Preliminary investigation on the mechanisms of multi-organ toxicity induced by prolonged inhalation exposure to silica nanoparticles

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Since respiratory exposure is the primary route of human exposure to SiNPs, this study systematically investigated their distribution and damaging effects in the lungs, heart, liver, and kidneys following tracheal drip injection. The results demonstrated that SiNPs distribute across these organs and induce mitochondrial damage, endoplasmic reticulum stress, and activate cell death pathways, including apoptosis, pyroptosis, and autophagy. The most significant damage occurred in the middle-dose group (6 mg/kg). The lungs, as the primary target organ, exhibited pronounced fibrotic changes, while fibrotic lesions were also observed in the heart, liver, and kidneys to varying degrees. These findings suggest that the observed injury mechanisms may collectively contribute to chronic inflammation and promote fibrosis. This study provides critical insights into the multi-organ toxicity of SiNPs, offering a foundation for their safety assessment. SiNPs Fibrosis Multiorgan Respiratory exposure Toxic mechanisms of action Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights Cross-organ toxicity of SiNPs was demonstrated for the first time by tracheal perfusion, revealing a synchronised progression of injury in the lung-cardiac-hepatic-renal system. Multi-mechanism decoding identified mitochondrial damage, ER stress, apoptosis, pyroptosis, and autophagy crosstalk as core drivers of SiNPs-induced lesions. Long-term exposure modeling establishes tracheal perfusion as a reliable approach to simulate population-level exposure and nanoparticle deposition patterns. 1. Introduction Nanotechnology has rapidly advanced in recent years, with applications in environmental governance, healthcare, precision agriculture and food engineering 1 . As the production and use of nanomaterials grow, the risk of human exposure to silica nanoparticles (SiNPs) is increasing. However, research on the chronic toxic effects of long-term exposure to SiNPs remains limited. Emerging toxicological evidence reveals that pulmonary inhalation constitutes the predominant exposure pathway for SiNPs, not only triggering inflammatory cascades and aberrant proliferation within alveolar epithelium but also facilitating particle translocation into systemic circulation via compromising the integrity of the blood- lung barrier 2 , and potentially reach the heart, liver, kidneys, and other organs. Yet, the specific mechanisms underlying systemic chronic toxic responses from respiratory exposure to SiNPs are not well understood, and comprehensive knowledge of their body distribution and multi-organ effects is lacking Nanoparticles can cross biological barriers and enter physiological systems 3 . SiNPs enter the body through the respiratory tract, making the lungs a primary target. Exposure to SiNPs induces local and systemic inflammation in the lungs 4 . After crossing the blood-gas barrier, the heart becomes the next target organ 5 . The liver, the largest solid organ, is the main site of SiNPs accumulation, regardless of exposure route, as it processes most toxins 6 . The kidneys, as the principal metabolic organ, are responsible for excreting nanomaterials 7 . Therefore, studying the toxic effects and pathogenesis of SiNPs exposure on the lungs, heart, liver, and kidneys is crucial for assessing the risks associated with human exposure to SiNPs. Recent studies have shown that long-term exposure to inhaled SiNPs activates inflammatory responses not only in the lungs but also in the heart, liver, and kidneys, leading to tissue fibrosis in mice 8 . The mechanisms behind this multi-organ fibrosis require further investigation. Current research identifies several pathways contributing to organ fibrosis. Notably, SiNPs primarily invade mitochondria, damaging their structure and function across various cell types 9 , which results in excessive reactive oxygen species (ROS) generation and mitochondrial dysfunction 10 . This dysfunction disrupts cellular energy metabolism and can trigger endoplasmic reticulum (ER) stress, exacerbating cellular damage 11 . ER stress activates downstream signaling pathways through the unfolded protein response (UPR) 12 and may induce autophagy 13 . Excessive autophagy induction is emerging as a potential mechanism of SiNPs toxicity, contributing to disease pathogenesis 14 . Chronic SiNPs exposure can inhibit autophagosome degradation by blocking autophagic flux, disrupting cellular homeostasis 15 16 . Studies indicate that ER stress synergistically promotes SiNPs-induced apoptosis, particularly in human alveolar epithelial cells 17 . ER stress, an internal apoptotic pathway 18 , interacts with other pathways like the caspase pathway and is commonly associated with fibrosis-related diseases 19 . Furthermore, SiNPs may induce pyroptosis by activating NLRP3 inflammatory vesicles 20 , resulting in a cell death mode characterized by intense inflammation 21 . Processes such as apoptosis, pyroptosis, and autophagy can overwhelm the system when large numbers of cells die suddenly, as seen in infections, chronic inflammation, and tissue damage. This sudden cell death leads to the massive release of cellular contents, known as danger-associated molecular patterns (DAMPs) 22 , which trigger a robust immune response to recruit phagocytes and promote tissue repair. In contrast, during chronic inflammation, myofibroblasts often avoid cell death, leading to aberrant wound healing and excessive extracellular matrix production, thus driving fibrosis 23 . Further experiments are needed to determine whether mitochondrial damage 24 , ER stress 25 , apoptosis 24 , pyroptosis 21 , and autophagy 26 are also involved in the multiorgan fibrosis induced by SiNPs exposure. While the single organ toxicity of SiNPs has been studied, the mechanisms underlying their multi-organ toxicity, particularly regarding fibrosis induced by mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, remain inadequately explored. This study aims to systematically evaluate the distribution and damaging effects of SiNPs in the lungs, heart, liver, and kidneys through tracheal drip injections of varied SiNPs concentrations. We will assess the expression of marker proteins related to mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, autophagy, and fibrosis to investigate the potential mechanisms of the toxic effects of SiNPs on these organs following respiratory exposure. 2. Materials and methods 2.1 Preparation and characterisation of SiNPs particles TEM was used to image a 1.0 mg/mL suspension of SiNPs (Nanocomposix) that was dried at 20°C Average particle size was calculated using ImageJ software. Mean particle size and zeta potential of SiNPs in saline were determined after 1, 2, 4, 8 and 24 h at 20°C using a zeta potential particle size analyser (Malvern, UK). 2.2 Animal experimental design Fifty-two SPF (Specific pathogen free) grade, 7–8 week old, healthy male C57BL/6J mice were randomly divided into 4 groups: control (saline) and low, medium and high dose groups (3, 6 and 12 mg/kg-bw), and SiNPs suspension was administered by intratracheal drip under anaesthesia (chlorpromazine) once per weekday for a total of 12 times. SiNPs Dose conversions were performed according to occupational exposure limits for hazardous substances in the workplace (5 mg/m 3 , GBZ 2.1–2019) and respiratory physiological parameters of mice 27 . All animal experimental operations were reviewed and approved through strict laboratory animal ethics (Animal Ethics Approval Number: 2400643). 2.3 Histopathological examination Histopathological analysis was performed according to standard protocols. Briefly, tissue blocks of major organs i.e. lungs, heart, liver and kidneys were fixed in 10% neutral buffered formalin, routinely dehydrated and degreased and then embedded in paraffin blocks. The 5 µm thick paraffin sections were sliced and fixed on slides. Afterwards, tissue sections were stained with hematoxylin-eosin (H&E; China) for histopathological lesion observation or Masson trichrome staining (Masson; China) for pathological fibrosis assessment. Finally, the slides were scanned with a tissue multilabel panoramic imaging analysis system (Nanozoomer S60; Japan). 2.4 TEM Observations of Tracheal Drops of SiNPs into the Lungs 2.5% glutaraldehyde was prefixed and 1% osmium tetroxide was refixed. The samples were then rinsed three times with 0.1 MPB and dehydrated with 30%, 50%, 70%, 80%, 90%, and 100% alcohol and 100% acetone successively, after which the dehydrating agent and Epon-812 embedding agent, in the ratio of 3:1, 1:1, and 1:3, respectively, were sequentially infiltrated, and the samples were embedded with Epon-812 Pure Embedding Agent in order to create a block of cells or tissues. Ultrathin sections (60–90 nm) were obtained by an ultrathin slicer (Ultracut UCT, Leica, Germany). They were then stained with lead citrate and uranyl acetate and examined by TEM (JEM-1400FLASH, JEOL, Japan). 