Human retinal capillary endothelial cells (HRCECs) were acquired from the Cell Bank of Shanghai Academy of Chinese Sciences (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) was purchased from HyClone (Logan, United States), fetal bovine serum (FBS) was bought from GIBCO (Grand Island, United States). Cell Counting Kit-8 (CCK-8) and PBS were obtained from meilun biotechnology co., Ltd. (Dalian, China). ATP Assay Kit, Mitochondrial membrane potential kit and ROS assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Apoptosis Detection Kit and cycle detection kit were provided by KeyGen Biotechnology co., Ltd. (Nanjing, China). Matrigel was provided by Corning (New York, United States). Primary antibody of p-NF-κB, NF-κB, p-P38, P38, BCL-XL, BCL-2 and GAPDH were bought from Cell Signaling Technology (Danvers, United States).

Root of Polygonum cuspidatum Sieb. et Zucc., fruit of Forsythia suspensa, whole plant of Sedum sarmentosum Bunge, whole plant of Siegesbeckia orientalis L., rattan stem of Spatholobus suberectus Dunn, root of Glycyrrhiza uralensis Fisch, root of Stragalus membranaceus (Fisch.) Bunge and root of Ligusticum chuanxiong hort. were provided by the ophthalmology Department of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Table 1). These Chinese herbal medicines were put into the decocting pot, and extracted with 10 times volume pure water for 1 hour, two times in total. The decocting liquid were concentrated with a rotating evaporator after filtering with a 4-layer gauze. After that, the liquid was further pre-freezed at −50°C for 5 h for a vacuum dry in the material tray of the freeze-dryer. The dryer was heated up and finally fixed to −40°C for lyophilized 72 h to make a lyophilized powder which was stored in a refrigerator at −20°C for later use. The stock solution of the HZQMY extract was prepared with first grade pure water to the concentration of 0.1 g/ml, and then it was filtered and sterilized by 0.22 μm microporous membrane. For temporary use, the medium containing 10% FBS was diluted to the working concentrations.

1.0 g of HZQMY extract was put into a 25 ml volumetric flask, and then 50% methanol was added to scale, ultrasonic treatment for 30 min, cooling, 50% methanol to make up the amount of loss reduction, fully mixed, filtrated with a 0.45 μm microporous membrane to obtain the test solution, and 10 μL was taken for mass spectrometry detection.

Agilent 6530 four-stage rod-time-of-flight mass spectrometry (Q-TOF-MS) system was performed, chromatographic separations were carried out on an ACQUITY UPLC HSS T3 (2.1 × 150 mm, 1.7 μm) column at 25°C and the drying gas (N₂) was 13 L/min. Gradient elution with mobile phase A (0.1 formic acid water), mobile phase B (acetonitrile): 99% A in 0–2 min; from 99 to 34% A in 2–20 min; from 34 to 5% A in 20–28 min. The mass spectrometry was performed in positive and negative ion scanning patter with a scanning range of 100–1700 m/z and a capillary voltage of 4000 V. The intercepting cone voltage is 60 V.

HRCECs were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂. DR model was established by induction of HRCECs with high glucose in vitro (Liu et al., 2020). The cells were divided into normal group (cells were cultured in normal-glucose (5.5 mM) media, NG), high glucose model group (cells were cultured in high-glucose (35 mM) media, HG), and HZQMY different dose groups. Except normal group, all the other groups of cells were given 35 mM glucose to imitate high-glucose environment. Different concentrations of HZQMY extract were used to treat the cells. Cells were collected for subsequent experiments following treatment with HZQMY extract for 24 h or 48 h.

HRCECs were seeded into 96-well plates (5 × 10³ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then different concentrations of HZQMY extract (10, 25, 50, 100, 150 μg/ml) were added in 96-well plates for 24 and 48 h. Discarding the old medium, 100 μL of 10% CCK-8 kit was added into each well for incubation at 37°C for 30 min. OD value was detected at 450 nm with a microplate analyzer. Cell survival rate (%) = (OD value of experimental group–OD value of blank group)/(OD value of control group–OD value of blank group) ×100%.

HRCECs were seeded into 6-well plates (600 cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then added with different concentrations of the extract and cultured for 8 days. The cells were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 15 min. Clone formation rate (%) = (amount of clones/number of inoculated cells) ×100%.

HRCECs were seeded into 6-well plates (5 × 10⁵ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then each group was added with different concentrations of the extract for 24 h culture. Cells were harvested, 1 ml of precooled 70% ethanol was added to mix well, and fixed at 4°C for 2 h. The cells were collected, mixed with 0.5 ml PI into the sample tube, and incubated in the dark for 30 min. The red fluorescence was detected by flow cytometry at 488 nm, and the light scattering was recorded at the same time. The Beckman flow cytometer was used for detection.

60 µL of the dissolved matrigel (9–12 mg/ml) was added in a well in 96-well plate and then placed in the incubator at 37°C for 1 h. HRCECs were cultured under different concentrations of HZQMY with or without HG. Subsequently, 50 μL of cell suspension was collected and added into the pre-solidified matrigel for 8 h in the 37°C incubator. The tube formation was observed under an optical microscope, and photographed. ImageJ was used to calculate the number of lumens and the length of tubules.

