD 处理人唇腺腺泡细胞，生理盐水作为安慰剂。苏木精-伊红染色观察各组小鼠颌下腺的组织病理学变化。酶联免疫吸附试验检测各组小鼠颌下腺抗 SS 抗原 A（SS-A）、抗 SS 抗原 B（SS-B）、M3R 和α-胞衬蛋白的血清水平。免疫荧光染色观察腺泡细胞中 AQP5 和 M3R 的空间定位。逆转录聚合酶链反应和 Western blot 检测各组小鼠颌下腺组织和细胞中蛋白激酶 A（PKA）、环磷酸腺苷（cAMP）、Epac1、AQP5、M3R、核因子 kappa-B（NF-κB）和肿瘤坏死因子（TNF）-α的表达。
与正常小鼠相比，免疫后 1-6 周，模型组小鼠的体重、5 分钟唾液分泌量、30 分钟泪液分泌量和泪膜破裂时间均降低（均 P<0.05），而饮水量增加（均 P<0.05）。在模型组中，颌下腺腺体出现萎缩，伴有不同大小的腺泡、数量减少、排列疏松，导管扩张，伴有不同程度的淋巴细胞浸润。SGD 组小鼠的情况有所改善。与正常组相比，各剂量 SGD 处理的腺泡细胞中 AQP5 和 M3R 的阳性表达更高；各剂量 SGD 处理的动物血清 SS-A、SS-B 和α-胞衬蛋白水平更低，M3R 水平更高（均 P<0.05）。与模型组和毛果芸香碱组相比，中、高剂量 SGD 处理的小鼠或细胞中 NF-κB 和 TNF-α 的 mRNA 和蛋白水平均降低（均 P<0.05），而 PKA、Epac1、AQP5 和 M3R 的水平均升高（均 P<0.05）。
SGD 通过调节 cAMP-PKA 信号通路，增加 AQP5 和 M3R 水平，可改善 SS 症状。
