This study used amine-modified zinc oxide nanoparticles (NH2-ZnONPs). The morphology and structure of ZnONPs were observed by transmission electron microscopy (TEM) (Fig. 1A). The average hydrodynamic diameter and polydispersion of the ZnONPs were measured by using dynamic light scattering (DLS) (Fig. 1B), which showed that the mean diameter of ZnONPs was approximately 35.65 ± 7.93 nm. The DLS data revealed that the hydrodynamic size of ZnONPs was 45.6 ± 11.3 nm in aqueous solution and that ZnONPs had a positive surface charge of 25.4 mV (Fig. 1D). When the ZnONPs were dispersed in culture medium (DMEM with 10% fetal bovine serum), the mean particle size was approximately threefold the primary size (145.1 ± 2.6 nm) (Fig. 1C, D), which could be taken up by HaCaT cells, as demonstrated by fluorescence microscopy analysis of HaCaT cells after exposure to 10 μg/mL R6G-ZnONPs (Additional file 1: Figure 1A). In addition, the results of flow cytometry analysis showed that the cellular FSC and SSC signals increased, indicating that the internal complexity was increased after ZnONPs exposure (Additional file 1: Figure 1B). Morphologically, HaCaT cells showed swelling and spherical shapes after exposure to ZnONPs and UVB. In contrast, the addition of PT significantly protected HaCaT cells against these morphological changes (Fig. 2A). Cell viability studies were undertaken, with trypan blue assay used to obtain the toxicological profiling of UVB when using ZnONPs and PT alone or in combination to treat HaCaT cells. Cells were first tested with treatments of only ZnONPs or PT by using a series of dosages. The HaCaT cells showed significant dose- and time-dependent cytotoxicity in the ZnONP treatment of 7.5–17.5 μg/mL (Additional file 1: Figure 2A). Regarding the effects of PT, 2 μM PT treatment yielded no significant toxicity at any time point (24, 48, or 72 h), whereas 3 μM PT yielded significant cytotoxicity at 24 h (Additional file 1: Figure 2B). Therefore, we applied 2 μM PT in our further experiments. After the cells were exposed to ZnONPs and UVB for 24 h, a dose-dependent decrease in cell viability was observed (Fig. 2B). As expected, PT (2 μM) significantly protected cells against ZnONPs and UVB-induced cell death. These findings indicated that ZnONP and UVB exposure induced severe cytotoxicity and that PT had the ability to rescue cell death (Fig. 2C). For in-depth investigation of the mechanisms, we applied ZnONPs at a 10 μg/mL concentration combined with UVB (68 mJ) in our further experiments.
Fig. 1Characterization of ZnONPs. A The shape and size of ZnONPs used in this study were determined by transmission electron microscopy. B The hydrodynamic size of ZnONPs in distilled water were characterized by dynamic light scattering (DLS). C The hydrodynamic size of ZnONPs in complete culture medium (DMEM with 10% FBS) were characterized by nanoparticle tracking analysis (NTA). D Characterization of the diameter, hydrodynamic diameter in distilled water and complete medium, polydispersity index (P.I.) and zeta potential of ZnONPs. Values are presented as the mean ± standard deviation averaged over three replicates
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Fig. 2Morphology change and viability of HaCaT cells treated with ZnONPs and UVB. A Morphological changes after treatment with ZnONPs (10 μg/mL), UVB + ZnONPs (68 mJ/cm2 + 10 μg/mL) and UVB + ZnONPs + PT (2 μM). B Cell viability assay showing the dose-dependent cytotoxicity of combined treatment with ZnONPs (0–15 μg/mL) and UVB (68 mJ/cm2). C PT treatment (0–2 μM) followed by ZnONPs (10 μg/mL) + UVB (68 mJ/cm2) significantly increased the viability of HaCaT cells. D TEM image of HaCaT cells after ZnONPs (10 μg/mL) treatment for 3 h. The autophagosome double-membrane structures (blue arrow) engulfed ZnONPs (black arrow), the mitochondrial outer membranes were swollen, and the inner cristae were highly degenerated (red arrow). Values are presented as the mean ± SD (n = 3). *p < 0.05, control group versus treatment groups. #p < 0.05, the UVB + ZnONPs groups versus the UVB + ZnONPs + PT groups. Abbreviations N nucleus, A autophagosome, Z ZnONPs, M mitochondria