HRCECs were seeded into 24-well plates (5 × 10⁴ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then each group was added with different concentrations of the extract for 24 h culture. Subsequently, the cells were washed by PBS three times, 150 µL of 4% paraformaldehyde was added to each well and fixed at room temperature for 30 min, and remove the fixing solution, add 150 µL of 0.1% Triton X-100 for 10 min. Finally, 150 µL DAPI solution was added in each well for incubation at room temperature for 10 min in dark. The samples were photographed by Operetta CLS high-content analysis system.

HRCECs were seeded into 6-well plates (5 × 10⁵ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then each group was added with different concentrations of drugs for 24 h culture. After that, the cells were digested with trypsin without Ethylene Diamine Tetraacetic Acid (EDTA) and centrifuged with a 15 ml centrifuge tube at 2000 rpm for 10 min at 4°C, and the supernatant was discarded. 100 μL 1 × FITC binding solution was added to mix well, then 5 μL FITC dye was added for 10 min incubation at room temperature in dark. Last, 5 μL PI dye was added for 5 min incubation in dark. The Beckman flow cytometer was used for detection.

HRCECs were seeded into a 96-well plate (1 × 10⁵ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then each group was added with different concentrations of the extract for 6 h. DCFH-DA was added for 1 h incubation in a 37°C cell incubator. The Beckman flow cytometer was used for detection.

ATP concentration was detected by the ATP Assay Kit. HRCECs were seeded into 6-well plates (5 × 10⁵ cells/well) for 12 h in a 5% CO₂ incubator at 37°C, then added with different concentrations of HZQMY extract to culture for 24 h. Cells were lysed to isolate total protein, then centrifuged at 12,000 g and 4°C for 10 min. Next, 20 µL sample or standard solution were added to 100 µL ATP detection solution, mixed, and luminescence was measured with a multifunctional enzyme plate analyzer.

HRCECs were seeded into 24-well plates (5 × 10⁴ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then added with different concentrations of the extract for 24 h culture. Apoptotic cells should show green fluorescence after JC-1 staining, while normal cells show red fluorescence. The relative ratio of red-green fluorescence is often used to measure the proportion of mitochondrial depolarization, which was used as one of the early detection indicators of cell apoptosis. The fluorescence quantification was carried out using ImageJ software.

HRCECs were seeded into 6-well plates (5 × 10⁵ cells/well) for 24 h in a 5% CO₂ incubator at 37°C, then each group was added with different concentrations of the extract for 24 h culture. Cells were collected and total proteins were extracted from HRCECs using RIPA lysate and PMSF (RIPA: PMSF = 100:1). Protein concentration was measured by using the BCA Protein Concentration Assay Kit. The proteins were separated by 10% SDS-polyacrylamide electrophoresis and transferred to PVDF membrane, which was then sealed with QuickBlock™ Blocking Buffer at room temperature for 1 h. After that, the membrane was incubated with the primary antibody overnight at 4°C, followed by washing 3 times with TBST, and the rabbit/mouse antibody was incubated at room temperature for 1 h. Then the membrane was washed 3 times with TBST. Finally, the gray values of the protein bands were detected and photographed by Chemi Scope Mini, and GAPDH was used as internal reference.

HRCECs (cultured with 35 mM glucose) were seeded into 100 mm culture dishes and 100 µg/ml HZQMY extract was added until 60% confluent. After 24 h, cells were harvested and flash-frozen in liquid nitrogen. Identification and analysis were processed by Ouyi Biotechnology Co., Ltd. (Shanghai, China). The proteins were quantified by iTRAQ labeling, and based on UniProt, KEGG, go, KOG/cog databases, the annotated information of the identified proteins was extracted to mine the protein functions. Proteins with quantification changes >1.5 and p value <0.05 were considered as differentially expressed proteins. After the differentially expressed proteins were obtained, GO/KEGG enrichment analysis were performed to describe the functions of these proteins, and the involved interaction network analysis was also executed by using STRING database.

All data were analyzed by SPSS 22.0 statistical software, and the results were expressed as mean ± standard deviation. The comparison of the mean between two groups was performed by t-test, and the comparison of the mean between multiple groups was carried out by one-way analysis of variance. p < 0.05 was considered statistically significant.

HPLC-Q-TOF-MS was used to analyze the constituents of HZQMY, and a total of 27 compounds were identified by comparing databases and literatures, which are Forsythoside E (1), Heteroclitin D (2), Quercetin (3), Forsythoside (4), Pinoresinol 4-O-β-D-glucopyranoside (5), Quercitrin (6), Forsyithin (7), Arctiin (8), Polydatin (9), Liquiritin (10), Apigenin (11), Isorhamnetin (12), Calycosin-7-O-glucoside (13), Kaempferol (14), Afromosin (RG) (15), (-)-Catechin hydrate (16), Genistein (17), Eleutheroside A (18), trans-Anethole (19), Oleanic acid (20), Quercetin dihydrate (21), Physcion 8-β-D-glucoside (22), Hyperoside (23), 3-Hydroxy-4-methoxybenzoic acid (24), Luteolin (25), Formononetin (26) and Prunetin (27). The chromatogram of the compounds obtained is shown in Figure 1.

