Inhibitive effect of TAK-242 on Tenon's capsule fibroblasts proliferation in rat eyes

Abstract

Aim: To study the inhibition effect of TAK-242 on the proliferation of rat eye Tenon's capsule fibroblasts via the toll-like receptor 4 (TLR4) signaling pathway.

Methods: SD rat Tenon's capsule fibroblasts were extracted and cultured, then the cells were divided into normal control group, lipopolysaccharide (LPS) group (10 g/mL LPS) and TAK-242 group (1 µmol/L TAK-242, and 10 µg/mL LPS after 30min). The expressions of TLR4, transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) in each group were detected by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Cell proliferation was detected by cell counting kit-8 (CCK-8).

Results: Double immunofluorescent labeling in the extracted cells showed negative keratin staining and positive vimentin staining. Western blot showed that the LPS group had the highest expression of TLR4 and TGF-β1 (P<0.01). Enzyme linked immunosorbent assay (ELISA) also showed that the secretion of IL-6 was the highest in LPS group (P<0.01). But there was no significant difference in TLR4 and TGF-1, as well as IL-6 expressions between the TAK-242 group and the normal control group (P>0.05). RT-PCR showed that the IL-6 mRNA expression in LPS group was the highest in the three groups (P<0.01).

Conclusion: TAK-242 inhibits the proliferation of LPS-induced Tenon's capsule fibroblasts and the release of inflammatory factors by regulating the TLR4 signaling pathway, providing a new idea for reducing the scarring of the filter passage after glaucoma filtration surgery.

Keywords: TAK-242; Tenon's capsule fibroblasts; fibrosis; rat.

Figure 1. Primary culture of SD rat Tenon' s capsule fibroblasts (100×)

A: In the center, tissue fragments digested by trypsin. The cells migrate from the tissue fragments and divide and proliferate. The cells are spindle shaped or spindle-shaped, and a few are triangular. B: The cells are arranged in bundles or spirals after confluence.

Figure 2. Identification of keratin vimentin: double immunofluorescent labeling (400×)

A-C: Vimentin staining was positive, green fluorescence was seen in the cytoplasm (A), blue fluorescence was seen in the nucleus (B), and C was co-development of cytoplasm and nucleus. D-F: Keratin staining was negative, cytoplasm red fluorescence was not visible (D), the nucleus was blue fluorescence (E), and only the nucleus fluorescence could be seen in the cytoplasm co-development (F).

Figure 3. Expression of TLR4 protein in each group was detected by Western blot

After LPS intervention, TLR4 protein expression level in the LPS group increased significantly compared with that in the normal control group (^bP<0.01). In the TAK-242 group, TLR4 protein expression was significantly lower than that in the LPS group due to the intervention of TAK-242 (^bP<0.01). There was no significant difference between TAK-242 group and normal control group (P>0.05).

Figure 4. Western blot analysis of protein expression of TGF-β1 in each group

After the intervention of LPS, the protein expression level of TGF-β1 in the LPS group was significantly increased compared with that of the normal control group (^bP<0.01). In the TAK-242 group, the protein expression of TGF-β1 was significantly lower than that of the LPS group due to the intervention of TAK-242 (^bP<0.01). However, compared with the normal control group, the expression level of TGF-β1 protein in TAK-242 group decreased slightly, and the difference have no statistics significance (P>0.05).

Figure 5. ELISA detect the secretion level of IL-6 in each group

After LPS intervention, IL-6 secretion level in the LPS group was significantly increased compared with that in the normal control group (^bP<0.01). In the TAK-242 group, IL-6 secretion level was significantly lower than that in the LPS group due to the intervention of TAK-242 (^bP<0.01). There was no significant difference in IL-6 secretion level between the TAK-242 group and the normal control group (P>0.05).

Figure 6. RT-PCR detect the relative transcription levels of IL-6 mRNA in each group

A: Solubility curve. B: The amplification curve: a stands for LPS group; b stands for group TAK-242; c is the normal control group. All curves are unimodal distribution and the peak is concentrated. C: After the intervention of LPS, the relative transcription level of IL-6 mRNA in the LPS group was significantly raised compared with that in normal control group (^bP<0.01). In the TAK-242 group, the relative transcription level of Il-6 mRNA was significantly lower than that in the LPS group due to the intervention of TAK-242 (^bP<0.01). Compared with the normal control group, the relative transcription level of IL-6 mRNA increased in the TAK-242 group (^bP<0.01).

Figure 7. CCK-8 detect cell proliferation in each group

OD value in the LPS group increased significantly compared with that in the normal control group after LPS intervention (^aP<0.05). In the TAK-242 group, OD value decreased significantly compared with LPS group due to the intervention of TAK-242 (^aP<0.05). However, there was no significant difference in OD value between the TAK-242 group and the normal control group (P>0.05).