Kaempferol Alleviates Corneal Transplantation Rejection by Inhibiting NLRP3 Inammasome Activation and Macrophage M1 Polarization via Promoting Autophagy

Corneal transplantation rejection remains a major threat to the success rate in high-risk patients. Given the many side effects presented by traditional immunosuppressants, there is an urgency to clarify the mechanism of corneal transplantation rejection and to identify new therapeutic targets. Kaempferol is a natural avonoid that has been proven in various studies to possess anti-inammatory, antioxidant, anticancer, and neuroprotective properties. However, the relationship between kaempferol and corneal transplantation remains largely unexplored. To address this, both in vivo and in vitro, we established a model of corneal allograft transplantation in Wistar rats and an LPS-induced inammatory model in THP-1 derived human macrophages. In the transplantation experiments, we observed an enhancement in the NLRP3 / IL-1 β axis and in M1 macrophage polarization post-operation. In groups to which kaempferol intraperitoneal injections were administered, this response was effectively reduced. However, the effect of kaempferol was reversed after the application of autophagy inhibitors. Similarly, in the inammatory model, we found that different concentrations of kaempferol can reduce the LPS-induced M1 polarization and NLRP3 inammasome activation. Moreover, we conrmed that kaempferol induced autophagy and that autophagy inhibitors reversed the effect in macrophages. In conclusion, we found that kaempferol can inhibit the activation of the NLRP3 inammasomes by inducing autophagy, thus inhibiting macrophage polarization, and ultimately alleviating corneal transplantation rejection. Thus, our study suggests that kaempferol could be used as a potential therapeutic agent in the treatment of allograft rejection.


Introduction
Macrophages are involved in the initial stages of corneal transplantation rejection [6]. It is generally suggested that they can be divided into the M1 and M2 phenotypes induced by external stimulation [7]. M1 macrophages highly express CD80 and CD86 receptors and secrete IL-6, iNOS and other proin ammatory cytokines, whereas M2 macrophages highly express CD206 receptors and predominantly secrete IL-10 and other anti-in ammatory cytokines [8].
The NLR family pyrin domain-containing 3 (NLRP3) in ammasome is a member of the pathogen recognition receptors (PRRs) and is widely expressed in macrophages. It is involved in the in vivo recognition of danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) and the subsequent triggering of in ammatory responses [9]. Activation of the NLRP3 in ammasome transforms precursor Caspase-1 into Caspase-1, which then cleaves the precursor of IL-1 β into a mature form and releases it to the downstream immune in ammatory reaction cascade [10]. The NLRP3 in ammasome has been proven to participate in immune rejection post-transplantation in graftversus-host disease (GVHD), as well as heart, kidney, and skin transplantations [11,12]. Studies have shown that the NLRP3 in ammasome is associated with the M1 polarization of macrophages [13]. Autophagy is a process of self-digestion in higher eukaryotes, and is associated with many physiological and pathological processes [14]. Some recent studies have shown that enhanced myocardial autophagy can delay the occurrence of heart transplantation rejection [15]. Kaempferol (Ka) is a natural avonoid small molecule, and is the active component in various traditional Chinese medicines [16]. It has been proven to have anti-in ammatory, antioxidant, anticancer and neuroprotective effects [17]. However, the role and mechanism of kaempferol in corneal allograft rejection remain unknown. In this study, we explored the effects of kaempferol on corneal allograft rejection in rats and used an in ammatory model in human macrophages to further investigate whether these are in fact mediated by autophagy.
The animal experiment plan has been reviewed and approved by Nanfang Hospital Animal Ethic Committee, which is in line with the principles of animal protection, animal welfare and ethics, and the relevant provisions of the national experimental animal ethical welfare. The feeding and use of the experimental animals were in accordance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals.

Establishment of the Rat Corneal Allograft Model
The Wistar rats were divided into the following groups: control, corneal autograft, corneal allograft, kaempferol, vehicle control, and kaempferol + 3-MA (kaempferol combined with the autophagy inhibitor 3-methyladenine). The corneal allograft group was then further divided into three subgroups named Day 5, Day 9, and Day 14, according to the time of sampling post-operation.
Autogenous (Wistar→Wistar) and allogeneic keratoplasty (SD→Wistar) models were established according to the methodology of Williams et al. [18]. The rejection index (RI) was calculated according to the degree of opaci cation, edema, and vascularization of the corneal graft according to the scoring standards of Larkin [19] (0-4, 0-2, and 0-4, respectively). Rejection was de ned as RI≥5 or an opaci cation degree ≥ 3.

