The gene sequences of anti-TNF-α nanobody (Nb-TNFα) and anti-VEGF-A nanobody (Nb-VEGFA) were synthesized by Tsingke Biotechnology (Shanghai, China). The sequence information of Nb-TNFα and Nb-VEGFA were provided by our laboratory. The bispecific nanobody Nb-TV was obtained by linking Nb-TNFα and Nb-VEGFA via a G4S flexible linker. The TNF-α, VEGF-A, Nb-TNFα, Nb-VEGFA, and Nb-TV sequence were inserted into the pET-24a plasmid through the Nde I and Xho I restriction sites and then confirmed by sequencing, respectively. The constructed plasmid was then transferred into E. coli Rosetta gami2 (DE3) (Cat #EC1013, Weidibio, Shanghai, China). A 3% inoculum was inoculated into 1 L of culture. When the OD600 value of the culture reached 0.6, induction was carried out with 0.4 mM IPTG (Cat #367–93–1, Macklin, Shanghai, China) at 37 °C for 10 h. Subsequently, purification was performed using Ni-NTA affinity chromatography. The purified proteins were verified through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The sequence of Nb-TV was uploaded to I-TASSER for simulating its three-dimensional structure. Meanwhile, the sequences of Nb-TV, TNF-α, and VEGF-A were separately uploaded to the HDOCK server to simulate the docking scenarios of Nb-TV with TNF-α and of Nb-TV with VEGF-A.

Immune plate was coated with antigen (diluted with Na₂CO₃ (Cat #S818014, Macklin, Shanghai, China) buffer, pH9.6) at a concentration of 10 μg/mL per well overnight at 4 °C. After blocking with 5% skim milk (Cat #S917757, Macklin, Shanghai, China) at 37 °C for 1 h, Nb-TV, Nb-TNFα, and Nb-VEGFA diluted in phosphate buffered saline buffer (PBS) (Cat #P750064, Macklin, Shanghai, China) were added to the plate in a dilution series and then incubated at 37 °C for 2 h. After washing, HRP Anti-HA tag antibody (Cat #100028-MM10-H, Sinobiological, Beijing, China) was incubated at 37 °C for 2 h as a secondary antibody. After incubation with TMB (Cat #PR1200, Solarbio, Beijing, China), the reaction was terminated by H₂SO₄ (Cat #81936C, Adamas-life, Shanghai, China). The absorbance (OD) of each well was detected at 450 nm.

ARPE-19 cells were cultivated in DMEM/F12 medium (Cat #D6501, Solarbio, Beijing, China) supplemented with 10% FBS and 1% streptomycin-penicillin-amphotericin B. HUVEC cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) (Cat #FS201-02, Transgen, Beijing, China) and 1% streptomycin-penicillin-amphotericin B (Cat #P7630, Solarbio, Beijing, China). The culture was maintained at 37 °C under a 5% CO₂ atmosphere. The medium was refreshed every 2 days. The cells were detached and passaged using 0.25% trypsin-EDTA (Cat #11995, Solarbio, Beijing, China) in accordance with their actual growth status.

ARPE-19 (1.5 × 10⁴) cells were seeded into 96-well plate and incubated overnight. TNF-α (0.53 μM) was added to model and drug groups. The drug group received a dilution series of Nb-VEGFA, Nb-TNFα or Nb-TV. After 24 h incubation, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Cat #T6126, Macklin, Shanghai, China) was added to each well for 4 h. Then, a 150 μL of DMSO (Cat #D806645, Macklin, Shanghai, China) was added at room temperature for 15 min. OD490 nm was measured using a microplate reader. HUVEC (2 × 10⁴) cells were seeded in 96-well plate and incubated overnight. VEGF-A (0.02 μM) was added to model and drug groups. The drug group received a dilution series of Nb-VEGFA, Nb-TNFα or Nb-TV. Subsequent steps are as described above.