2.5 Small animal live imaging The mice were randomly divided into experimental and control groups with at least 3 mice in each group. Mice in the experimental group were given SiNPs with fluorescent labelling (FITC) (at a dose of 6 mg/kg bw) by tracheal drip (zhongkekeyou, China), and the control group was injected with an equal volume of saline. Mice were anaesthetised using isoflurane, placed in an anaesthesia induction chamber, and transferred to the imager platform after the mice were fully anaesthetised. Turn on the IVIS Lumina Ⅲ imaging system, warm up the instrument and set the imaging parameters, select the wavelength of 440–520 nm according to the fluorescent markers, fix the mice in the prone position on the imaging platform, make sure that their breathing was stable, and select the A field of view. Whole-body imaging was performed 24 hours after administration. Mice were ensured to be under stable anaesthesia before each imaging session. At the end of imaging, mice were executed and major organs (e.g., lungs, heart, liver, spleen, kidneys) were dissected. The organs were placed on the imaging platform and ex vivo imaging was performed using the same parameters to further verify the distribution of nanoparticles. 2.6. protein blotting assay Western blot determination of lung, heart, liver, and kidney tissue proteins were extracted by Protein Extraction Kit (Solarbio, China), and concentrations were measured by BCA protein assay (Solarbio, China). Primary antibodies included α-SMA, Caspase 1/P20/P10, Beclin1, Bcl2, Parkin, OPA1 antibody (1:1000, Proteintech, China), ATF6 (1:2000, Proteintech, China), VIM, LC3A/B, caspase-3, PERK (1:1000, CST, USA), P62, PINK1 (1:10,000, abcam, UK), IL-1β, XBP1, IRE1α antibodies (1:1000, Boster, China), GSDMD antibody (1:1000, Bioss, China), MFN1 (1:500, PTMAL, China), the NLRP3 (1:500, abclonal, China), collagen1 (1:500, WANLEI, China) were diluted with BSA. After sealing with 5% skimmed milk, the polyvinylidene difluoride (PVDF) membrane was incubated overnight to detect proteins. The membranes were then incubated with the corresponding HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies for 1 h at room temperature. Finally, the membranes were reacted with a chemiluminescent detection system (ECL Detection Kit) (Smart-Lifesciences, China) and visualised under a gel imaging system (Bio-Rad), with GAPDH as an internal control and quantified using ImageJ software. 2.7 Statistics Data are expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 5.0 software. One-way analysis of variance (ANOVA) was used to compare the data between the control and model groups. p < 0.05 was considered significant difference. 3. Results 3.1 Characterization of silica nanoparticles and their in vivo distribution As shown in Fig. 1 A and B, transmission electron microscopy revealed that SiNPs have a diameter of approximately 80 nm, with a good dispersion and nearly spherical shape. In saline, the hydrodynamic size and zeta potential of SiNPs were measured at about 86.38 ± 0.82 nm and − 25.64 ± 0.065 mV, respectively, indicating their stability and dispersion (Fig. 1 C and D). Electron microscopy of mouse lung tissues (Fig. 1 E) revealed a significant presence of SiNPs in the SiNPs group, accompanied by autophagic vacuoles and mitochondrial damage. Additionally, using fluorescently labeled SiNPs, we found that they were widely distributed in the lungs, heart, liver, and kidneys. 3.2 Mechanisms of silica nanoparticle toxicity in lung tissue To investigate the distribution of SiNPs in lung tissues, we conducted in vivo imaging on the lung tissues of mice exposed to fluorescently labeled SiNPs through the respiratory tract and confirmed their presence (Fig. 3 A). H&E and MASSON staining revealed varying effects across dose groups (Fig. 3 B): at low doses, peripheral alveoli were fused and dilated (blue arrows). At mid-dose, alveolar walls thickened, some alveoli atrophied and collapsed (green stars), and perivascular fibroblastic hyperplasia was observed (green arrows). At high doses, similar fusion and dilation were noted with some atrophied alveoli. MASSON staining indicated a gradual increase in collagen fibers, most pronounced in the mid-dose group. Western blot analysis of Col-1, α-SMA, and VIM protein levels confirmed elevated expression across all groups, with the mid-dose group showing the most fibrosis, supporting our pathological findings (Fig. 3 C). Mitochondrial damage assessment revealed increased expression of MFN1 and decreased expression of OPA1, PINK1, and Parkin, particularly in the mid-dose group (Fig. 3 D). ER stress markers ATF6, PERK, IRE1α, and XBP1s were elevated in all groups, with the mid-dose group showing the most significant increases (Fig. 3 E). We examined cell death markers and found decreased Bcl2 and increased IL-1β and Caspase-3 across all groups, especially at mid-dose (Fig. 3 F). Additionally, NLRP3, GSDMD-N, and Caspase-1 expressions increased in each group, more distinctly in the mid-dose group (Fig. 3 G). P62 and Beclin1 expressions rose across groups, while LC3 II/I levels decreased, again most noticeably in the mid-dose group (Fig. 3 H). Collectively, these results indicate that long-term exposure to SiNPs through the respiratory tract leads to their distribution in lung tissues, resulting in fibrosis, mitochondrial damage, heightened endoplasmic reticulum stress, increased apoptosis, focal death, and impaired autophagic flow. 3.3 Mechanisms of silica nanoparticle toxicity in cardiac tissue To investigate the distribution of SiNPs in cardiac tissues, we performed in vivo imaging on cardiac tissues from mice exposed to fluorescently labeled SiNPs via the respiratory tract, confirming their presence in the heart (Fig. 3 A). Hearts from all groups were analyzed using H&E staining to assess pathological changes, and collagen deposition was evaluated with Masson staining (Fig. 3 B). H&E staining revealed well-defined and aligned cardiomyocytes in the control group, while the low-dose group exhibited fiber deformation. The medium-dose group displayed significant histological abnormalities, including myocardial fiber destruction (black arrows), and the high-dose group showed signs of edema, irregular staining of myocardial fibers, and abnormal interstitial space distribution (yellow &). Masson staining indicated that collagen fibers (blue) were most pronounced in the medium-dose group, where fibrosis was further confirmed through Western blot analysis of Col-1, α-SMA, and VIM protein expression, showing significant elevation in the medium-dose group (Fig. 3 C). To evaluate mitochondrial damage, we examined related proteins and found increased expression of MFN1 across all groups, with decreased levels of OPA1, PINK1, and Parkin, particularly notable in the medium-dose group (Fig. 3 D). Endoplasmic reticulum stress was indicated by elevated levels of ATF6, PERK, IRE1α, and XBP1s in all groups, most prominently in the medium-dose group (Fig. 3 E). We also assessed marker proteins for apoptosis, necrosis, and autophagy (Fig. 3 F). Bcl2 expression decreased while IL-1β and Caspase-3 levels increased in all groups, especially in the medium-dose group. Enhanced expression of NLRP3, GSDMD-N, and Caspase-1 was noted in all groups, with the most significant increase in the medium-dose group (Fig. 3 G). Additionally, levels of P62 and Beclin1 increased across all groups, while LC3 II/I expression decreased, most notably in the medium-dose group (Fig. 3 H). These results suggest that chronic exposure to SiNPs via the respiratory tract results in their distribution in cardiac tissues, leading to fibrosis, mitochondrial damage, heightened endoplasmic reticulum stress, increased apoptosis, necrosis, and impaired autophagic flow. 3.4 Mechanisms of silica nanoparticle toxicity in liver tissue We analyzed the distribution of SiNPs in liver tissues of mice exposed via the respiratory tract using in vivo imaging, confirming their presence (Fig. 4 A). Histological analysis with H&E and Masson staining revealed normal hepatocyte morphology in the control group (Fig. 4 B). In the low-dose group, liver lobule structure was disrupted (orange arrows); the medium-dose group exhibited disordered lobule formation with obvious pseudolobules (green triangles), while the high-dose group showed significant inflammatory cell infiltration (blue stars). Masson staining indicated high collagen fiber expression in the medium and high-dose groups. To assess liver fibrosis, we measured Col-1, α-SMA, and VIM protein levels via Western blot and found elevated expression across all groups, most notably in the medium-dose group, aligning with pathological findings (Fig. 4 C). To investigate mitochondrial damage, we analyzed related proteins and found increased MFN1 levels, alongside decreased expression of OPA1, PINK1, and Parkin in all groups, with the most significant changes in the medium-dose group (Fig. 4 D). We also examined endoplasmic reticulum stress markers and noted elevated levels of ATF6, PERK, IRE1α, and XBP1s across all groups, particularly pronounced in the medium-dose group (Fig. 4 E). Examining markers of cell death, we observed decreased Bcl2 and increased levels of IL-1β and Caspase-3 in all groups, again more evident in the medium-dose group (Fig. 4 F). Additionally, markers of pyroptosis, NLRP3, GSDMD-N, and Caspase-1, were increased in all groups, especially in the medium-dose group (Fig. 4 G). For autophagy, we found that P62 and Beclin1 expression rose while LC3 II/I decreased across all groups, with the medium-dose group showing the most significant alterations (Fig. 4 H). These findings suggest that long-term exposure to SiNPs via the respiratory tract leads to their distribution in liver tissues, resulting in fibrosis, mitochondrial damage, increased endoplasmic reticulum stress, apoptosis, pyroptosis, and impaired autophagic flow. 3.5 Mechanisms of silica nanoparticle toxicity in renal tissues To assess the distribution of SiNPs in renal tissues, we conducted in vivo imaging on renal tissues from mice exposed to fluorescently labeled SiNPs via the respiratory tract and confirmed their presence (Fig. 5 A). Kidney tissues underwent H&E and MASSON staining (Fig. 5 B). The low dose showed interstitial edema (purple #) and inflammatory cell infiltration (blue star), while the medium dose displayed thickening of the glomerular capsule wall and matrix accumulation (green arrow). The high dose group exhibited solidly transformed glomeruli (blue arrows). MASSON staining revealed blue collagen matrix deposition in glomeruli and tubules, with the most severe fibrosis noted in the medium dose group. To confirm fibrosis in renal tissues, we analyzed the expression of Col-1, α-SMA, and VIM proteins via Western blotting, finding elevated levels across all groups, particularly pronounced in the medium dose group, consistent with histopathological findings (Fig. 5 C). To evaluate mitochondrial damage, we examined mitochondrial proteins and observed increased MFN1 expression and decreased levels of OPA1, PINK1, and Parkin in all groups, with the most significant changes in the medium dose group (Fig. 5 D). Assessment of endoplasmic reticulum stress showed an increase in ATF6, PERK, IRE1α, and XBP1s in all groups, especially evident in the medium dose group (Fig. 5 E). Given the recent interest in cell death mechanisms, we investigated marker proteins for apoptosis, pyroptosis, and autophagy. As shown in Fig. 5 F, Bcl2 levels decreased, while IL-1β and Caspase-3 levels increased in all groups, particularly in the medium dose group. Similarly, NLRP3, GSDMD-N, and Caspase-1 levels rose across all groups, with the most evident changes in the medium dose group (Fig. 5 G). Figure 5 H illustrates an increase in P62 and Beclin1 levels in all groups, alongside a decrease in LC3 II/I ratios, also more pronounced in the medium dose group. Overall, these findings indicate that prolonged exposure to SiNPs via the respiratory tract leads to their distribution in renal tissues, resulting in fibrosis, mitochondrial damage, elevated endoplasmic reticulum stress, apoptosis, increased focal death, and impaired autophagic flow. 4. Discussion Recent advancements in nanotechnology have heightened interest in the biosafety of various nanomaterials 28 . SiNPs are widely utilized due to their unique physicochemical properties, yet their potential toxicity and molecular mechanisms remain poorly understood. This study systematically assessed the distribution and harmful effects of SiNPs in the lungs, heart, liver, and kidneys through tracheal drip injection. Findings indicate that SiNPs exposure activates several cell death pathways, including mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, which may lead to chronic inflammation and fibrosis. The lungs emerged as the primary target organ, displaying significant fibrotic changes, while the heart, liver, and kidneys also exhibited varying degrees of fibrosis. This study uniquely demonstrates that SiNPs damage multiple pathways, including mitochondrial dysfunction, endoplasmic reticulum (ER) stress, apoptosis, pyroptosis, and autophagy, potentially leading to multi-organ fibrosis. Mitochondria, crucial for oxidative metabolism and biomolecule synthesis, are particularly vulnerable during pathological processes 29 . Our findings indicate that SiNPs exposure increases MFN1 expression while decreasing OPA1, PINK1, and Parkin levels, suggesting an imbalance in mitochondrial dynamics and impaired autophagy. The upregulation of MFN1 indicates enhanced mitochondrial fusion as a cellular adaptive response to damage, whereas the downregulation of OPA1 points to compromised inner membrane fusion, contributing to mitochondrial dysfunction. Additionally, reduced PINK1 and Parkin levels suggest impaired initiation of autophagy, preventing the efficient removal of damaged mitochondria. These results imply that SiNPs disrupt mitochondrial quality control and dynamics, aligning with previous mitochondrial damage findings 10 , 30 . Regarding ER stress, the study reveals increased expression of the three main stress markers (PERK, ATF6, and IRE1α), highlighting their role in SiNPs-induced multi-organ injury. While research on SiNPs and ER stress is limited, our results corroborate earlier studies 31 , 32 . All three pathways activate the transcription of chaperones and proteins involved in redox homeostasis, protein secretion, or cell death 33 . SiNPs exposure also resulted in decreased Bcl2 expression, an anti-apoptotic protein, leading to reduced mitochondrial membrane potential and cytochrome C release, which activates Caspase-9 and Caspase-3, inducing apoptosis 22 . Increased IL-1β and Caspase-3 expression further indicate that SiNPs trigger apoptosis and inflammatory responses via the mitochondria-dependent apoptotic pathway, consistent with prior in vitro studies 34 , and the present results further support the conclusion that SiNPs induce apoptosis. The elevated IL-1β levels suggest that SiNPs not only induce apoptosis but also activate inflammation, a potential driver of fibrosis. Cellular focal death is triggered by the activation of inflammatory vesicle sensors. In the classical pathway, the NLRP3 inflammasome serves as a platform to activate the protein hydrolase caspase-1, which further processes caspase-1 into its p33/p10 form 35 . Activated caspase-1 then activates GSDMD, allowing it to oligomerize and insert into the plasma membrane, creating pores that destabilize the membrane and lead to cell lysis, releasing DAMPs. Additionally, activated caspase-1 is crucial for converting precursors of IL-1β into active 17KDa cytokines 36 . This study found increased expression of NLRP3, GSDMD-N, and caspase-1 across all groups, suggesting that SiNPs induce multi-organ cellular pyroptosis, aligning with previous findings on cardiotoxicity 21 . Autophagy is a key catabolic process that transports proteins, cytoplasmic components, and organelles to the lysosome for degradation and recycling 33 . Our study found a significant increase in P62 and Beclin1 expression, along with a decrease in the LC3-II/LC3-I ratio in mouse organs. The rise in P62 and reduction in the LC3-II/LC3-I ratio indicate that autophagic flow may be inhibited, resulting in inadequate degradation of autophagic substrates. The increase in Beclin1 suggests enhanced autophagic initiation or accumulation of autophagosomes. Therefore, SiNPs may contribute to the development of pulmonary fibrosis by disrupting autophagic flow. Internalized particles are recognized by cells as foreign, activating autophagy. Electron microscopy revealed that SiNPs primarily localized within the cytoplasm, lysosomes, and autophagic vesicles. However, excessive uptake of SiNPs could overwhelm autophagy's degradation capacity, blocking the process. Previous studies, mainly focusing on early autophagy activation, contrast with our findings 15 , 37 , 38 . Our findings indicate a late-stage blockage, which is speculated that SiNPs may impair lysosomal function, preventing autophagosome-lysosome fusion, or activate the UPR through endoplasmic reticulum stress, increasing autophagosome formation but disrupting degradation. They may also induce mitochondrial damage, leading to elevated ROS production and further autophagy dysfunction. This area will be explored further in future research. This study's limitations include differences between animal models and human exposure, as well as the potential for optimizing dose selection. We found that SiNPs-induced fibrosis and protein damage peaked at the medium dose (6 mg/kg-bw) but decreased at the high dose (12 mg/kg-bw). While most current research has focused on short-term high doses, our study examined long-term low doses, and other short-term studies reported significant damage responses at the high dose (12 mg/kg-bw) 7 , 39 . Short-term high-dose exposure (12 mg/kg-bw) to SiNPs can cause rapid cellular damage and inflammatory responses, significantly increasing damage protein indicators. In contrast, long-term low to medium-dose exposure may lead to gradual fibrosis and damage due to chronic toxicity. Interestingly, the high-dose group might activate a protective mechanism in response to excessive damage, resulting in decreased protein expression. This could indicate an atypical dose-effect relationship due to inappropriate dosing, leading to cellular or tissue intolerance. Alternatively, SiNPs toxicity may not follow a typical dose-response pattern, with toxic effects emerging only after reaching a certain dose level. This suggests that controlling SiNPs doses within a specific range could minimize their toxic side effects. However, further experimental and epidemiological data are needed to support this finding. Future studies will include more intermediate doses to better determine the toxic dose threshold for SiNPs. Our current research has concentrated on a single mechanism of SiNPs-induced mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, but the interactions and synergistic effects among these mechanisms require further investigation. Future work will delve into these synergistic effects to elucidate their complex biological impacts. In conclusion, this study aims to systematically investigate the distribution and damaging effects of SiNPs in the lungs, heart, liver, and kidneys after tracheal drip administration, expanding on previous research that primarily examined damage to individual organs. We will also delve into the mechanisms of mitochondrial damage, ER stress, apoptosis, pyroptosis, and autophagy, hypothesizing their roles in fibrosis. This comprehensive exploration of the complex cellular effects and interrelations prompted by SiNPs surpasses the analysis of a single mechanism. By elucidating the multi-organ toxicity of SiNPs, our research will contribute valuable experimental data for the safety assessment of nanomaterials and provide a theoretical foundation for future intervention strategies. 