Establishment of the LPS-induced macrophage in ammatory model
The human monocytic leukemia cell line (THP-1) was purchased from the cell bank of the Chinese Academy of Sciences. The cells were maintained in RMPI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco, USA), at 37 °C, in a 5% CO 2 atmosphere. The cell line was centrifuged and resuspended in culture medium containing phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) (Sigma Aldrich, USA). After 48 h of induction with PMA, the adherent cells were considered M0 macrophages.

Administration of kaempferol and 3-MA
In the animal experiments, kaempferol (Selleck, USA) was rst prepared into a 200 mg/mL solution with DMSO and then diluted into a 10 mg/mL working solution with a vehicle (DMSO + polyethylene glycol + polyoxyethylene sorbitan mono-oleate + pure water) before injection. Rats in the kaempferol groups were intraperitoneally injected with 50 mg/kg of kaempferol working solution every day, starting at 3 days before the operation and continuing till the date of sampling or till the end of observation (which occurred either at the instance of graft rejection, or else 30 days after the operation). Rats in the vehicle control groups were injected with 5 mg/kg of the aforementioned vehicle daily. Rats in the kaempferol+ 3-MA group were intraperitoneally injected with 10 mg/kg of 3-MA solution (Selleck) 12 h before the kaempferol injection.
In the cell experiments, kaempferol was diluted to 40 mM with DMSO and incubated with macrophages  at a dilution rate of 20 to 200 μM, while 3-MA was diluted to 50 mM with ultrapure water and incubated with macrophages at 5 mM.
Cell Counting Kit-8 (CCK-8) Assay Macrophages were incubated with different concentrations of kaempferol (0, 20, 40, 100, and 200 μM) for 12 and 24 h. After that, the instructions of the cell-counting kit-8 (CCK-8 Kit; Dojindo, Japan) were followed for the assay. Absorbance was measured at 450 nm by uorometer, and the cell survival rate was calculated according to theinstrument manufacturer .

Reverse Transcriptase Real-Time Polymerase Chain Reaction (RT-PCR)
In accordance with the operating instructions, RNAiso Plus (Trizol) (TaKaRa, Japan) was used to extract total RNA from the rat corneas (one piece of cornea per group) and the macrophages. PrimeScript RT Master Mix (TaKaRa) was used to reverse transcribe the mRNA into cDNA. The PCR system was prepared according to the 2×SYBR Green qPCR Master Mix (Selleck) instructions. The LightCycler 480 system was performed to detect the mRNA content of each sample. Melting curves were analyzed to con rm the homogeneity of the product. The primers used were synthesized by Sangon Biotech and are shown in Table 1. Rat and human GAPDH (Sangon Biotech, China) are provided for internal reference.

Western Blot Analysis
Protein samples were obtained from the rat corneas (one piece of cornea per group) and the THP-1 macrophages, and were lysed using RIPA lysis buffer (Beyotime, China). The protein concentrations were then determined using a BCA protein assay kit (Beyotime), wherein 20-30 μg of protein was loaded each time and separated by 10-15% SDS-PAGE, then transferred to poly(vinylidene uoride) (PVDF) membranes. The membranes were then blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The dilution ratios of primary antibodies were as follows: the dilution ratio for anti-NLRP3, anti-IL-1β, anti-caspase-1, anti-microtubuleassociated protein light chain 3 (LC3), and anti-p62 (Abclonal, China) was 1:800; the dilution ratio for antiβ-actin (Fude antibody, China) was 1:10,000. Following incubation with the A niPure Goat Anti-Rabbit IgG (H+L) and A niPure Goat Anti-Mouse IgG (H+L) (Fude antibody), the lms were exposed and scanned using ECL reagent (A nity Biosciences, USA). ImageJ software (Rawak Software, Germany) was used to analyze the band gray value. The representative blots are shown in the gures.