ARPE-19 (8 × 10³) cells were seeded in 96-well plate, and co-treated with Nb-TNFα or Nb-VEGFA or Nb-TV and TNF-α for 10 h. Cells were fixed and permeabilized with 4 °C pre-cooled anhydrous methanol for 15 min, then blocked with 1% bovine serum albumin (BSA) (Cat #SW3015, Solarbio, Beijing, China) at 37 °C for 1 h, and incubated with Rabbit anti-NF-κB p65 antibody (Cat #ER0815, HUABIO, Hangzhou, China) diluted in 1% BSA at 4 °C overnight. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Cat #ab150077, Abcam, United Kingdom) as secondary antibody. The nuclear was stained with DAPI (Cat #C0060, Solarbio, Beijing, China) for 10 min at room temperature. Images were captured using a fluorescence inverted microscope (XDS-200C, Olympus, Japan). For each group, at least four random fields of view (>100 cells) were collected, and the average fluorescence intensity of the images was analyzed using ImageJ software. HUVEC (5 × 10³) cells were seeded in 96-well plate, and co-treated with Nb-TNFα or Nb-VEGFA or Nb-TV and TNF-α for 10 h. Primary antibodies were Rabbit anti-p38 alpha/MAPK14 k antibody (Cat #ET1602-26 HUABIO, Hangzhou, China) and iNOS polyclonal antibody (Cat #YP-Ab17172, UpingBio, Hangzhou, China). The following steps are the same as above.

ARPE-19 were seeded in 6-well plate. Cells were co-treated with Nb-TNFα or Nb-VEGFA or Nb-TV and TNF-α for 24 h. Total RNA extracted from the cells was reverse-transcribed into cDNA using BeyoRT™ III cDNA First Strand Synthesis System (Cat #D7178S, Beyotime, Shanghai, China). qPCR analysis was performed with 2×Color SYBR Green qPCR Master Mix (Low ROX) (Cat #G8047-1, Adamas-life, Shanghai, China). The primers used are shown as follows: for IL-6, forward 5′-TCT​GCG​CAG​CTT​TAA​GGA​GT-3′ and reverse 5′-CCC​AGT​GGA​CAG​GTT​TCT​GA-3′; for IL-8, forward 5′-TACTCCAAAC CTTTCCACCCC-3′ and reverse 5′-CCC​AGT​TTT​CCT​TGG​GGT​CC-3′; for MCP-1, forward 5′-CAA​GCA​GAA​GTG​GGT​TCA​GG-3′ and reverse 5′-TGG​GGA​AAG​CTA​GGG​GAA​AAT-3′; for GAPDH, forward 5′-CCA​CTC​CTC​CAC​CTT​TGA​CG-3′ and reverse 5′-TAG​CCA​AAT​TCG​TTG​TCA​TAC​CAG​G -3′.

ARPE-19 were treated with VEGF-A and different nanobodies: Nb-TNFα, Nb-VEGFA, and Nb-TV respectively for 24 h. The cells were harvested and lysed with radioimmunoprecipitation assay buffer (RIPA) (1% phenylmethanesulfonyl fluoride (PMSF)) (Cat #R0010, Solarbio, Beijing, China) on ice for 30 min. The protein-containing supernatant was collected by centrifugation at 12400 g at 4 °C. 30 μg of each sample was subjected to SDS-PAGE and transferred onto a PVDF membrane (Cat #FFP24, Beyotime, Shanghai, China). The PVDF membrane was blocked with 5% skim milk powder in TBST buffer (Cat #T917679, Macklin, Shanghai, China) at room temperature for 2 h and then incubated with the following primary antibodies: anti GAPDH (Cat #10494-1-AP, Proteintech, Chicago, United States), anti-IL-6 (Cat #WL02841, Wanleibio, Shenyang, China), anti-IL-8 (Cat #WL03074, Wanleibio, Shenyang, China), anti MCP-1 (Cat #P13500, CUSABIO, Wuhan, China) and anti-Caspase-1 (Cat #ET1608-69, HUABIO, Hangzhou, China) overnight at 4 °C, followed by incubation with the diluted secondary antibody at room temperature for 1 h. Color exposure was performed using the ECL kit (Cat #36208 ES, Yeasen, Shanghai, China) and photographed with a gel imaging system. ImageJ was used to analyze the grayscale of the bands.