5. Conclusion This study found that chronic respiratory exposure to SiNPs can damage multiple organs, including the heart, lungs, liver, and kidneys. It identified potential mechanisms of toxicity, including mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, providing a crucial basis for safety assessments and future intervention strategies. Declarations Competing interests The authors declare they have no confict of interest. Funding This study was supported by Medical Research Joint Fund for High Quality Development of health in Guizhou Province (grant number 2024GZYXKYJJXM0013). Author Contribution Jiaqi Ban：Conceptualization. Lihong Ao、He-Qun Gu: validation. Qiong Tian：subsidiary. Jiaqi Ban, Jun Li, Yungeng Wei, Xiu He, Hua Zhao: writing-review and editing, supervision. All authors have read and agreed to the published version of the manuscript. References Murugadoss S, Lison D, Godderis L, Van Den Brule S, Mast J, Brassinne F, Sebaihi N, Hoet PH. Toxicology of Silica Nanoparticles: An Update. Arch Toxicol. 2017;91(9):2967–3010. https://doi.org/10.1007/s00204-017-1993-y . 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The Role of DRP1 Mediated Mitophagy in HT22 Cells Apoptosis Induced by Silica Nanoparticles. Ecotoxicol Environ Saf. 2024;272:116050. https://doi.org/10.1016/j.ecoenv.2024.116050 . Chen F, Sun J, Wang Y, Grunberger JW, Zheng Z, Khurana N, Xu X, Zhou X, Ghandehari H, Zhang J. Silica Nanoparticles Induce Ovarian Granulosa Cell Apoptosis via Activation of the PERK-ATF4-CHOP-ERO1α Pathway-Mediated IP3R1-Dependent Calcium Mobilization. Cell Biol Toxicol. 2023;39(4):1715–34. https://doi.org/10.1007/s10565-022-09776-4 . Wang J, Li Y, Duan J, Yang M, Yu Y, Feng L, Yang X, Zhou X, Zhao Z, Sun Z. Silica Nanoparticles Induce Autophagosome Accumulation via Activation of the EIF2AK3 and ATF6 UPR Pathways in Hepatocytes. Autophagy. 2018;14(7):1185–200. https://doi.org/10.1080/15548627.2018.1458174 . Senft D, Ronai ZA, UPR. Autophagy, and Mitochondria Crosstalk Underlies the ER Stress Response. Trends Biochem Sci. 2015;40(3):141–8. https://doi.org/10.1016/j.tibs.2015.01.002 . Chen F, Jin J, Hu J, Wang Y, Ma Z, Zhang J. Endoplasmic Reticulum Stress Cooperates in Silica Nanoparticles-Induced Macrophage Apoptosis via Activation of CHOP-Mediated Apoptotic Signaling Pathway. IJMS 2019, 20 (23), 5846. https://doi.org/10.3390/ijms20235846 Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL, Gomez GA, Holley CL, Bierschenk D, Stacey KJ, Yap AS, Bezbradica JS, Schroder K. Caspase-1 Self-Cleavage Is an Intrinsic Mechanism to Terminate Inflammasome Activity. J Exp Med. 2018;215(3):827–40. https://doi.org/10.1084/jem.20172222 . Wang K, Sun Q, Zhong X, Zeng M, Zeng H, Shi X, Li Z, Wang Y, Zhao Q, Shao F, Ding J. Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis. Cell. 2020;180(5):941–e95520. https://doi.org/10.1016/j.cell.2020.02.002 . Zhang Q, Xin M, Yang S, Wu Q, Xiang X, Wang T, Zhong W, Helder MN, Jaspers RT, Pathak JL, Xiao Y. Silica Nanocarrier-Mediated Intracellular Delivery of Rapamycin Promotes Autophagy-Mediated M2 Macrophage Polarization to Regulate Bone Regeneration. Mater Today Bio. 2023;20:100623. https://doi.org/10.1016/j.mtbio.2023.100623 . Ma Y, Liang Q, Wang F, Yan K, Sun M, Lin L, Li T, Duan J, Sun Z. Silica Nanoparticles Induce Pulmonary Autophagy Dysfunction and Epithelial-to-Mesenchymal Transition via P62/NF-κB Signaling Pathway. Ecotoxicol Environ Saf. 2022;232. https://doi.org/10.1016/j.ecoenv.2022.113303 . Wang M, Li J, Dong S, Cai X, Cai X, Simaiti A, Yang X, Zhu X, Zhu X, Luo J, Jiang L-H, Jiang L-H, Du B, Yu P, Yang W. Silica Nanoparticles Induce Lung Inflammation in Mice via ROS/PARP/TRPM2 Signaling-Mediated Lysosome Impairment and Autophagy Dysfunction. Part Fibre Toxicol. 2020;17(1). https://doi.org/10.1186/s12989-020-00353-3 . Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6208873","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427728251,"identity":"23631d99-ddce-40cf-a5c4-d9006b409ff4","order_by":0,"name":"Jia-Qi Ban","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Qi","middleName":"","lastName":"Ban","suffix":""},{"id":427728252,"identity":"fb0bbf06-6d7b-4217-9d0c-3f039674ab0d","order_by":1,"name":"Li-Hong Ao","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li-Hong","middleName":"","lastName":"Ao","suffix":""},{"id":427728253,"identity":"948bd822-fe59-4a61-ad18-5cfd5cacb4e7","order_by":2,"name":"He-Qun Gu","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"He-Qun","middleName":"","lastName":"Gu","suffix":""},{"id":427728254,"identity":"1258dd9f-ff8d-4c66-82f3-2d2d65cad754","order_by":3,"name":"Yun-Geng Wei","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yun-Geng","middleName":"","lastName":"Wei","suffix":""},{"id":427728255,"identity":"40ae280e-f087-4e47-b161-8013efac5b7e","order_by":4,"name":"Qiong Tian","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Tian","suffix":""},{"id":427728256,"identity":"d4047084-49d0-4daa-a154-f90b2a51e5a7","order_by":5,"name":"Xiu He","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiu","middleName":"","lastName":"He","suffix":""},{"id":427728257,"identity":"dc4ccafe-0c1c-4a83-b149-2a706c3ed78f","order_by":6,"name":"Hua Zhao","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Zhao","suffix":""},{"id":427728258,"identity":"8672d585-5ae3-474d-840e-50cc1d1696b9","order_by":7,"name":"Jun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYLCCBzw2PPz8DaRoSeBJk5GccYAkLQyHbQwaEohULR+Rfk0iQeY8jwHDAcYPH3OI0GJ4I6dMIoHnNo85cwOz5MxtxGiZkZN2A6TFsuEAGzMvCVrO8RgcSCBSi7xE+jGglgMkaDHgecP+I4EnmUdyxsFm4vwi357+2OBjj509P3/zwQ8fibLlQo4BA2MPiMnYQIR6kC39xx8wMPwgTvEoGAWjYBSMUAAAwgQ4Vp0d7jYAAAAASUVORK5CYII=","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-03-12 05:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6208873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6208873/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78501177,"identity":"0d742f8f-8fe4-4612-9a02-c484a71640da","added_by":"auto","created_at":"2025-03-14 06:36:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":804335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterisation and distribution of silica nanoparticles.\u003c/strong\u003e (A, B) Transmission electron microscopy images showing the diameter and morphology of SiNPs, with scale bars of 200 nm and 100 nm. (C, D) Hydrodynamic size and zeta potential of SiNPs in saline: mean diameter was 86.38 ± 0.82 nm and mean potential was -25.64 ± 0.065 mV, expressed as mean ± standard deviation. (E) Detection of SiNPs in lung tissues using transmission electron microscopy, with scale bars of 2 μm and 500 nm; black arrows indicate SiNPs, autophagic vacuoles (AV), mitochondrial vacuolisation (Mt), and SiNPs in lysosomes (Ly). (F) In vivo imaging of SiNPs distribution in mice.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/cfb745d7cf0cf0de68c5a0e0.png"},{"id":78501558,"identity":"395286a4-8bca-473c-baaa-3a2b8f631cbe","added_by":"auto","created_at":"2025-03-14 06:44:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":942415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms of toxic effects of silica nanoparticles in lung tissue. \u003c/strong\u003e(A) SiNPs distribution in mouse lungs revealed through in vivo imaging. (B) Pathological results of H\u0026amp;E and MASSON staining of mouse lung tissues, at ×200 magnification; scale bar = 100 μm. (C) Protein bands in lung tissues related to fibrosis and corresponding quantitative graph. (D) Protein bands linked to mitochondrial damage in lung tissues and quantitative graph. (E) Endoplasmic reticulum stress indicated in lung tissues with a quantitative graph. (F) Protein bands related to apoptosis in lung tissues and quantitative plots. (G) Protein bands associated with pyroptosis in lung tissues and quantitative plots. (H) Protein bands pertinent to autophagy in lung tissues and quantitative plots. (Data from at least five independent experiments. Statistical significance: * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns indicates no significant difference.)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/4d6bb5ce819f13c344b37df3.png"},{"id":78501559,"identity":"8c34a6b7-d42c-47e5-ac3c-56ba336c6f54","added_by":"auto","created_at":"2025-03-14 06:44:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1033429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms of toxic effects of silica nanoparticles in cardiac tissue. \u003c/strong\u003e(A) In vivo imaging of SiNPs distribution in the heart of mice. (B) H\u0026amp;E and Masson staining results of mouse heart tissue; H\u0026amp;E magnification ×200, scale bar = 100 μm; Masson magnification ×400, scale bar = 50 μm. (C) Protein bands and quantitative graphs for fibrosis-associated indicators in cardiac tissues. (D)Protein bands and quantitative graphs for mitochondrial damage-associated indicators in cardiac tissues. (E)Protein bands and quantitative graphs for endoplasmic reticulum stress-associated indicators in cardiac tissues. (F) Protein bands and quantitative plots for apoptosis-related indicators. (G) Protein bands and quantitative plots for pyroptosis-related indicators. (H) Protein bands and quantitative plots for autophagy-related indicators. (Data from at least five independent experiments; significance levels: *\u003cem\u003e p\u003c/em\u003e \u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001, ns indicates no significant difference.)