Fluorescence Microscopy
The corneal tissue sections were permeabilized with 0.5% Triton X-100, then blocked with goat serum (Solarbio, China) for 60 min. anti-NLRP3 and anti-CD80 (Santa Cruz Biotechnology, USA) were prepared at a dilution ratio of 1:100 and incubated with the cornea sections at 4 °C overnight. After incubating with the secondary antibodies at room temperature for 1 h, an anti-fade solution (containig DAPI) (Solarbio) was added, and the sections were observed under a uorescence microscope.
Cell immuno uorescence staining was carried out by counting and inoculating the THP-1 cells in a confocal culture dish. Paraformaldehyde (4%) was added, and the cells were xed for 15 minutes. The remaining steps were the same as described above for immuno uorescence staining of the cornea.
To observe the induction of autophagy, autophagosome-lysosome living cell dye DALGreen (Dojindo) was added to the macrophages cultured in the confocal dish for 30 min according to the manuscript. The remaining steps were the same as above for the cornea and cell immuno uorescence methods. Relative uorescence intensity was analyzed by ImageJ software.

Flow Cytometry
Macrophages were digested with trypsin-EDTA (Gibco) in a 37 °C incubator for 5 min and then centrifuged and resuspended with PBS. Fluorescent antibodies directed against CD molecules FITC-CD11b, PE-CD80, and APC-CD206 (eBioscience, USA) were added, and the macrophages were evaluated using ow cytometry. The gating strategy incorporated live cells and singlet gates prior to gating on CD11b, after which CD80 and CD206 were detected. The results were analyzed using Flowjo (BD Falcon, USA).

Statistical Analysis
The Kaplan-Meier statistical method was used to analyze graft survival times, the time required till corneal graft rejection occurred or the observation period reached day 30 post-operation.
All data were expressed as the mean ± SD and one-way analysis of variance (ANOVA) was used for statistical analysis. SPSS 20.0 (IBM, USA) was used for statistical analysis. Statistical signi cance was set at p < 0.05.

Kaempferol inhibited NLRP3 in ammasome 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 postoperation). 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 di cult to distinguish; RI reached 5, which indicated rejection ( Figure 1A).
In order to investigate whether the NLR family pyrin domain-containing 3 (NLRP3) in ammasome 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 signi cantly 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 fth 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 signi cantly 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 in ammasome in the rat corneal allograft model.

Activation of autophagy is critical for kaempferol's inhibition of the NLRP3 in ammasome 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 signi cantly 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 signi cantly 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 signi cantly 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 in ammasome 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 in ammasome and alleviation of transplant rejection.
Kaempferol inhibits M1 polarization in cornea by inhibiting the activation of the NLRP3 in ammasome 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 signi cantly 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 signi cantly 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 signi cantly 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 in ammasome and M1 polarization, we studied the expression of NLRP3 and CD86 in each group using immuno uorescence. Compared with the control group, the uorescence intensity of CD86 and NLRP3 of the allograft group was signi cantly increased, with co-staining shown correlation between NLRP3 and CD86. The relative uorescence intensity of CD86 and NLRP3 in the cornea in the kaempferol group was decreased ( Figure  3C). These results suggest that M1 polarization and NLRP3 in ammasome 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 signi cantly downregulated the expression of all mRNAs ( Figure 4B).
Next, we observed the effect of kaempferol on the proportion of M1 and M2 macrophages using ow 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 signi cantly increased compared with the control group. Following kaempferol treatment, however, the above effect was signi cantly 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 in ammasome 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 signi cantly downregulated it ( Figure 5A). Additionally, kaempferol at all concentrations signi cantly 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 signi cantly 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 signi cantly 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 signi cant 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 uorescence intensity of DALGreen and LC3 were signi cantly increased after kaempferol administration. Conversely, DALGreen and LC3 uorescence intensity was weakened after 3-MA was added ( Figure 6C).
In line with what our in vivo ndings indicated, the results of the above tests con rm that kaempferol can indeed enhance the autophagy of macrophages.

Autophagy inhibitors can inhibit the effects of kaempferol on macrophage polarization and NLRP3 in ammasome activation in vitro
Finally, we considered the effect of autophagy inhibitors on kaempferol's inhibition of the NLRP3 in ammasome 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 in ammasome ( Figure 7A).
In terms of what concerns macrophage polarization, the results of ow cytometry showed that, compared with the kaempferol group, the M1/M2 ratio in the kaempferol + 3-MA group increased signi cantly, 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 uorescence 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 in ammasome was via inducing autophagy.