HUVEC were treated with VEGF-A and different nanobodies: Nb-TNFα, Nb-VEGFA, and Nb-TV respectively for 24 h. Primary antibodies were Anti-ERK1/2 rabbit pAb (Cat #WL01864, Wanleibio, Shenyang, China), Anti-ERK1 (phospho-T202/Y204) + ERK2 (phospho-T185/Y187) antibody (Cat #BM3950, BOSTER, Wuhan, China) and GAPDH (Cat #10494-1-AP, Proteintech, Chicago, United States). The following steps are the same as before.

HUVEC were seeded in 12-well plates. Once the cells grew to contact inhibition, a scratch was created using a 200 μL pipette tip. The floating cells were removed by washing with PBS. Cells were co-treated with Nb-TNFα or Nb-VEGFA or Nb-TV and VEGF in basal medium (containing 1% FBS), and the cells were further cultured for 36 h. Photographs were taken at 0 h, 12 h, 24 h, and 36 h.

A 96-well cell culture plate was coated with 50 μL of growth factor-reduced Mogengel Matrix (Cat #AC-M082703, ACROBiosystems, Beijing, China) and solidified at 37 °C for 1 h. HUVEC cells were seeded on the plate at a density of 3 × 10⁴. The experiment was carried out in the presence of VEGF-A and different nanobodies: Nb-TNFα, Nb-VEGFA and Nb-TV, respectively. The cells were further incubated for 4 h. The angiogenesis situation was analyzed by the ImageJ plugin Angiogenesis Analyzer.

Thirty 6-week-old male C57BL/6 mice (Cat #N000013, Gempharmatech, Shanghai, China) were randomly divided into a control group (n = 6) and a diabetic retinopathy (DR) group (n = 24). After a 6-h fasting period, the mice in the DR group were intraperitoneally injected with streptozotocin (STZ) (Cat #S6089, Macklin, Shanghai, China) freshly dissolved in sodium citrate (Cat #S818273, Macklin, Shanghai, China) buffer (pH 4.2–4.5) at a dose of 55 mg/kg for 5 consecutive days. The control group received an equal volume of sodium citrate solution via intraperitoneal injection. After the final injection, blood glucose levels were regularly monitored. The successful establishment of the DR model was indicated when the blood glucose value exceeded 16.7 mmol/L. Mice in DR group and control group were fed normal diet for 2 months. Then the DR group was randomly divided into DR group (eye drop PBS), Nb-TNF-α group (eye drop 0.2 mg/mL Nb-TNFα), Nb-VEGFA group (eye drop 0.2 mg/mL Nb-VEGFA) and Nb-TV group (eye drop 0.2 mg/mL Nb-TV). The control group received the same amount of PBS. Retinas were harvested and therapeutic efficacy was evaluated at 2 weeks and 4 weeks after nanobody treatment, respectively.

Under a dissecting microscope, the retina was carefully dissected. An appropriate amount of digestive enzyme solution was added, and enzymatic digestion was carried out at 37 °C for 30 min. Subsequently, centrifugation was performed to remove the enzyme and excess tissues. The retina was evenly spread on glass slides and dried at a low temperature. The slides were immersed in an iodic acid solution for oxidation for 10 min, then placed in Schiff’s reagent for staining in the dark for 30 min. Next, the slides were put into hematoxylin staining solution for staining for 10 min. Subsequently, the slides were immersed in a differentiation solution for 20 s, and then placed in a bluing solution for 2 min until the cell nuclei turned blue. The slides were placed successively in 70%, 80%, 90%, 95%, and 100% alcohol for dehydration. An appropriate amount of mounting medium was dropped on the slides and they were covered with coverslips. Finally, they were observed under a microscope.