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/2cf0f93fbace1f295a7f471d.png"},{"id":78501560,"identity":"451e47fe-971c-478f-be54-bcbf2e99577f","added_by":"auto","created_at":"2025-03-14 06:44:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1028949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism of toxic effects of silica nanoparticles in liver tissue. \u003c/strong\u003e(A) In vivo imaging of SiNPs distribution in the liver. (B)H\u0026amp;E and MASSON staining results of mouse liver tissues (magnification ×400; scale bar = 50 μm). (C)Protein bands and quantitative graphs of fibrosis-related indexes in liver tissues. (D)Protein bands and quantitative graphs of mitochondrial damage-related indexes in liver tissues. (E)Protein bands and quantitative plots of endoplasmic reticulum stress-related indexes in liver tissues. (F)Protein bands and quantitative plots of apoptosis-related indicators in liver tissue. (G)Protein bands and quantitative plots of pyroptosis-related indicators in liver tissue. (H)Protein bands and quantitative plots of autophagy-related indicators in liver tissue. (Data from at least five independent experiments; statistical significance: * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.0001, ns indicates no significant difference).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/4238797388845f69d527747c.png"},{"id":78501180,"identity":"5dcf8d77-187c-44e3-b836-bd05e5cbd167","added_by":"auto","created_at":"2025-03-14 06:36:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1058366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms of toxic effects of silica nanoparticles in renal tissues. \u003c/strong\u003e(A) Distribution of SiNPs in the kidney observed through in vivo imaging of mice. (B) H\u0026amp;E and Masson staining results of mouse kidney tissues at 400x magnification; scale bar = 50 μm. (C)Protein bands and quantitative graphs of fibrosis indicators in renal tissues. (D)Protein bands and quantitative graphs of mitochondrial damage indicators in renal tissues. (E)Protein bands and quantitative graphs of endoplasmic reticulum stress indicators in renal tissues. (F)Protein bands and quantitative graphs of apoptosis indicators in renal tissues. (G)Protein bands and quantitative graphs of pyroptosis indicators in renal tissues. (H)Protein bands and quantitative graphs of autophagy indicators in renal tissues. (Data from at least five independent experiments; statistical significance is denoted as * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.0001, ns indicates no significant difference).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/817371d61c89917ec277b4df.png"},{"id":87822472,"identity":"49c1b8c6-9b0a-4a58-b2ea-c675bf43cb9c","added_by":"auto","created_at":"2025-07-29 11:17:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5989387,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/403fa218-0ce7-429b-b5db-660e3ad9caaf.pdf"},{"id":78501172,"identity":"e7c75e4c-90a3-459f-9c7c-989601abe7d4","added_by":"auto","created_at":"2025-03-14 06:36:36","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1061551,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-6208873/v1/0aec1c6697bb67d0c5f4bff1.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preliminary investigation on the mechanisms of multi-organ toxicity induced by prolonged inhalation exposure to silica nanoparticles","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eCross-organ toxicity of SiNPs was demonstrated for the first time by tracheal perfusion, revealing a synchronised progression of injury in the lung-cardiac-hepatic-renal system.\u003c/li\u003e\n \u003cli\u003eMulti-mechanism decoding identified mitochondrial damage, ER stress, apoptosis, pyroptosis, and autophagy crosstalk as core drivers of SiNPs-induced lesions.\u003c/li\u003e\n \u003cli\u003eLong-term exposure modeling establishes tracheal perfusion as a reliable approach to simulate population-level exposure and nanoparticle deposition patterns.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eNanotechnology has rapidly advanced in recent years, with applications in environmental governance, healthcare, precision agriculture and food engineering \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As the production and use of nanomaterials grow, the risk of human exposure to silica nanoparticles (SiNPs) is increasing. However, research on the chronic toxic effects of long-term exposure to SiNPs remains limited. Emerging toxicological evidence reveals that pulmonary inhalation constitutes the predominant exposure pathway for SiNPs, not only triggering inflammatory cascades and aberrant proliferation within alveolar epithelium but also facilitating particle translocation into systemic circulation via compromising the integrity of the blood- lung barrier \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and potentially reach the heart, liver, kidneys, and other organs. Yet, the specific mechanisms underlying systemic chronic toxic responses from respiratory exposure to SiNPs are not well understood, and comprehensive knowledge of their body distribution and multi-organ effects is lacking\u003c/p\u003e \u003cp\u003eNanoparticles can cross biological barriers and enter physiological systems \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. SiNPs enter the body through the respiratory tract, making the lungs a primary target. Exposure to SiNPs induces local and systemic inflammation in the lungs \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. After crossing the blood-gas barrier, the heart becomes the next target organ \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The liver, the largest solid organ, is the main site of SiNPs accumulation, regardless of exposure route, as it processes most toxins \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The kidneys, as the principal metabolic organ, are responsible for excreting nanomaterials \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Therefore, studying the toxic effects and pathogenesis of SiNPs exposure on the lungs, heart, liver, and kidneys is crucial for assessing the risks associated with human exposure to SiNPs.\u003c/p\u003e \u003cp\u003eRecent studies have shown that long-term exposure to inhaled SiNPs activates inflammatory responses not only in the lungs but also in the heart, liver, and kidneys, leading to tissue fibrosis in mice \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The mechanisms behind this multi-organ fibrosis require further investigation. Current research identifies several pathways contributing to organ fibrosis. Notably, SiNPs primarily invade mitochondria, damaging their structure and function across various cell types \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, which results in excessive reactive oxygen species (ROS) generation and mitochondrial dysfunction \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This dysfunction disrupts cellular energy metabolism and can trigger endoplasmic reticulum (ER) stress, exacerbating cellular damage \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. ER stress activates downstream signaling pathways through the unfolded protein response (UPR) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and may induce autophagy \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Excessive autophagy induction is emerging as a potential mechanism of SiNPs toxicity, contributing to disease pathogenesis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Chronic SiNPs exposure can inhibit autophagosome degradation by blocking autophagic flux, disrupting cellular homeostasis \u003csup\u003e15 16\u003c/sup\u003e. Studies indicate that ER stress synergistically promotes SiNPs-induced apoptosis, particularly in human alveolar epithelial cells \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. ER stress, an internal apoptotic pathway \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, interacts with other pathways like the caspase pathway and is commonly associated with fibrosis-related diseases \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Furthermore, SiNPs may induce pyroptosis by activating NLRP3 inflammatory vesicles \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, resulting in a cell death mode characterized by intense inflammation \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Processes such as apoptosis, pyroptosis, and autophagy can overwhelm the system when large numbers of cells die suddenly, as seen in infections, chronic inflammation, and tissue damage. This sudden cell death leads to the massive release of cellular contents, known as danger-associated molecular patterns (DAMPs) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, which trigger a robust immune response to recruit phagocytes and promote tissue repair. In contrast, during chronic inflammation, myofibroblasts often avoid cell death, leading to aberrant wound healing and excessive extracellular matrix production, thus driving fibrosis \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Further experiments are needed to determine whether mitochondrial damage \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, ER stress \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, apoptosis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, pyroptosis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and autophagy \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e are also involved in the multiorgan fibrosis induced by SiNPs exposure.\u003c/p\u003e \u003cp\u003eWhile the single organ toxicity of SiNPs has been studied, the mechanisms underlying their multi-organ toxicity, particularly regarding fibrosis induced by mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, remain inadequately explored. This study aims to systematically evaluate the distribution and damaging effects of SiNPs in the lungs, heart, liver, and kidneys through tracheal drip injections of varied SiNPs concentrations. We will assess the expression of marker proteins related to mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, autophagy, and fibrosis to investigate the potential mechanisms of the toxic effects of SiNPs on these organs following respiratory exposure.