Conclusions
Macrophages are the main components of the innate immune system and constitute the rst barrier of immune defense [20]. Studies have shown them to be pluripotent cells with high plasticity and functional diversity that can polarize towards different phenotypes [21]. Before the phenomenon of macrophage polarization was found, the role of APC headed by macrophages in corneal transplantation immunity was collectively referred to as the costimulatory pathway. This mainly includes the interaction between CD28 on T cells and B7 molecules (CD80, CD86) on APCs [22]. It is believed that activated macrophages are in ammatory cells with an antigen presentation function and a costimulatory function [23]. However, after the emergence of the concept of macrophage polarization, studies suggest that M1 macrophage cell membranes highly express HLA-DR, CD80, CD86, and other protein markers, and participate in proin ammatory reactions [24]. Therefore, we have reason to think that M1 macrophage polarization is involved in corneal transplantation rejection, and that M1 macrophages are the main force responsible for secreting chemokines and presenting antigens. Our experiment corroborates these hunches, showing that M1 cytokines increased following corneal allograft transplantation.
Our study also investigated the potential therapeutic effect of kaempferol on macrophage polarization in corneal transplantation. We found that in groups to which kaempferol was administered, M1 cytokines were down regulated. This indicated that kaempferol could inhibit the aggregation of M1 macrophages after corneal transplantation. Additionally, using an in ammation model in macrophages, we showed that kaempferol at different concentrations had the same inhibitory effect on M1 polarization in human cells in vitro.
The NLR family, pyrin domain-containing 3 (NLRP3) in ammasome is composed of the NLRP receptor, apoptosis-related spot-like protein (ASC), and pro-caspase-1 [25]. Many studies have shown that the NLRP3 in ammasome can induce macrophages into the M1 phenotype. For example, knockout of the NLRP3 gene or use of NLRP3 inhibitor CY09 can effectively inhibit M1 polarization [26]. Similarly, the NLRP3 in ammasome induces M1 polarization in oral in ammation, ischemic stroke, and other diseases [27,28]. In our study, we con rmed that the NLRP3 in ammasome and downstream cytokine IL-1β were aggregated in the corneal epithelium post-transplantation in the rat model, indicating once again that the NLRP3/IL-1β axis is involved in the early stage of corneal allograft rejection.
Scholars have de ned that reactive oxygen species (ROS) is the bridge between the NLRP3 in ammasome and M1 macrophage polarization [29]. In the process of mitochondrial dysfunction, released ROS-activated NLRP3 in ammasome and pro-capase-1 (an important component of the NLRP3 in ammasome) cleave into caspase-1, which contributes to the production and secretion of M1 proin ammatory cells [30]. Meanwhile, M1 macrophage polarization can in turn promote the activation of the NLRP3 in ammasome, contributing to the production of high-level proin ammatory cells.
In ammatory factors and ROS can increase and maintain the in ammatory response [13]. In ophthalmology research, studies have shown the increase in ROS to be involved in the occurrence of corneal allograft rejection. The inhibition of mitochondrial damage, however, prolonged the survival of corneal grafts [31]. Therefore, we speculated that mitochondrial damage may be the cause of NLRP3 in ammasome activation after corneal transplantation. In our animal and cell experiments, we demonstrated that kaempferol at different concentrations can reduce the activation of the NLRP3 in ammasome and the expression of IL-1β and caspase-1.
Autophagy promotes the degradation of misfolded proteins, abnormal organelles, and other waste materials while maintaining cell metabolic function [32]. Microtubule-associated protein light chain 3 LC3 and p62 are critical proteins in autophagy. In particular, cytoplasmic soluble LC3 (LC3I) is hydrolyzed during the autophagic process to form the membrane-bound LC3 (LC3II), with the increase in LC3II/I thus being indicative of enhancement of autophagy [33,34]. P62 binds to ubiquitinated proteins and interacts with LC3, mediating ubiquitinated protein transport in the autophagic progress [35]. Autophagy plays an important role in the regulation of the in ammatory response [36,37]. The decrease in autophagy leads to a large number of depolarized mitochondria and leaked substances, including mitochondrial DNA and ROS [38,39]. In addition, there is increasing evidence to suggest that moderating autophagy can promote the polarization of macrophages from the M1 to the M2 phenotype, while the ensuing de ciency of autophagy-related genes could drive the upregulation of M1 polarization [40][41][42].