The eyeballs of mice were harvested and fixed in an eyeball fixative solution (Cat #G1109, Servicebio, Wuhan, China) for 24 h. Subsequently, the fixed eyeballs were dehydrated and embedded in paraffin. Sections with a thickness of 4 μm were cut parallel to the eye axis, flattened in 45 °C warm water, picked up with anti-drop slides, baked (60 °C) in an oven for 6 h, and then prepared for the next procedure.

The paraffin sections of the retina were blocked with a 3% bovine serum albumin (BSA) solution at room temperature for 30 min. The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C. After being washed three times with PBS, the diluted fluorescent-labeled secondary antibodies were added and incubated at room temperature in the dark for 50 min. The cell nuclei were stained with DAPI solution and incubated at room temperature in the dark for 10 min. Finally, the fluorescent images were captured using a scanner.

Paraffin sections were first subjected to deparaffinization in a dilution series of xylene (Cat #X821391, Macklin, Shanghai, China) solutions. Subsequently, the sections were rehydrated step-by-step in a dilution series of ethanol (Cat #E821482, Macklin, Shanghai, China) solutions and finally through distilled water. Hematoxylin staining was then performed on the sections, followed by a 20-min rinse with tap water. After that, eosin staining was carried out. The stained sections were dehydrated using a series of alcohol solutions with increasing concentrations. Post-dehydration, the sections were dried in a fume hood until they became transparent. Then, neutral gum (Cat #N861409, Macklin, Shanghai, China) was applied to the center of the tissue for mounting, and the mounted sections were further dried in the fume hood. Finally, the retinal sections were examined under a light microscope.

Data were statistically analyzed and presented using Prism 9.5 (GraphPad Software, San Diego, CA, United States). Data were presented as mean ± standard deviation (SD) from at least three independent experiments, each performed on different days with distinct cell passages. Each independent experiment included at least three technical replicates per condition. The two-sample t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons among multiple groups, followed by the Tukey’s post hoc test. *: compared with negative control; #: compared with TNF-α/VEGF-A treated control. P value of <0.05 was considered significant.

As presented in Figure 1A, the schematic diagram of Nb-TV construction was illustrated. Nb-TNFα was located at the N-terminus, while Nb-VEGFA was situated at the C-terminus, with three repeated G4S serving as the linker in between, and the His-Tag at the C-terminal end being utilized as the purification tag. Figure 1B respectively displayed the three-dimensional structures of Nb-TNFα, Nb-VEGFA and Nb-TV simulated by I-TASSER (Figure 1B). It was observed that the structures of Nb-TNFα and Nb-VEGFA within Nb-TV exhibited no significant differences from those of the individual Nb-TNFα and Nb-VEGFA. Subsequently, the HDOCK server was employed to simulate the docking of Nb-TV with TNF-α or VEGF-A (Figure 1C). It was found that Nb-TV could bind to either TNF-α or VEGF-A alone. When both antigens were present simultaneously, Nb-TV was still able to bind to VEGF-A even after binding to TNF-α, suggesting that Nb-TV possessed dual-targeting activity (Figure 1D). The SDS-PAGE results indicated that the purified TNF-α, VEGF-A, Nb-TNFα, Nb-VEGFA and Nb-TV presented single bands, and their purities were all greater than 95% (Figure 1E).