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Mitochondrial homeostasis plays a vital role in the cellular response to stress. Dysfunction of mitochondria and the release of mtROS in cell is a key upstream event involved in NLRP3 activation [16]. We first investigated the effects of ZnONPs and UVB on mitochondrial homeostasis by using TEM to observe the ultrastructure of mitochondria. After treatment with ZnONPs, the mitochondria showed swelling of the external membrane, and the mitochondrial cristae were highly degenerated, indicating severe mitochondrial damage (Fig. 2D). Second, the mitochondrial membrane potential (MMP) results showed that MMP distinctly decreased after ZnONPs (10 μg/mL) and UVB (68 mJ/cm2) treatment for 24 h (Fig. 3A, B), and PT (2 μM) significantly inhibited the MMP decrease caused by ZnONPs and UVB exposure. Third, we employed MitoSox, a specific mitochondrial ROS probe, to further demonstrate that the administration of ZnONPs and UVB increased mtROS in HaCaT cells in 3 h, and PT treatment also inhibited mtROS generation (Fig. 3C, D). Last, we tested whether ZnONPs induce total ROS generation. As shown in Fig. 3E, F, the intracellular ROS level significantly increased with ZnONPs and UVB treatment in 3 h, and PT treatment attenuated ROS generation in cells. These results indicated that ZnONPs and UVB exposure could induced mitochondrial damage and ROS production.
Fig. 3ZnONP- and UVB-induced Mitochondrial damage and NLRP3 inflammasome activation in HaCaT cells. A Mitotracker Deep Red was employed to examine ZnONP-induced mitochondrial membrane potential (MMP) loss via flow cytometry after 24 h exposure. B Histograms represent the percentage of MMP loss. C mtROS generation were examined by flow cytometry in HaCaT cells after ZnONPs and UVB treatment in 3 h. D Histograms represent the fluorescence intensity of mtROS. E ROS generation were examined by flow cytometry in HaCaT cells after ZnONPs and UVB treatment in 3 h. F Histograms represent the fluorescence intensity of ROS. G, H Western blot analysis of the levels of the NLRP3 inflammasome proteins NLRP3, ASC, pro-caspase-1 and cleaved caspase-1 in HaCaT cells. GAPDH was used as a loading control. I LDH release in ZnONP-treated HaCaT cells. Values are presented as the mean ± SD (n = 3). *p < 0.05, control group versus treatment groups. #p < 0.05, the UVB + ZnONPs groups versus the UVB + ZnONPs + PT groups
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To determine whether the NLRP3 inflammasome was activated in HaCaT cells after ZnONPs and UVB treatment, we detected the expression of the NLRP3 inflammasome proteins NLRP3, ASC, pro-caspase-1 and cleaved caspase-1. As shown in Fig. 3G, H, the levels of NLRP3, ASC, and cleaved caspase-1 were increased in ZnONP- and UVB-treated HaCaT cells, indicating activation of the NLRP3 inflammasome. Based on the observation that caspase-1 was activated in HaCaT cells, we hypothesized that pyroptosis could have occurred. Pyroptosis is dependent on GSDMD activation and will cause pore formation on the cell membrane [16, 36]. To test this hypothesis, we first employed LDH and PI/Annexin V assays to evaluate cellular rupture caused by pore formation on the cell membrane [37, 38]. In addition, to further distinguish the pyroptosis membrane rupture from classic apoptosis, we applied staurosporine (STS, 50 nM), a well-known apoptosis inducer, to serve as a negative control of pyroptosis. The results showed that both LDH release and PI signals increased significantly in ZnONPs and UVB group compared to the control and the STS groups (Figs. 3I, 4A, C). Then, we measured the protein expression of pyroptosis markers in ZnONP- and UVB-treated HaCaT cells and found increased expression of pro-GSDMD and GSDMD-NT (Fig. 4B, D–F). Interestingly, the increase in inflammasome and pyroptosis proteins induced by ZnONPs and UVB was reversed by PT (Fig. 4B, D–F). Moreover, we applied immunofluorescence staining with the GSDMD antibody, and the results further confirmed the abovementioned findings that GSDMD expression was increased after ZnONP- and UVB-treatment. The PT treatment inhibited GSDMD expression (Fig. 4G). To clarify the link between the NLRP3 inflammasome and pyroptosis, we used sh-caspase1 to knock down the expression of the key NLRP3 inflammasome effector caspase-1. Compared with nontarget vesicle control, sh-caspase1 resulted in a specific and significant reduction in the protein level of caspase-1. After caspase-1 inhibition, the PI/Annexin V assay showed a decrease in cell death (Additional file 1: Figure 3A, C). In addition, the pyroptosis proteins caspase 4, caspase 5 and GSDMD were also decreased after caspase-1 silencing (Additional file 1: Figure 3B, D–F). Collectively, these results demonstrated that ZnONPs and UVB triggered NLRP3 inflammasome and pyroptosis activation in keratinocytes and that PT treatment attenuated NLRP3 inflammasome and pyroptosis activation.
