Qi-Ju-Di-Huang-Pill delays the progression of diabetic retinopathy

Ethnopharmacological relevance: Qi-Ju-Di-Huang-Pill (QJDH pill) is a Chinese decoction. Although it is commonly used to treat eye conditions, such as diabetic retinopathy (DR), its exact mechanism of action is unknown.

Aim of the study: To investigate the specific mechanism by which QJDH pill slows the progression of diabetic retinopathy (DR) based on animal and cellular experiments.

Material and methods: The major components of QJDH pill were characterized by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLCMS/MS). C57BL/6J mice were randomly divided into five groups as follows: normal group (control group), model group (STZ group), low-dosage QJDH pill group (QJDH-L group), medium-dosage QJDH pill group (QJDH-M group) and high-dosage QJDH pill group (QJDH-H group). Changes in water intake, urination, food intake, and body mass were monitored weekly, while changes in blood glucose were monitored monthly. Fluorescein fundus angiography (FFA), optical coherence tomography angiography (OCTA), and optical coherence tomography (OCT) were utilized to analyze the changes in fundus imaging indications. Hematoxylin & eosin (H&E) and transmission electron microscopy (TEM) were employed to examine histopathologic and ultrastructural changes in retina. The levels of interleukin-6 (IL-6), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF) in peripheral blood were detected using Enzyme-linked immunosorbent assay (ELISA). The mouse retina apoptotic cells were labeled with green fluorescence via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (Tunel). The protein levels of Bcl-2-Associated X (Bax), B cell lymphoma 2 (Bcl-2), Caspase-3, PI3K, phosphorylated PI3K (p-PI3K), protein kinase B (Akt) and phosphorylated Akt (p-AKT) were quantified by Western blot (WB). The retinal pigment epithelium (RPE) cells were cultured and classified into five groups as follows: normal glucose group (NG group), high glucose group (HG group), high glucose + QJDH pill group (HG + QJDH group), high glucose + inhibitor group (HG + LY294002 group), and high glucose + inhibitor + QJDH pill group (HG + LY294002 + QJDH group). Cell viability and Apoptosis were detected via Cell Counting Kit-8 (CCK8) and then analyzed by flow cytometry.

Results: In vivo experiments revealed that the QJDH pill effectively reduced blood glucose, symptoms of increased water intake, elevated urination, increased food intake and decreased body mass in DR mice. QJDH pill also slowed the development of a series of fundus imaging signs, such as retinal microangiomas, tortuous dilatation of blood vessels, decreased vascular density, and thinning of retinal thickness, downregulated IL-6, IL-17, TNF-α, and VEGF levels in peripheral blood, and inhibited retinal cell Apoptosis by activating the PI3K/Akt signaling pathway. Moreover, in vitro experiments showed that high glucose environment inhibited RPE cell viability and activated RPE cell Apoptosis pathway. In contrast, lyophilized powder of QJDH pill increased RPE cell viability, protected RPE cells from high glucose-induced damage, and decreased Apoptosis of RPE cells by activating the PI3K pathway.

Conclusion: QJDH pill induces hypoglycemic, anti-inflammatory effects, anti-VEGF and anti-retinal cell Apoptosis by activating PI3K/Akt signaling pathway, and thus can protect the retina and slow the DR progression.

Apoptosis; Diabetic retinopathy; Mechanism studies; PI3K/AKT; QJDH pill (Qi-Ju-Di-Huang Pill).