The binding capacity of Nb-TV was assessed via indirect ELISA. In the case of the antigen TNF-α (as shown in Figure 2A), the Kd value of Nb-TNFα was determined to be 0.619 μM, while that of Nb-TV was 0.596 μM. For the antigen VEGF-A (depicted in Figure 2B), the affinity Kd value of Nb-VEGFA was 0.333 μM and that of Nb-TV was 0.283 μM. The binding capacity of Nb-TV to TNF-α was 1.04 times greater than that of Nb-TNFα, and its binding capacity to VEGF-A was 1.18 times higher than that of Nb-VEGFA. These results suggest that the bispecific nanobody not only enhances specificity but also guarantees that the binding activity of the internal single-targeted nanobodies to their respective target antigens remains unaltered.

TNF-α, an early inflammatory mediator, activates inflammation-related pathways, triggers reactions, causes cell damage and cycle arrest, inhibiting proliferation (Subhi and Lykke Sørensen, 2017). Upon treatment with 0.53 μM TNF-α on ARPE-19, a significant reduction in cell activity was observed, with a decrease of approximately 30%. Nb-VEGFA, which lacks binding affinity for TNF-α, failed to ameliorate the TNF-α-induced cell proliferation inhibition (Figure 3A). In contrast, under the influence of Nb-TV and Nb-TNFα, the cell viability exhibited a concentration-dependent increase (Figures 3B,C). At a concentration of 0.025 μM Nb-TNFα (Figure 3B), a remarkable improvement in ARPE-19 cell viability was noted, with a half-maximal effective concentration of 0.473 μM. Under identical experimental conditions, 0.5 μM Nb-TV effectively restored the TNF-α-induced inhibition of ARPE-19 cell proliferation. The half-maximal effective concentration was determined to be 0.505 μM, and the final cell activity was comparable to that of the control group (Figure 3C). The calculated effect ratio between Nb-TV and Nb-TNFα was 1.07, indicating equivalent efficacy in reversing the TNF-α-induced ARPE-19 cell proliferation inhibition, and both treatments demonstrated significant effects.

TNF-α functions by activating the NF-κB signaling pathway. The activated NF-κB is released from the NF-κB/IκB complex and translocate into the nucleus, thereby regulating the transcription of downstream genes. The inhibitory effect of Nb-TV on the TNF-α/NF-κB signaling pathway was analyzed by immunofluorescence assay (Figures 3D,E). In the control group, p65 was mainly distributed in the cytoplasm of ARPE-19 cells. Under TNF-α stimulation alone, p65 underwent nuclear translocation, and the proportion of nuclear p65 to the total cellular p65 was 57.73%, indicating the activation of the NF-κB pathway. When co-stimulated with Nb-TNFα or Nb-TV and TNF-α, the nuclear translocation of p65 was significantly inhibited, and the proportions of nuclear p65 were only 14.34% and 16.83%, respectively. These results suggest that both Nb-TNFα and Nb-TV are capable of blocking the activation of the NF-κB signaling pathway by TNF-α. TNF-α activates the NF-κB pathway, thereby leading to the upregulation of the expression of downstream genes IL-6, IL-8 and MCP-1. These cytokines are involved in both inflammatory responses and angiogenesis. As determined by qPCR and Western blot, treatment with TNF-α caused a significant increase in the levels of IL-6, IL-8 and MCP-1 in ARPE-19 cells. In contrast, in the groups co-treated with Nb-TNFα or Nb-TV and TNF-α, the levels of IL-6, IL-8 and MCP-1 were markedly reduced at both transcriptional and translational levels. These results indicate that Nb-TV is capable of ameliorating the inflammatory response (Figures 3F–H).

In an inflammatory environment, activation of the NF-κB/NLRP3/Caspas-1 pathway induces retinal pigment epithelial cell apoptosis and impairs retinal barrier integrity (Lu et al., 2018; Hao et al., 2019). Western blot analysis of Caspase-1 expression in ARPE-19 cells revealed that TNF-α treatment significantly upregulated its expression. In contrast, when co-treated with Nb-TNFα or Nb-TV, the Caspase-1 level was remarkably lower than that in the TNF-α alone group (Figures 3I,J). These results indicate that Nb-TV is able to effectively protect the survival of ARPE-19 cells in an inflammatory environment. Overall, Nb-TV exerts an anti-inflammatory effect by targeting and binding to TNF-α, which inhibits the activation of the NF-κB inflammatory pathway and reduces the release of IL-6, IL-8, and MCP-1. Meanwhile, it suppresses the destruction of the retinal pigment epithelium by blocking the NF-κB/NLRP3/Caspas-1 pathway.