Fig. 4Effect of ZnONP- and UVB-triggered HaCaT cell pyroptosis. A Annexin V and PI were employed to examine ZnONP induced HaCaT cell pyroptosis via flow cytometry. C Histograms represent the percentage of Q1 + Q2 regions, indicating PI positive cells. B–F The levels of the pyroptosis proteins caspase 4, caspase 5, GSDMD and cleaved GSDMD-NT in HaCaT cells were detected by Western blotting, and GAPDH was used as a loading control. G HaCaT cell immunofluorescence staining with an anti-GSDMD antibody after a 24 h treatment with ZnONPs (10 μg/mL), UVB (68 mJ/cm2) + ZnONPs (10 μg/mL) or UVB (68 mJ/cm2) + ZnONPs (10 μg/mL) + PT (2 μM). Arrows indicate GSDMD puncta. Values are presented as the mean ± SD (n = 3). *p < 0.05, control group versus treatment groups. #p < 0.05, the UVB + ZnONPs groups versus the UVB + ZnONPs + PT groups
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ROS generation is one of the mechanisms by which the NLRP3 inflammasome can be activated [1]. In order to assess the role of ROS in NLRP3 inflammasome activation, HaCaT cells were pretreated with NAC (1 mM). NAC pretreatment significantly decreased the intracellular ROS level and alleviated cell death compared to ZnONPs and UVB treatment alone in 3 h (Fig. 5A, B). After NAC treatment, the PI/Annexin V assay showed a decrease in cell death (Fig. 5C, D) and LDH release was also decrease significantly (Fig. 5H). Moreover, immunoblotting assays showed that NAC reduced the expression of NLRP3, ASC, pro-caspase-1, cleaved caspase-1 and GSDMD in HaCaT cells after treatment with ZnONPs combined with UVB (Fig. 5F, G). Immunofluorescence staining also showed that NAC reduced the number of puncta with positive staining for the pyroptosis protein GSDMD (Fig. 5E). These results indicated that ROS played a crucial role in ZnONP- and UVB-activated NLRP3 inflammasome and pyroptosis.
Fig. 5ZnONP-induced NLRP3 activation and pyroptosis are mediated by ROS in HaCaT cells. A Inhibitory effect of NAC on the ZnONP- and UVB-elicited production of ROS. B Histograms represent the fluorescence intensity of ROS. C Annexin V and PI were employed to examine ZnONP-induced HaCaT cell pyroptosis via flow cytometry. D Histograms represent the percentage of Q1 + Q2 regions, indicating PI positive cells. E Immunofluorescence staining with an anti-GSDMD antibody of HaCaT cells treated with UVB (68 mJ/cm2) + ZnONPs (10 μg/mL), UVB (68 mJ/cm2) + ZnONPs (10 μg/mL) + NAC (1 mM) for 24 h. Arrow indicate GSDMD puncta. F, G Western blot analysis of the effects of NAC on the ZnONP-induced NLRP3 inflammasome and pyroptosis proteins NLRP3, caspase-1, cleaved caspase-1, ASC, GSDMD and cleaved GSDMD-NT in HaCaT cells. H LDH release in ZnONP-treated HaCaT cells treated with NAC. Values are presented as the mean ± SD (n = 3). *p < 0.05, control group versus treatment groups. #p < 0.05, the UVB + ZnONPs groups versus the UVB + ZnONPs + NAC groups
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Recent studies have highlighted the cross link between inflammasome and autophagy. Autophagy activation can limit NLRP3 inflammasome through the intracellular degradation system of autophagy [22]. On the other hands, autophagy dysfunction is considered an early indicator of nanomaterial interactions with cells, and autophagosome accumulation could serve as a general phenotype induced by nanoparticles [17]. As shown in Fig. 2D, the subcellular organelles were morphologically normal in untreated control cells with rare autophagic vacuoles, while a large number of double-membrane autophagosomes were obviously observed in ZnONP-treated HaCaT cells. Moreover, autophagosome-engulfed ZnONPs were also observed by TEM. These data show autophagosome accumulation under ZnONPs treatment. Autophagic flux was assessed using acridine orange (AO) staining (Fig. 