As shown in Figure 2, when compared with NG group (5.5 mmol/L), the cell viability significantly increased in HG group (35 mmol/L). However, 50–150 μg/ml HZQMY significantly reduced the cell viability increased by high glucose after 24 h incubation (Figures 2A,B), and the inhibitory effect was more pronounced at 48 h (Figures 2C,D). These results suggested that high glucose can improve cell viability, while HZQMY can reverse the promotion effect in a time and dose-dependent manner.

To test the proliferation effect of HZQMY on HRCECs, cell colony formation and cycle assay were performed. Figures 2E,G show that high concentration of glucose can promote cell proliferation, while HZQMY could reverse this effect in a dose-dependent manner. Decreased cell proliferation is always associated with changes in the phase of the cell cycle, and the flow cytometry analysis displayed that the G₂/M phase accounted for a higher proportion in the HZQMY group than in the HG group (Figures 2F,H). The results suggested that HZQMY inhibits cell proliferation by arresting the cell cycle in the G₂/M phase.

Angiogenesis is a characteristic of endothelial cells. Tube formation in vitro was used to simulate angiogenesis in vivo, and the length of angiogenic branches was finally compared with ImageJ. The results (Figures 3A–C) displayed that the number of tubes and the length of tube branches were decreased after HZQMY treatment, which indicated that HZQMY can slightly inhibit the angiogenesis of HRCECs induced by high glucose.

In order to determine whether the inhibitory effect of HZQMY on cell proliferation was related to the induction of cell apoptosis, the nucleus was stained with DAPI to analyze the nuclear morphology. As shown in Figure 4A, after HZQMY treatment, the number of cells decreased significantly, nuclear pyknosis and apoptotic bodies were formed in a dose-dependent manner.

Flow cytometry was used to quantitatively analyze the proportion of cells in different stages of apoptosis in each group after Annexin V and PI double staining. Annexin V is used as an indicator of early apoptosis, and PI acts as an indicator of late apoptosis. As shown in Figure 4B, with the increase of HZQMY concentration, the sum of early and late apoptosis significantly increased, with the NG group was 9.94%, the HG group was 5.61%, the HG + HZ-50 group was 28.60%, the HG + HZ-100 group was 48.95%, which suggested that HZQMY could induce cell apoptosis in a dose-dependent manner.

Mitochondria are important organelles responsible for energy generation, which are closely related to ATP synthesis (Tang et al., 2018). What’s more, the production of ROS has also closely relation with mitochondria (Indo et al., 2007). Therefore, MMP and the levels of ROS and ATP are often used to evaluate the functional status of mitochondria. To verify whether HZQMY affected the production of ATP and ROS, the cellular ROS levels were detected by DCFH-DA and ATP content was assessed by ATP assay kit. As shown in Figures 5A,C, after treatment with 100 μg/ml HZQMY for 24 h, the production of ROS in cells increased sharply. Compared with the NG group (Figure 5D), high glucose increased ATP levels in HRCECs, while HZQMY significantly reduced its production.

JC-1 is a fluorescent probe, which can act as an indicator of early cell apoptosis, and is often used in the detection of MMP. In the NG group, the cytoplasm of the cells showed red fluorescence, indicating high MMP. While the cells are injured, MMP will decrease, the cytoplasmic red fluorescence of the cells will reduce, and the green fluorescence will increase. The ratio of red fluorescence to green fluorescence (R/G) can be used for quantitative comparison. As shown in Figure 5B, the red fluorescence significantly decreased and the green fluorescence markedly increased. The higher the concentration of HZQMY is, the lower the ratio of R/G is (Figure 5E), indicating promotive effects of HZQMY on MMP collapse.

HZQMY regulated the proteins of P38 and NF-κB signaling pathway to inhibit proliferation of HRCECs.

To explore the mechanism of inhibitory effect of HZQMY on the proliferation of HRCECs in high glucose environment, we studied its mitochondrial apoptosis pathway. The decreased MMP, ROS and ATP levels proved that mitochondrial function was impaired.

As we all know, P38 is a classic pathway of apoptosis. In our study, P-P38 could be upregulated by HZQMY, and BCL-2 and BCL-XL could be downregulated by it, as shown in Figures 6A,C–E. Since NF-κB signal pathway plays a significant role in cell proliferation (Mu et al., 2020). Then western blot assay was used to detect the protein expression of NF-κB signaling pathway. As shown in Figures 6A,B, HZQMY downregulated the expression of p-NF-κB in HRCECs cultured with high glucose. All these results indicated that HZQMY suppressed cell proliferation possibly through regulating P38 and NF-κB signal transduction pathway.