VEGF-A is essential in angiogenesis, binding to VEGFR2 on vascular endothelium to initiate related pathways. The activation of these signal pathways promotes the cell cycle and cell proliferation. The MTT results showed that treatment with VEGF-A for 24 h remarkably promoted the proliferation of HUVEC cells. Different concentrations of Nb-TNFα had no influence on the proliferation-promoting effect of VEGF-A (Figure 4A). In contrast, both Nb-VEGFA and Nb-TV had inhibitory effects on the proliferation-promoting effect of VEGF-A in a concentration-dependent manner. The half-maximal effective concentration of Nb-VEGFA was 0.467 μM, and that of Nb-TV was 0.363 μM (Figures 4B,C).

VEGF-A promotes cell proliferation and participates in angiogenesis through Erk1/2 signal transduction. Western blot was utilized to analyze the phosphorylation levels of Erk1/2 in HUVEC cells under different treatment conditions. The level of phosphorylated Erk1/2 in the VEGF-A group was 30 times that of the control group. In contrast, the phosphorylation levels of Erk1/2 in the groups co-treated with VEGF-A and Nb-VEGFA or Nb-TV were lower than that of the control group, which indicates that Nb-TV can effectively inhibit VEGF-A-induced vascular cell proliferation (Figures 4D,E).

Endothelial cell migration is a crucial step in new blood vessel formation. The binding of VEGF-A to vascular endothelial growth factor receptor 2 (VEGFR2) triggers the activation of the p38 MAPK pathway, resulting in the nuclear translocation of phosphorylated p38, which subsequently promotes endothelial cell migration and maintains the structure and function of blood vessels. Immunofluorescence assays were employed to analyze the cellular distribution of phosphorylated p38 in HUVECs after different treatments (Figures 4F,G). In the control group, phosphorylated p38 was mainly distributed in the cytoplasm. After 24-h exposure to VEGF-A, the nuclear level of phosphorylated p38 was remarkably elevated and was double that of the control. Nb-TNFα exerted no influence on the nuclear translocation of phosphorylated p38 induced by VEGF-A. The binding of Nb-VEGFA or Nb-TV with VEGF-A impeded the binding of VEGF-A to VEGFR2, thus suppressing the nuclear translocation of phosphorylated p38, and the nuclear level of phosphorylated p38 was comparable to that of the control. This finding was further corroborated by scratch assays (Figures 4H,I). VEGF-A markedly enhanced HUVEC cell migration, whereas Nb-VEGFA and Nb-TV counteracted the migration-stimulating activity of VEGF-A.

To clarify the anti-angiogenic effect of Nb-TV, a tube formation assay was conducted (Figure 5A). Upon exposure to VEGF-A, the number of junction points of HUVECs was augmented by 64.3% (Figure 5B), the number of meshes was doubled (Figure 5C), and the total segment length was increased by 88.3% (Figures 5C,D), suggesting that VEGF-A could remarkably promote the in vitro angiogenesis of HUVECs. With the treatment of Nb-VEGFA or Nb-TV, the migration of HUVECs was suppressed and angiogenesis was considerably diminished, with merely 1-2 meshes being formed (Figure 5C), and the total segment length was also notably shortened (Figure 5D). Nb-TV reduced the number of junction points by 84.4%, the number of meshes by 91.7%, and the total segment length by 80%, powerfully evidencing that Nb-TV possesses the inhibitory capacity against the in vitro angiogenesis of HUVECs induced by VEGF-A.