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation and characterisation of SiNPs particles\u003c/h2\u003e \u003cp\u003eTEM was used to image a 1.0 mg/mL suspension of SiNPs (Nanocomposix) that was dried at 20\u0026deg;C Average particle size was calculated using ImageJ software. Mean particle size and zeta potential of SiNPs in saline were determined after 1, 2, 4, 8 and 24 h at 20\u0026deg;C using a zeta potential particle size analyser (Malvern, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animal experimental design\u003c/h2\u003e \u003cp\u003eFifty-two SPF (Specific pathogen free) grade, 7\u0026ndash;8 week old, healthy male C57BL/6J mice were randomly divided into 4 groups: control (saline) and low, medium and high dose groups (3, 6 and 12 mg/kg-bw), and SiNPs suspension was administered by intratracheal drip under anaesthesia (chlorpromazine) once per weekday for a total of 12 times. SiNPs Dose conversions were performed according to occupational exposure limits for hazardous substances in the workplace (5 mg/m\u003csup\u003e3\u003c/sup\u003e, GBZ 2.1\u0026ndash;2019) and respiratory physiological parameters of mice \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. All animal experimental operations were reviewed and approved through strict laboratory animal ethics (Animal Ethics Approval Number: 2400643).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Histopathological examination\u003c/h2\u003e \u003cp\u003eHistopathological analysis was performed according to standard protocols. Briefly, tissue blocks of major organs i.e. lungs, heart, liver and kidneys were fixed in 10% neutral buffered formalin, routinely dehydrated and degreased and then embedded in paraffin blocks. The 5 \u0026micro;m thick paraffin sections were sliced and fixed on slides. Afterwards, tissue sections were stained with hematoxylin-eosin (H\u0026amp;E; China) for histopathological lesion observation or Masson trichrome staining (Masson; China) for pathological fibrosis assessment. Finally, the slides were scanned with a tissue multilabel panoramic imaging analysis system (Nanozoomer S60; Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 TEM Observations of Tracheal Drops of SiNPs into the Lungs\u003c/h2\u003e \u003cp\u003e2.5% glutaraldehyde was prefixed and 1% osmium tetroxide was refixed. The samples were then rinsed three times with 0.1 MPB and dehydrated with 30%, 50%, 70%, 80%, 90%, and 100% alcohol and 100% acetone successively, after which the dehydrating agent and Epon-812 embedding agent, in the ratio of 3:1, 1:1, and 1:3, respectively, were sequentially infiltrated, and the samples were embedded with Epon-812 Pure Embedding Agent in order to create a block of cells or tissues. Ultrathin sections (60\u0026ndash;90 nm) were obtained by an ultrathin slicer (Ultracut UCT, Leica, Germany). They were then stained with lead citrate and uranyl acetate and examined by TEM (JEM-1400FLASH, JEOL, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Small animal live imaging\u003c/h2\u003e \u003cp\u003eThe mice were randomly divided into experimental and control groups with at least 3 mice in each group. Mice in the experimental group were given SiNPs with fluorescent labelling (FITC) (at a dose of 6 mg/kg bw) by tracheal drip (zhongkekeyou, China), and the control group was injected with an equal volume of saline. Mice were anaesthetised using isoflurane, placed in an anaesthesia induction chamber, and transferred to the imager platform after the mice were fully anaesthetised. Turn on the IVIS Lumina Ⅲ imaging system, warm up the instrument and set the imaging parameters, select the wavelength of 440\u0026ndash;520 nm according to the fluorescent markers, fix the mice in the prone position on the imaging platform, make sure that their breathing was stable, and select the A field of view. Whole-body imaging was performed 24 hours after administration. Mice were ensured to be under stable anaesthesia before each imaging session. At the end of imaging, mice were executed and major organs (e.g., lungs, heart, liver, spleen, kidneys) were dissected. The organs were placed on the imaging platform and ex vivo imaging was performed using the same parameters to further verify the distribution of nanoparticles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. protein blotting assay\u003c/h2\u003e \u003cp\u003eWestern blot determination of lung, heart, liver, and kidney tissue proteins were extracted by Protein Extraction Kit (Solarbio, China), and concentrations were measured by BCA protein assay (Solarbio, China). Primary antibodies included α-SMA, Caspase 1/P20/P10, Beclin1, Bcl2, Parkin, OPA1 antibody (1:1000, Proteintech, China), ATF6 (1:2000, Proteintech, China), VIM, LC3A/B, caspase-3, PERK (1:1000, CST, USA), P62, PINK1 (1:10,000, abcam, UK), IL-1β, XBP1, IRE1α antibodies (1:1000, Boster, China), GSDMD antibody (1:1000, Bioss, China), MFN1 (1:500, PTMAL, China), the NLRP3 (1:500, abclonal, China), collagen1 (1:500, WANLEI, China) were diluted with BSA. After sealing with 5% skimmed milk, the polyvinylidene difluoride (PVDF) membrane was incubated overnight to detect proteins. The membranes were then incubated with the corresponding HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies for 1 h at room temperature. Finally, the membranes were reacted with a chemiluminescent detection system (ECL Detection Kit) (Smart-Lifesciences, China) and visualised under a gel imaging system (Bio-Rad), with GAPDH as an internal control and quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistics\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analyses were performed using GraphPad Prism 5.0 software. One-way analysis of variance (ANOVA) was used to compare the data between the control and model groups. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of silica nanoparticles and their in vivo distribution\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B, transmission electron microscopy revealed that SiNPs have a diameter of approximately 80 nm, with a good dispersion and nearly spherical shape. In saline, the hydrodynamic size and zeta potential of SiNPs were measured at about 86.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 nm and \u0026minus;\u0026thinsp;25.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065 mV, respectively, indicating their stability and dispersion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). Electron microscopy of mouse lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) revealed a significant presence of SiNPs in the SiNPs group, accompanied by autophagic vacuoles and mitochondrial damage. Additionally, using fluorescently labeled SiNPs, we found that they were widely distributed in the lungs, heart, liver, and kidneys.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Mechanisms of silica nanoparticle toxicity in lung tissue\u003c/h2\u003e \u003cp\u003eTo investigate the distribution of SiNPs in lung tissues, we conducted in vivo imaging on the lung tissues of mice exposed to fluorescently labeled SiNPs through the respiratory tract and confirmed their presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). H\u0026amp;E and MASSON staining revealed varying effects across dose groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB): at low doses, peripheral alveoli were fused and dilated (blue arrows). At mid-dose, alveolar walls thickened, some alveoli atrophied and collapsed (green stars), and perivascular fibroblastic hyperplasia was observed (green arrows). At high doses, similar fusion and dilation were noted with some atrophied alveoli. MASSON staining indicated a gradual increase in collagen fibers, most pronounced in the mid-dose group. Western blot analysis of Col-1, α-SMA, and VIM protein levels confirmed elevated expression across all groups, with the mid-dose group showing the most fibrosis, supporting our pathological findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Mitochondrial damage assessment revealed increased expression of MFN1 and decreased expression of OPA1, PINK1, and Parkin, particularly in the mid-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). ER stress markers ATF6, PERK, IRE1α, and XBP1s were elevated in all groups, with the mid-dose group showing the most significant increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We examined cell death markers and found decreased Bcl2 and increased IL-1β and Caspase-3 across all groups, especially at mid-dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Additionally, NLRP3, GSDMD-N, and Caspase-1 expressions increased in each group, more distinctly in the mid-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). P62 and Beclin1 expressions rose across groups, while LC3 II/I levels decreased, again most noticeably in the mid-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Collectively, these results indicate that long-term exposure to SiNPs through the respiratory tract leads to their distribution in lung tissues, resulting in fibrosis, mitochondrial damage, heightened endoplasmic reticulum stress, increased apoptosis, focal death, and impaired autophagic flow.