6A). AO staining was measured the number of acidic vesicular organelles (AVOs), such as autophagolysosomes in cells [39]. Autophagy activation can be assessed by measuring the change in intracellular AO fluorescence. The intracellular AO fluorescence level significantly increased with ZnONPs and UVB treatment, and PT treatment significantly decreased the AO fluorescence intensity (Fig. 6B). We then used Western blotting to analyze the autophagic flux proteins LC3B and p62 and showed that the expression of LC3B-II and p62 was increased under ZnONPs and UVB treatment, whereas PT treatment decreased LC3B and p62 expression (Fig. 6D). These results imply that ZnONPs and UVB might have blocked autophagic flux, resulting in autophagy dysfunction, and PT treatment reversed this dysfunction. To confirm the blockage of autophagic flux by ZnONPs and UVB treatment, we used LAMP-1 (lysosome marker) and LC3B (autophagosome marker) double immunofluorescence staining to monitor co-localization of lysosome and autophagosome in autophagic flux. The ZnONPs and UVB treatment group showed separate green and red fluorescence, indicating that lysosome and autophagosome were unable to fuse and autophagic flux was blocked. The PT treatment group showed significant yellow fluorescence, which indicated the successful fusion of autolysosomes, showing that PT treatment restored the blocked autophagic flux (Fig. 6E).
Fig. 6ZnONP-induced autophagy dysfunction in HaCaT cells. A Acridine orange staining was employed to examine the number of acidic vesicular organelles (AVOs), such as autophagolysosomes, in cells. Flow cytometry analysis demonstrated the effect of PT against ZnONP- and UVB-induced keratinocyte autophagolysosomes. The FL3 positive region (Q1 + Q2 regions) indicated the AO positive staining. B Histograms represent the percentage of acridine orange positive cells. C, D Western blot analysis of the effect of ZnONPs and UVB on the autophagy proteins LC3B and p62 in HaCaT cells. E Immunofluorescence staining with anti-LC3B/anti-LAMP1 antibodies in HaCaT cells treated with ZnONPs (10 μg/mL), UVB (68 mJ/cm2) + ZnONPs (10 μg/mL) or UVB (68 mJ/cm2) + ZnONPs (10 μg/mL) + PT (2 μM) for 24 h. Arrows indicate LC3B/LAMP1 co-localized puncta. Values are presented as the mean ± SD (n = 3). *p < 0.05, control group versus treatment groups. #p < 0.05, the UVB + ZnONPs groups versus the UVB + ZnONPs + PT groups
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Our previous study indicated that ZnONPs and UVB induced the NLRP3 inflammasome in HaCaT cells [40]. A recent study indicated that cells can propagate exosomes loading with NLRP3 inflammasome protein complex via and affect other cellular behaviors [41]. Thus, we hypothesized that ZnONPs and UVB may promote NLRP3 inflammasome and pyroptosis propagation by inflammasome protein loading in exosomes. To determine the importance of exosome cargo for inflammasome propagation, we used ultracentrifugation to isolate exosomes from the medium of HaCaT cells treated with ZnONPs and UVB. Figure 7A demonstrates the presence of the canonical exosome proteins CD63, TSG101 and Flotillin-1 and the decrease of the intracellular protein HSP70, which are typical exosome features. The TEM images show that the exosomes exhibited a cup-shaped morphology (Fig. 7B). NTA analysis demonstrated a size distribution of particles between approximately 125 and 175 nm, which is consistent with the size range of exosomes (Fig. 7C). Western blot analysis was used to determine whether exosomes play a role in inflammasome propagation, and the results showed that ZnONPs and UVB exposure enhanced the expression of exosome loaded NLRP3 inflammasome proteins NLRP3, caspase-1 and the pyroptosis protein GSDMD when normalized with flotillin-1 (housekeeping protein) (Fig. 7D, E).
