3.1. Kaempferol inhibited NLRP3 inflammasome expression in corneal allografts and prolonged graft survival time
Starting at 7 days post-operation, neovascularization commenced around the graft in the autograft group. However, the graft remained transparent throughout the observation window (i.e., 30 days post-operation). In the allograft group, corneal edema and neovascularization began on day 7, after which the graft began to appear turbid and edematous. On day 14, acute corneal edema and a large amount of neovascularization invaded the graft, exceeding the radius by 50%. The graft was turbid, obviously edematous, and the pupil contour was difficult to distinguish; RI reached 5, which indicated rejection (Figure 1A).
In order to investigate whether the NLR family pyrin domain-containing 3 (NLRP3) inflammasome is involved in the rejection of corneal allografts, we studied its expression in the cornea. The expression of NLRP3 and IL-1β mRNA in the allograft group was found to be significantly higher than that in the control and autograft groups on days 5, 9, and 14 post-operation (Figure 1B). The peak expression of mRNAs was observed on day 5 post-operation.
However, in the kaempferol group, corneal edema subsided on the fifth day after operation. The cornea was still transparent on days 5, 9, and 14 post-operation, with no new blood vessel growth in the graft. Some of the grafts began to be rejected from day 26, while half remained with a RI<3 during the 30 day observation period (Figure 1C). The mRNA expression of NLRP3 and IL-1β at day 5 was significantly lower in the kaempferol group than in the vehicle control group (Figure 1D). The same applied for the protein expression of NLRP3 and IL-1β (Figure 1E).ndicate that kaempferol inhibited the expression of the NLRP3 inflammasome in the rat corneal allograft model.
- Activation of autophagy is critical for kaempferol’s inhibition of the NLRP3 inflammasome and alleviation of graft-rejection
To investigate whether autophagy is involved in the therapeutic effect of kaempferol on corneal allograft rejection, the autophagy inhibitor 3-methyladenine (3-MA) was used.
In the group of rats to which kaempferol only was administered, expression of the autophagy genes Beclin1, LC3, and ATG5 was upregulated and that of p62 mRNA was downregulated compared with that in the control and vehicle control groups, indicating the activation of autophagy. Following treatment with a combination of kaempferol and 3-MA, the expression of these mRNAs was significantly reversed, indicating that autophagy was inhibited (Figure 2A). Parallelly, all the grafts was rejected at day 14 in the kaempferol+3-MA group (Figure 2B). Compared with the allograft (12.6 ± 1.08 days), vehicle control (11.4 ± 0.97 days), and kaempferol + 3-MA (10.5 ± 0.71) groups, the corneal graft survival time of the kaempferol group was significantly prolonged to (27.7 ± 1.49) days (Figure 2C).
After the combined application of kaempferol and 3-MA, the mRNA and protein expression of NLRP3 and IL-1 β was significantly higher than that in the kaempferol group on day 5 post-operation (Figure 2D, E), indicating that the inhibitory effect of kaempferol on the NLRP3 inflammasome was weakened by the addition of the autophagy inhibitor. Combined with the above results, this suggests that autophagy is critical for kaempferol’s inhibition of the NLRP3 inflammasome and alleviation of transplant rejection.
- Kaempferol inhibits M1 polarization in cornea by inhibiting the activation of the NLRP3 inflammasome
Firstly, to verify the role of macrophage polarization in the occurrence of rejection after corneal transplantation, we used RT-PCR to detect the expression of cornea M1 cytokines in the isograft and allograft groups. The mRNA expression levels of M1 cytokines iNOS, IL-6, TNF-α, CXCL-10 in the allograft group were significantly upregulated on days 5, 9, and 14 post-operation compared with the isograft group (Figure 3A).
Secondly, we explored the effect of kaempferol on macrophage polarization after corneal transplantation. On day 5 post-operation, the mRNA expression levels of the M1 cytokines in the kaempferol group were significantly lower than those in the allograft group. This indicates that kaempferol treatment can effectively inhibit cornea M1 polarization. However, after the combined use of 3-MA, the mRNA expression levels of the M1 cytokines were significantly up-regulated, which suggests that the effect of kaempferol on macrophage poarization is related to autophagy (Figure 3B).
To further explore the relationship between the expression of the NLRP3 inflammasome and M1 polarization, we studied the expression of NLRP3 and CD86 in each group using immunofluorescence. Compared with the control group, the fluorescence intensity of CD86 and NLRP3 of the allograft group was significantly increased, with co-staining shown correlation between NLRP3 and CD86. The relative fluorescence intensity of CD86 and NLRP3 in the cornea in the kaempferol group was decreased (Figure 3C). These results suggest that M1 polarization and NLRP3 inflammasome expression both increase after allograft keratoplasty and that kaempferol can effectively reduce this response.