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Mechanisms of silica nanoparticle toxicity in cardiac tissue\u003c/h2\u003e \u003cp\u003eTo investigate the distribution of SiNPs in cardiac tissues, we performed in vivo imaging on cardiac tissues from mice exposed to fluorescently labeled SiNPs via the respiratory tract, confirming their presence in the heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Hearts from all groups were analyzed using H\u0026amp;E staining to assess pathological changes, and collagen deposition was evaluated with Masson staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). H\u0026amp;E staining revealed well-defined and aligned cardiomyocytes in the control group, while the low-dose group exhibited fiber deformation. The medium-dose group displayed significant histological abnormalities, including myocardial fiber destruction (black arrows), and the high-dose group showed signs of edema, irregular staining of myocardial fibers, and abnormal interstitial space distribution (yellow \u0026amp;). Masson staining indicated that collagen fibers (blue) were most pronounced in the medium-dose group, where fibrosis was further confirmed through Western blot analysis of Col-1, α-SMA, and VIM protein expression, showing significant elevation in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To evaluate mitochondrial damage, we examined related proteins and found increased expression of MFN1 across all groups, with decreased levels of OPA1, PINK1, and Parkin, particularly notable in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Endoplasmic reticulum stress was indicated by elevated levels of ATF6, PERK, IRE1α, and XBP1s in all groups, most prominently in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We also assessed marker proteins for apoptosis, necrosis, and autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Bcl2 expression decreased while IL-1β and Caspase-3 levels increased in all groups, especially in the medium-dose group. Enhanced expression of NLRP3, GSDMD-N, and Caspase-1 was noted in all groups, with the most significant increase in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Additionally, levels of P62 and Beclin1 increased across all groups, while LC3 II/I expression decreased, most notably in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These results suggest that chronic exposure to SiNPs via the respiratory tract results in their distribution in cardiac tissues, leading to fibrosis, mitochondrial damage, heightened endoplasmic reticulum stress, increased apoptosis, necrosis, and impaired autophagic flow.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Mechanisms of silica nanoparticle toxicity in liver tissue\u003c/h2\u003e \u003cp\u003eWe analyzed the distribution of SiNPs in liver tissues of mice exposed via the respiratory tract using in vivo imaging, confirming their presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Histological analysis with H\u0026amp;E and Masson staining revealed normal hepatocyte morphology in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In the low-dose group, liver lobule structure was disrupted (orange arrows); the medium-dose group exhibited disordered lobule formation with obvious pseudolobules (green triangles), while the high-dose group showed significant inflammatory cell infiltration (blue stars). Masson staining indicated high collagen fiber expression in the medium and high-dose groups. To assess liver fibrosis, we measured Col-1, α-SMA, and VIM protein levels via Western blot and found elevated expression across all groups, most notably in the medium-dose group, aligning with pathological findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To investigate mitochondrial damage, we analyzed related proteins and found increased MFN1 levels, alongside decreased expression of OPA1, PINK1, and Parkin in all groups, with the most significant changes in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We also examined endoplasmic reticulum stress markers and noted elevated levels of ATF6, PERK, IRE1α, and XBP1s across all groups, particularly pronounced in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Examining markers of cell death, we observed decreased Bcl2 and increased levels of IL-1β and Caspase-3 in all groups, again more evident in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Additionally, markers of pyroptosis, NLRP3, GSDMD-N, and Caspase-1, were increased in all groups, especially in the medium-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). For autophagy, we found that P62 and Beclin1 expression rose while LC3 II/I decreased across all groups, with the medium-dose group showing the most significant alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These findings suggest that long-term exposure to SiNPs via the respiratory tract leads to their distribution in liver tissues, resulting in fibrosis, mitochondrial damage, increased endoplasmic reticulum stress, apoptosis, pyroptosis, and impaired autophagic flow.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Mechanisms of silica nanoparticle toxicity in renal tissues\u003c/h2\u003e \u003cp\u003eTo assess the distribution of SiNPs in renal tissues, we conducted in vivo imaging on renal tissues from mice exposed to fluorescently labeled SiNPs via the respiratory tract and confirmed their presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Kidney tissues underwent H\u0026amp;E and MASSON staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The low dose showed interstitial edema (purple #) and inflammatory cell infiltration (blue star), while the medium dose displayed thickening of the glomerular capsule wall and matrix accumulation (green arrow). The high dose group exhibited solidly transformed glomeruli (blue arrows). MASSON staining revealed blue collagen matrix deposition in glomeruli and tubules, with the most severe fibrosis noted in the medium dose group. To confirm fibrosis in renal tissues, we analyzed the expression of Col-1, α-SMA, and VIM proteins via Western blotting, finding elevated levels across all groups, particularly pronounced in the medium dose group, consistent with histopathological findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To evaluate mitochondrial damage, we examined mitochondrial proteins and observed increased MFN1 expression and decreased levels of OPA1, PINK1, and Parkin in all groups, with the most significant changes in the medium dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Assessment of endoplasmic reticulum stress showed an increase in ATF6, PERK, IRE1α, and XBP1s in all groups, especially evident in the medium dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Given the recent interest in cell death mechanisms, we investigated marker proteins for apoptosis, pyroptosis, and autophagy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, Bcl2 levels decreased, while IL-1β and Caspase-3 levels increased in all groups, particularly in the medium dose group. Similarly, NLRP3, GSDMD-N, and Caspase-1 levels rose across all groups, with the most evident changes in the medium dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH illustrates an increase in P62 and Beclin1 levels in all groups, alongside a decrease in LC3 II/I ratios, also more pronounced in the medium dose group. Overall, these findings indicate that prolonged exposure to SiNPs via the respiratory tract leads to their distribution in renal tissues, resulting in fibrosis, mitochondrial damage, elevated endoplasmic reticulum stress, apoptosis, increased focal death, and impaired autophagic flow.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRecent advancements in nanotechnology have heightened interest in the biosafety of various nanomaterials \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. SiNPs are widely utilized due to their unique physicochemical properties, yet their potential toxicity and molecular mechanisms remain poorly understood. This study systematically assessed the distribution and harmful effects of SiNPs in the lungs, heart, liver, and kidneys through tracheal drip injection. Findings indicate that SiNPs exposure activates several cell death pathways, including mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, which may lead to chronic inflammation and fibrosis. The lungs emerged as the primary target organ, displaying significant fibrotic changes, while the heart, liver, and kidneys also exhibited varying degrees of fibrosis.\u003c/p\u003e \u003cp\u003eThis study uniquely demonstrates that SiNPs damage multiple pathways, including mitochondrial dysfunction, endoplasmic reticulum (ER) stress, apoptosis, pyroptosis, and autophagy, potentially leading to multi-organ fibrosis. Mitochondria, crucial for oxidative metabolism and biomolecule synthesis, are particularly vulnerable during pathological processes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that SiNPs exposure increases MFN1 expression while decreasing OPA1, PINK1, and Parkin levels, suggesting an imbalance in mitochondrial dynamics and impaired autophagy. The upregulation of MFN1 indicates enhanced mitochondrial fusion as a cellular adaptive response to damage, whereas the downregulation of OPA1 points to compromised inner membrane fusion, contributing to mitochondrial dysfunction. Additionally, reduced PINK1 and Parkin levels suggest impaired initiation of autophagy, preventing the efficient removal of damaged mitochondria. These results imply that SiNPs disrupt mitochondrial quality control and dynamics, aligning with previous mitochondrial damage findings \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Regarding ER stress, the study reveals increased expression of the three main stress markers (PERK, ATF6, and IRE1α), highlighting their role in SiNPs-induced multi-organ injury. While research on SiNPs and ER stress is limited, our results corroborate earlier studies \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. All three pathways activate the