During the progression of PDR, in addition to the emergence of neovascularization, a significant increase in vascular permeability can be observed. The binding of VEGF-A to VEGFR2 modulates vascular permeability through nitric oxide synthase (NOS). High levels of VEGF-A result in an increase in the expression of iNOS, which leads to an elevation in vascular permeability and subsequently undermines vascular integrity. As illustrated in Figures 5E,F, the expression of iNOS in HUVECs treated with VEGF-A was upregulated by 20 times. This will cause an increase in vascular permeability and is unfavorable for the maintenance of the normal structure of blood vessels. Meanwhile, in the groups treated with Nb-VEGFA and Nb-TV, the expression level of iNOS was basically the same as that in the control group, suggesting that after binding to VEGF-A, Nb-VEGFA and Nb-TV can effectively maintain the permeability of blood vessels.

Animal experiments were conducted in accordance with the design presented in Figure 6A. After continuous injection of STZ, the mice in the DR group manifested typical diabetic characteristics (Figure 6B). Compared with the control group, their blood glucose levels increased steadily and exceeded 16.7 mmol/L in the second week. Meanwhile, their food intake increased significantly, yet their body weight remained almost unchanged.

After 2-week and 4-week treatments, the Nb-TV group demonstrated the optimal therapeutic effect, with significantly decreased levels of TNF-α and VEGF-A in the retinas of the mice (Figures 6C,D). Compared to the DR group, after 2 weeks of treatment, the TNF-α level decreased by 30.47%, and the VEGF-A level decreased by 35.48%. After 4 weeks of treatment, the TNF-α level decreased by 73.29%, and the VEGF-A level decreased by 53.63%. Simultaneously, treatment with Nb-TNFα led to a 61.26% reduction in TNF-α, and treatment with Nb-VEGFA resulted in a 57.44% decrease in VEGF-A.

Notably, during the two - week treatment period, Nb-TNFα had no impact on the VEGF-A level, and Nb-VEGFA had no effect on the TNF-α level. However, after 4 weeks of treatment, Nb-TNFα caused a 22.86% decrease in the VEGF-A level, and Nb-VEGFA led to a 19.44% reduction in the TNF-α level. This indicates a close relationship between TNF-α and VEGF-A, suggesting that the inhibition of one factor can influence the other.

Furthermore, angiogenesis was observed by means of PAS staining. As shown in Figure 6E, compared with the control group, a large number of non - functional grown angiogenesis (red arrows) were generated in the retinas of the DR group. After 2 weeks of treatment with Nb-TV, the number of new blood vessels decreased by 38.83% compared with the DR group (Figures 6E,F). At the same time, Nb-VEGFA decreased the number of new blood vessels by 34.10%, while Nb-TNFα did not show the effect of inhibiting angiogenesis. After 4 weeks of treatment, Nb-TV effectively reduced the number of new blood vessels by 58.06%, Nb-VEGFA reduced it by 51.60%, and Nb-TNFα reduced it by 19.35%.

Finally, the pathological structure of the retinal tissues in mice was analyzed by HE staining (Figure 6G). In the retinas of the control group mice, the tissue structure was clear, the ganglion cells were arranged closely, and the inner nuclear layer (INL) and outer nuclear layer (ONL) were intact and compact. In the retinas of the DR group, the inner and outer nuclear layers were loose, the cells were arranged disorderly, and dilated new blood vessels (red arrows) could be seen. After 2 weeks of treatment with Nb-TV, the above-mentioned pathological changes were improved. After 4 weeks of treatment, the diseased retinas were effectively restored.

In conclusion, Nb-TV effectively reduces the levels of TNF-α and VEGF-A in the retinas of DR mice, decreases retinal angiogenesis, and improves the pathological changes of the retinal structure. Based on the above results, it is considered that Nb-TV has an effective therapeutic effect on DR.