Fig. 7Exosomes release NLRP3 inflammasome complex propagates ZnONP- and UVB-induced keratinocytes inflammation. A Western blot analysis of isolated exosomes. The presence of canonical exosome proteins and the HSP70 decreasing indicate a pure exosome preparation. B TEM imaging of exosomes, arrows indicated the exosomes with cup-shaped morphology. C NTA analysis demonstrated a size distribution of particles that was consistent with the size range of exosomes. D, E Western blot analysis of exosomes isolated from the medium of HaCaT cells treated with ZnONPs and UVB. The NLRP3 inflammasome and pyroptosis proteins NLRP3, pro-caspase-1 and GSDMD were detected, Flotillin-1 was used as a housekeeping protein (loading control). F, G Western blot analysis of NLRP3, caspase-1 and GSDMD expression in HaCaT cells after treatment with PT and exosomes isolated from control cells or cells exposed to ZnONPs and UVB. H, I Western blot analysis of exosomes isolated from HaCaT cells after treatment with rapamycin (autophagy inducer), chloroquine (autophagy inhibitor), ZnONPs and UVB. The NLRP3 inflammasome and pyroptosis proteins NLRP3, caspase-1 and GSDMD were detected, Flotillin-1 was used as a housekeeping protein (loading control). J A Transwell culture system was used to mimic the cellular milieu. HaCaT cells were exposed to foreign DioC18-labeled exosomes from donor HaCaT cells that were exposed to ZnONPs and UVB with or without Rapa or Rapa + CQ treatment. K Histograms represent the relative fluorescence intensity of DioC18 staining. L DioC18-labeled exosomes propagation was measured via flow cytometry. DioC18 positive HaCaT cells (Q2-UR + LR regions) indicate were exposed to foreign DioC18-labeled exosomes from donor HaCaT cells that were exposed to ZnONPs and UVB with or without Rapa or Rapa + CQ treatment. M Histograms represent the DioC18 positive cells. Values are presented as the mean ± SD (n = 3). *p < 0.05, the control group versus the treatment groups. #p < 0.05, the UVB + ZnONPs group versus the UVB + ZnONPs + PT group, Exosome (UVB + ZnONPs) group versus Exosome (UVB + ZnONPs) + PT group, UVB + ZnONPs + Rapa group versus UVB + ZnONPs + CQ group, UVB + ZnONPs + Rapa group versus UVB + ZnONPs + Rapa + CQ group
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To test whether exosomes transfer NLRP3 inflammasome and pyroptosis proteins between cells, HaCaT cells were treated with ZnONPs and UVB for 24 h, and then we collected exosomes from the medium. When we exposed cells to exosomes from either ZnONP- and UVB-treated HaCaT cells or untreated cells, Western blot analysis revealed significantly higher NLRP3, caspase-1 and GSDMD protein expression in the former group than in the latter group (Fig. 7F, G). This increase in NLRP3, caspase-1 and GSDMD was not observed when we added 0.1% Triton X-100 to the exosome solution to denature exosome function before the treatment (Additional file 1: Figure 4A). Collectively, these findings suggest that ZnONPs and UVB exposure induced cell-to-cell transmission of NLRP3 inflammasome and pyroptosis proteins via exosomes, which further propagated inflammasome and pyroptosis activation. Treatment with PT decreased the release of inflammasome- and pyroptosis-loaded exosomes.