We found that autophagy is key to kaempferol inhibiting the NLRP3 in ammasome and M1 polarization, both in vivo and in vitro. In our animal model of corneal transplantation, kaempferol promoted an increase in autophagy-related mRNA in corneal tissue, as well as the conversion of LC3I to LC3II in macrophages, and reduction in the accumulation of p62. To further explore the role of autophagy in kaempferol treatment, we used the autophagy inhibitor 3-methyladenine (3-MA), which selectively inhibits phosphatidylinositol-3 kinase (PI3K) and thus inhibits the initial stage of autophagy [43]. We found that 3-MA attenuated the protective effect of kaempferol on corneal allograft rejection. Similarly, in vitro, 3-MA blocked the effects of kaempferol, signi cantly increasing the proportion of M1 macrophages and activating the NLRP3 in ammasome. Finally, we observed the induction of autophagy using DALGreen, a lipid soluble uorescent dye that enters the cell membrane and can be used to detect autophagosomes [44]. We found that the uorescence intensity of DALGreen and LC3 was signi cantly enhanced by kaempferol, and the staining sites were highly consistent. As expected, 3-MA weakened the uorescence intensity. This further con rmed that promotion of autophagy is in fact the mechanism by which kaempferol inhibits macrophage polarization and NLRP3 in ammasome activation.
In conclusion, our study demonstrated that kaempferol can inhibit the activation of the NLRP3 in ammasome and M1 polarization via activating autophagy, thus delaying the occurrence of corneal allograft rejection in vivo and reducing in ammation in macrophages in vitro. We acknowledge that there are some shortcomings in our experiments; for example, we mainly focused on the role of kaempferol on macrophages. Previous studies suggested that kaempferol promotes the proliferation of CD4þFoxP3þ regulatory T cells, thereby affecting islet transplantation rejection [45]. In addition, we found that the rats inoculated with kaempferol had partial rejection near the end of the observation. Therefore, the role of kaempferol and other immune cells in corneal transplantation should be explored, and we consider that further study on the administration mode and dosage of kaempferol may have a deeper impact on the graft survival rate. We believe that these qualities of chemo-preventive activity and in vivo-tolerance in kaempferol highlight it as a novel therapeutic option for the prevention of transplantation rejection in humans.   vehicle and kaempferol group was taken at day 5 and made into frozen sections, CD86 (green) and NLRP3 (red) were detected by uorescence microscopy. DAPI (blue) was used as a nuclear dye. Scale bar, 50μm. One-way ANOVA was used and values are shown as means ± SD, n = 3-4 for each group. *p < 0.05, **p < 0.01 vs control group; +p < 0.05, ++p < 0.01 compared with allograft group; ##p < 0.01 compared with kaempferol group.  (20, 40, 100μM) were incubated with LPS for 24h, and the relative mRNA expression of M1 cytokines between control, LPS, kaempferol (20, 40, 100μM) group were detected by RT-PCR. (C) The ratio of M1 and M2 macrophages between control, LPS and kaempferol (40μM) group were detected by ow cytometry. CD11b+ cells were recognized as macrophages, within CD80+ cells were recognized as M1 macrophages and CD206+ cells were recognized as M2 macrophages. One-way ANOVA was used and values are shown as means ± SD, n = 3-4 for each group. **p < 0.01 vs control group; +p < 0.05, ++p < 0.01 vs LPS group. were test by uorescence microscopy. Scale bar, 200μm. One-way ANOVA was used and values are shown as means ± SD, n = 3-4 for each group. *p < 0.05, **p < 0.01 vs control group; +p < 0.05, ++p < 0.01 vs LPS group. intensity of LC3B (red) and DALGreen (green) between LPS, kaempferol (40μM) and kaempferol+3-MA group were detected by uorescence microscopy. Scale bar, 200μm. One-way ANOVA was used and values are shown as means ± SD, n = 3-4 for each group. *p<0.05, **p < 0.01 vs control group; +p < 0.05, ++p < 0.01 vs LPS group; ##p < 0.01 vs kaempferol group.