- Kaempferol decreases LPS -induced M1 polarization in vitro
In order to further study the mechanism of kaempferol inhibition of M1 macrophage polarization, we directly carried out cell experiments on macrophages. The results of the cell-counting kit-8 (CCK-8) assay revealed that kaempferol acted in a dose-dependent manner in both the 12 h and 24 h groups. However, the number of surviving cells was still more than 50% after 24 h administration with 200 µM of kaempferol (Figure 4A).
Three concentrations of kaempferol had been tested: 20 µM, 40 µM, and 100 µM. After LPS treatment, the relative mRNA expression of M1-associated cytokines IL-6, iNOS, TNF-α and CXCL-10 was upregulated. According with the result in vivo, kaempferol at different concentrations significantly downregulated the expression of all mRNAs (Figure 4B).
Next, we observed the effect of kaempferol on the proportion of M1 and M2 macrophages using flow cytometry. The expression of CD11b, CD80, and CD206 was used to detect the distinct macrophage phenotypes. Among them, CD11b+ CD80+ cells were considered to be M1 macrophages, whereas CD11b+ CD206+ cells were considered to be M2 macrophages. The results showed that the ratio of M1 to M2 in the LPS group significantly increased compared with the control group. Following kaempferol treatment, however, the above effect was significantly reversed, and the M1: M2 ratio was decreased (Figure 4C). These results suggest that kaempferol can reduce the polarization of M1 macrophages induced by LPS.
- Kaempferol reduces the activation of the NLRP3 inflammasome in macrophages in vitro
The RT-PCR results showed that the mRNA expression of NLRP3, Caspase-1, and ASC was upregulated after LPS treatment in macrophages. Subsequent kaempferol treatment at different concentrations significantly downregulated it (Figure 5A). Additionally, kaempferol at all concentrations significantly decreased the protein levels of NLRP3, pro-IL-1β, IL-1β, pro-Caspase-1, and Caspase-1 (which had been increased by LPS) (Figure 5B). Furthermore, NLRP3 protein expression and CD86 positive cells decreased significantly after kaempferol treatment, which was consistent with the experimental results in vivo (Figure 5C).
- Kaempferol promotes autophagy in vitro
Autophagy inhibitor 3-MA was used to reduce autophagy in macrophages. The results of RT-PCR showed that kaempferol at different concentrations significantly upregulated the mRNA expression of LC3, and downregulated that of p62 (Figure 6A). Accordingly, western blotting revealed the same pattern, and the LC3 II/I ratio was increased compared with that of the LPS group. The combination of the autophagy inhibitor 3-MA and kaempferol resulted in a significant decrease in the LC3II/I ratio and an increase in p62 expression, thus reversing the effects of kaempferol alone (Figure 6B).
DALGreen and LC3B co-staining was also produced. The results of the co-staining showed that the relative fluorescence intensity of DALGreen and LC3 were significantly increased after kaempferol administration. Conversely, DALGreen and LC3 fluorescence intensity was weakened after 3-MA was added (Figure 6C).
In line with what our in vivo findings indicated, the results of the above tests confirm that kaempferol can indeed enhance the autophagy of macrophages.
3.7. Autophagy inhibitors can inhibit the effects of kaempferol on macrophage polarization and NLRP3 inflammasome activation in vitro
Finally, we considered the effect of autophagy inhibitors on kaempferol’s inhibition of the NLRP3 inflammasome and of macrophage polarization.
Following treatment with 3-MA, the expression of NLRP3, pro-Caspase-1, Caspase-1, pro-IL-1β and IL-1β protein increased relative to that in the kaempferol-only group, indicating that 3-MA inhibits the inhibitory effect of kaempferol on the NLRP3 inflammasome (Figure 7A).
In terms of what concerns macrophage polarization, the results of flow cytometry showed that, compared with the kaempferol group, the M1/M2 ratio in the kaempferol + 3-MA group increased significantly, indicating that autophagy participates in the process of macrophage polarization (Figure 7B). In further support of this, results of NLRP3/CD86 co-staining showed that the fluorescence intensity of NLRP3 and the proportion of CD86 positive cells in kaempferol groups increased after 3-MA treatment (Figure 7C). Together, these results demonstrate that the effects of kaempferol on macrophage polarization and the NLRP3 inflammasome was via inducing autophagy.