Recent studies indicated autophagy is a key regulator of exosomal biogenesis. Autophagosomes fuses with multivesicular bodies (MVBs), a late endosome organelle, to form hybrid organelles termed amphisomes, which can subsequently fuse with lysosomes for content degradation [26, 42]. We hypothesized that the ZnONP- and UVB-induced autophagy dysfunction might increase cellular exosome release. To test this hypothesis, we used the autophagy inducer rapamycin and the autophagy inhibitor chloroquine (CQ) in combination with ZnONPs and UVB exposure. Western blot analysis showed that treatment with rapamycin in combination with ZnONPs and UVB decreased the relative expression of exosome loaded NLRP3 inflammasome proteins NLRP3, caspase-1 and the pyroptosis protein GSDMD when normalized with flotillin-1 (housekeeping protein) (Fig. 7H, I). In contrast, when we blocked autophagic flux by using CQ, the exosomal release of NLRP3, caspase-1, and the pyroptosis protein GSDMD was significantly increased (Fig. 7H, I). We further analyzed the exosome concentration changes by using Transwell assays, and we established coculture systems that mimicked the cellular milieu wherein HaCaT cells were exposed to exosomes on the apical surface. The results demonstrated that an increasing number of HaCaT cell-propagated exosomes were internalized by other HaCaT cells when the upper chamber of the Transwell was exposed to ZnONPs and UVB. Pretreatment with rapamycin significantly decreased exosome release compared to ZnONPs and UVB exposure alone. In contrast, CQ treatment significantly increased exosome release (Fig. 7K). These data indicated that treatment with CQ, an autophagy inhibitor, enhanced ZnONP- and UVB-induced autophagy dysfunction and autophagosome accumulation and enhanced exosome release from HaCaT cells. The autophagy inducer rapamycin decreased exosome release.
We next sought to determine whether UVB irradiation of mouse skin enhanced ZnONP-induced skin inflammation and to assess the effectiveness of topical PT treatment in the inhibition of acute skin inflammation. SKH-1 hairless mice were first administered a single dose of UVB radiation. Twenty minutes after radiation, 2 μg/cm2 ZnONPs alone or in combination with PT cream were applied to the mice, as demonstrated in Additional file 1: Figure 5A, B. The results indicated that skin thickness fold and skin redness were increased in mice exposed to UVB radiation and ZnONPs (Fig. 8A) (Additional file 1: Figure 5A, B). Moreover, after UVB irradiation and ZnONPs exposure, skin wrinkling and hyperplasia were observed by histological staining. Transepidermal water loss (TEWL, measured by standard evaporimetry), which reflects skin barrier function, was also increased in UVB irradiation- and ZnONP-exposed mice (Fig. 8B). In contrast, treatment with the topical PT cream (100 μM) following UVB radiation and ZnONPs exposure (Fig. 8C) decreased the skin thickness fold, skin redness and TEWL.
Fig. 8ZnONPs and UVB activate the NLRP3 inflammasome-induced pyroptosis in the skin. A H&E histology of mouse skin at 72 h after the acute inflammation test with ZnONPs and UVB showed dermal swelling and inflammatory cell infiltration (red arrows). ZnONPs and UVB treatment significantly increased the dermal thickness, as shown by H&E staining A, C. PT treatment significantly suppressed ZnONP- and UVB-induced increases in dermal thickness (scale bar represents 200 μm). Transepidermal water loss (TEWL) in SKH:HR-1 mice treated with control (untreated), ZnONPs (2 mg), UVB (150 mJ/cm2) and PT (100 µM) alone or in different combinations. B Epidermis thickness in mice treated with control (untreated), ZnONPs (2 mg), UVB (150 mJ/cm2) and PT (100 µM) alone or in different combinations. D–G The expression of the NLRP3 inflammasome and pyroptosis proteins NLRP3, ASC, caspase-1, cleaved caspase-1 and GSDMD following exposure to ZnONPs (2 mg), UVB (150 mJ/cm2) and PT (100 µM). Values are presented as the mean ± SD (n = 3). *p < 0.05, control group versus treatment groups. #p < 0.05, the UVB + ZnONPs groups versus the UVB + ZnONPs + PT groups
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To confirm the previously identified in vitro toxicity mechanism, we next investigated the expression of the NLRP3 inflammasome proteins NLRP3, ASC, pro-caspase-1 and cleaved caspase-1 and the pyroptosis protein GSDMD in mice skin tissue. As shown in Fig. 8D–G, the expression of NLRP3, ASC, cleaved caspase-1 and GSDMD was increased in the ZnONP- and UVB-treated group but was significantly decreased in the PT cream-treated group. These data indicated that in an in vivo experiment, cotreatment with UVB radiation and ZnONPs induced acute skin damage, activated the NLRP3 inflammasome and induced skin pyroptotic cell death. Topical treatment with PT alleviated the severe skin inflammation and damage triggered by UVB radiation and ZnONPs exposure through the inhibition of NLRP3 inflammasome complex. The expression of ASC and cleaved-caspase-1 expression were significantly decrease, whereas the NLRP3 expression was only slightly decrease without statistical significance after the treatment of PT due to unknown reason. These findings corresponded with the in vitro experimental data.