transcription of chaperones and proteins involved in redox homeostasis, protein secretion, or cell death \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSiNPs exposure also resulted in decreased Bcl2 expression, an anti-apoptotic protein, leading to reduced mitochondrial membrane potential and cytochrome C release, which activates Caspase-9 and Caspase-3, inducing apoptosis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Increased IL-1β and Caspase-3 expression further indicate that SiNPs trigger apoptosis and inflammatory responses via the mitochondria-dependent apoptotic pathway, consistent with prior in vitro studies \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and the present results further support the conclusion that SiNPs induce apoptosis. The elevated IL-1β levels suggest that SiNPs not only induce apoptosis but also activate inflammation, a potential driver of fibrosis. Cellular focal death is triggered by the activation of inflammatory vesicle sensors. In the classical pathway, the NLRP3 inflammasome serves as a platform to activate the protein hydrolase caspase-1, which further processes caspase-1 into its p33/p10 form \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Activated caspase-1 then activates GSDMD, allowing it to oligomerize and insert into the plasma membrane, creating pores that destabilize the membrane and lead to cell lysis, releasing DAMPs. Additionally, activated caspase-1 is crucial for converting precursors of IL-1β into active 17KDa cytokines \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This study found increased expression of NLRP3, GSDMD-N, and caspase-1 across all groups, suggesting that SiNPs induce multi-organ cellular pyroptosis, aligning with previous findings on cardiotoxicity \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Autophagy is a key catabolic process that transports proteins, cytoplasmic components, and organelles to the lysosome for degradation and recycling \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our study found a significant increase in P62 and Beclin1 expression, along with a decrease in the LC3-II/LC3-I ratio in mouse organs. The rise in P62 and reduction in the LC3-II/LC3-I ratio indicate that autophagic flow may be inhibited, resulting in inadequate degradation of autophagic substrates. The increase in Beclin1 suggests enhanced autophagic initiation or accumulation of autophagosomes. Therefore, SiNPs may contribute to the development of pulmonary fibrosis by disrupting autophagic flow. Internalized particles are recognized by cells as foreign, activating autophagy. Electron microscopy revealed that SiNPs primarily localized within the cytoplasm, lysosomes, and autophagic vesicles. However, excessive uptake of SiNPs could overwhelm autophagy's degradation capacity, blocking the process. Previous studies, mainly focusing on early autophagy activation, contrast with our findings \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our findings indicate a late-stage blockage, which is speculated that SiNPs may impair lysosomal function, preventing autophagosome-lysosome fusion, or activate the UPR through endoplasmic reticulum stress, increasing autophagosome formation but disrupting degradation. They may also induce mitochondrial damage, leading to elevated ROS production and further autophagy dysfunction. This area will be explored further in future research.\u003c/p\u003e \u003cp\u003eThis study's limitations include differences between animal models and human exposure, as well as the potential for optimizing dose selection. We found that SiNPs-induced fibrosis and protein damage peaked at the medium dose (6 mg/kg-bw) but decreased at the high dose (12 mg/kg-bw). While most current research has focused on short-term high doses, our study examined long-term low doses, and other short-term studies reported significant damage responses at the high dose (12 mg/kg-bw) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Short-term high-dose exposure (12 mg/kg-bw) to SiNPs can cause rapid cellular damage and inflammatory responses, significantly increasing damage protein indicators. In contrast, long-term low to medium-dose exposure may lead to gradual fibrosis and damage due to chronic toxicity. Interestingly, the high-dose group might activate a protective mechanism in response to excessive damage, resulting in decreased protein expression. This could indicate an atypical dose-effect relationship due to inappropriate dosing, leading to cellular or tissue intolerance. Alternatively, SiNPs toxicity may not follow a typical dose-response pattern, with toxic effects emerging only after reaching a certain dose level. This suggests that controlling SiNPs doses within a specific range could minimize their toxic side effects. However, further experimental and epidemiological data are needed to support this finding. Future studies will include more intermediate doses to better determine the toxic dose threshold for SiNPs. Our current research has concentrated on a single mechanism of SiNPs-induced mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, but the interactions and synergistic effects among these mechanisms require further investigation. Future work will delve into these synergistic effects to elucidate their complex biological impacts.\u003c/p\u003e \u003cp\u003eIn conclusion, this study aims to systematically investigate the distribution and damaging effects of SiNPs in the lungs, heart, liver, and kidneys after tracheal drip administration, expanding on previous research that primarily examined damage to individual organs. We will also delve into the mechanisms of mitochondrial damage, ER stress, apoptosis, pyroptosis, and autophagy, hypothesizing their roles in fibrosis. This comprehensive exploration of the complex cellular effects and interrelations prompted by SiNPs surpasses the analysis of a single mechanism. By elucidating the multi-organ toxicity of SiNPs, our research will contribute valuable experimental data for the safety assessment of nanomaterials and provide a theoretical foundation for future intervention strategies.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study found that chronic respiratory exposure to SiNPs can damage multiple organs, including the heart, lungs, liver, and kidneys. It identified potential mechanisms of toxicity, including mitochondrial damage, endoplasmic reticulum stress, apoptosis, pyroptosis, and autophagy, providing a crucial basis for safety assessments and future intervention strategies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare they have no confict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by Medical Research Joint Fund for High Quality Development of health in Guizhou Province (grant number 2024GZYXKYJJXM0013).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiaqi Ban：Conceptualization. Lihong Ao、He-Qun Gu: validation. Qiong Tian：subsidiary. Jiaqi Ban, Jun Li, Yungeng Wei, Xiu He, Hua Zhao: writing-review and editing, supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMurugadoss S, Lison D, Godderis L, Van Den Brule S, Mast J, Brassinne F, Sebaihi N, Hoet PH. Toxicology of Silica Nanoparticles: An Update. Arch Toxicol. 2017;91(9):2967\u0026ndash;3010. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00204-017-1993-y\u003c/span\u003e\u003cspan address=\"10.1007/s00204-017-1993-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu H, Zhu Y, Zhu L, Wang D, Lv S, Li X, Guo C, Li Y. 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Part Fibre Toxicol. 2020;17(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12989-020-00353-3\u003c/span\u003e\u003cspan address=\"10.1186/s12989-020-00353-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SiNPs, Fibrosis, Multiorgan, Respiratory exposure, Toxic mechanisms of action","lastPublishedDoi":"10.21203/rs.3.rs-6208873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6208873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increasing use of silica nanoparticles (SiNPs) has raised concerns about their biotoxicity. Since respiratory exposure is the primary route of human exposure to SiNPs, this study systematically investigated their distribution and damaging effects in the lungs, heart, liver, and kidneys following tracheal drip injection. The results demonstrated that SiNPs distribute across these organs and induce mitochondrial damage, endoplasmic reticulum stress, and activate cell death pathways, including apoptosis, pyroptosis, and autophagy. The most significant damage occurred in the middle-dose group (6 mg/kg). The lungs, as the primary target organ, exhibited pronounced fibrotic changes, while fibrotic lesions were also observed in the heart, liver, and kidneys to varying degrees. These findings suggest that the observed injury mechanisms may collectively contribute to chronic inflammation and promote fibrosis. This study provides critical insights into the multi-organ toxicity of SiNPs, offering a foundation for their safety assessment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Preliminary investigation on the mechanisms of multi-organ toxicity induced by prolonged inhalation exposure to silica nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-14 06:36:32","doi":"10.21203/rs.3.rs-6208873/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa4e671e-46e1-4726-a68b-363544e438f9","owner":[],"postedDate":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-29T11:08:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-14 06:36:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6208873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6208873","identity":"rs-6208873","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}