Study On Cardiovascular Toxicity Of Silica Nanoparticles And Related Mechanisms In Rats After Intratracheal Instillation

Silica nanoparticle is currently the most commonly used engineerednanomaterials in the world, which has been found extensive applications in material,cosmetics, biomedical and biotechnological fields. So, silica nanoparticle could easilyinto human body through inhalation, ingestion, dermal penetration and injection formedical purposes. But inhalation is considered the major route of exposure fornanoparticles. Many toxicological studies made it clear that nanoparticles of varioustypes can cause lung inflammatory responses and epithelial cell hyperplasia. Andsilica nanoparticles could pass through the alveolar-capillary barrier into systemiccirculation. But, few studies on silica nanoparticles cause biological effects of thecardiovascular system, and the mechanism of biological effects is not yet clear. In thisstudy, the male Wistar rats were intratracheally instilled with the three silicananoparticles (30,60, and90nm) and one fine silica particles (600nm) at three dosesof2,5and10(mg/Kg·bw) every other day for a total of15times. To investigate thecardiovascular toxicity of different sizes amorphous silica nanoparticles in rats afterintratracheal instillation. To provide experimental basis for the evaluation of thebiological effects of cardiovascular caused by nanomaterials.Transmission Electron Microscopy, Inductively Coupled Plasma-MassSpectrometry, the dynamic light scattering were used to characterize the differentsized silica particles. To determine whether the particles and media werecontaminated by endotoxin, the Tachypleus Amebocyte Lysate (TAL) test wasperformed. General toxicity of the Wistar rats was evaluated by body weight, heartorgan coefficient, and WBC, RBC, HGB, and PLT in peripheral blood. Distribution of silica nanoparticles in hearts and serum were measured by ICP-OES. Morphologicalchange in heart tissues were detected by HE staining. Levels of TNF-α, IL-6, IL-1β,ROS, ICAM-1, VCAM-1, ET-1, P-selectin, D-dimer, iNOS, eNOS, CK-MB, andhs-CRP were measured by ELISA according to the manufacturers’ protocols. Levelsof SOD, GSH-Px, GSH, MDA, LDH, NO, and NOS were measured by biochemicalassay. We used TUNEL method to detect the cardiomyocyte apoptosis. Expression ofTGF-β1, Bax, Bcl-2, and Caspase-3in heart tissue were determined usingimmunohistochemistry.1Characterization of silica particlesSi600, Nano-Si90, and Nano-Si60were mostly ellipsoidal particles, and uniformdistribution, exhibited very good monodispersity in the experiment, while Nano-Si30agglomerated slightly. Silica particles used in the present study had a higher purity,and the hydrodynamic sizes of four amorphous silica particles did not containendotoxin.2General toxic effects of silica nanoparticles in ratsCompared with normal control, the body weight change of different doses of foursize silica particles had a downward trend, but the heart organ coefficient had nosignificant change. Compared with the control group, there was a significant increasein WBC and PLT levels (P＜0.05). And the result showed a dose-dependent andsize-dependent manner. A significant decreases in RBC number and theconcentrations of HGB were found in Nano-Si90、Nano-Si60、Nano-Si30groups andthe high-dose of the Si600groups compared with the control group. But, nosignificant changes in the RBC number and HGB level were found in the four sizessilica particles.3Effects of silica nanoparticles on the morphology of heartIn the low-and middle-dose group of Si600, the rats cardiac muscle fibersarranged in neat rows, no fracture, normal cell gap and found no edema; but in thehigh dose group of Si600, The rat cardiomyocytes gap increases myocardial fiber fracture and a small amount of hyperplasia. In the three size silica particles group,myocardial tissue cell gap appears to increase the myocardial fiber breakage, cellboundary is unclear, the high-dose group rat myocardium visible fibrous tissuehyperplasia, myocardial cells pyknotic condensation myocardial cells stripesdisappear. The results show that the trend of increasing with decreasing silica scaleeffect dose increases, the degree of myocardial cell damage.4The change of the electron microscopy myocardial tissueThe results show that: the control rats cardiac muscle fibers arranged in neat rows, theintegrity of the mitochondrial membrane and intercalated disk, and no edema, normalendometrial crest density. The myocardial muscle silk fibers arranged in neat rows,the intercalated disc integrity, mitochondrial swelling, local cavitation, cristaedisappear in Si600exposed groups. The myocardial muscle silk fibers visible fracture,the intercalated disk dissociation, swelling of mitochondria, mitochondrialcondensation, partial cavitation, endometrial cristae disappear, increased interstitialcollagen fibers; mitochondria and endothelial higher electron density of the particlesobserved in the cytoplasm in Nano-silica exposure groups.5Oxidative stress for rats after intratracheal instillation of silica particlesCompared with the control group, the ROS levels of four sizes particles elevatedsignificantly (P＜0.05). At the same size particle groups, increasing the dose of silicaparticles, could cause significant elevation of the ROS levels (P＜0.05). All theseresults manifested that the levels of ROS in serum increased in both dose-dependentand size-dependent manner. There was a significant increase in the MDA levels in themiddle-and high-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groups, andthe high-dose groups of Si600groups compared with the control group (P＜0.05).The MDA levels in the high-dose groups of Nano-Si60and Nano-Si30groups werehigher than that in the middle and low-dose groups (P＜0.05). In the high-dose groupsof Nano-Si90and Si600groups, there was a significant increase of the MDA levelscompared with the low-dose group (P＜0.05). Compared with the control group, the SOD levels of the middle and high-dose groups of Nano-Si60, Nano-Si30groups andthe high-dose groups of Nano-Si90groups decreased significantly (P＜0.05). Therewas a significant decrease in the GSH-Px levels in the high–dose groups of four sizeparticles groups compared with the control group (P＜0.05). But there was nosignificant change in all experimental groups of four sizes particles groups.6Inflammatory reaction for rats after intratracheal instillation of silica particlesThere was a significant increase in hs-CRP, TNF-α, IL-1β and IL-6levels in allexpression groups of four sizes silica particles compared with the control group (P＜0.05). At the same size particle groups, increasing the dose of silica particles, couldcause significant elevation of the hs-CRP levels (P＜0.05). The hs-CRP levels inserum in the middle-and high-dose groups of four sizes particles were significantlyup-regulated when compared with the low-dose groups (P＜0.05), the hs-CRP levelsin serum in the high-dose groups of four sizes particles were higher than those of inthe middle-dose groups of four sizes particles (P＜0.05). And a significant increase inthe levels of TNF-α and IL-1β measured in serum were found in high-dose groups ofNano-Si90, Nano-Si60and Nano-Si30groups compared with low-dose groups (P＜0.05).But at the same dose groups, the hs-CRP levels in serum in the middle-andhigh-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groups were significantlyup-regulated when compared with those of the Si600groups (P＜0.05). A significantincrease in hs-CRP levels were found in the middle-and high-dose groups ofNano-Si30groups compared with Nano-Si90and Nano-Si60groups (P＜0.05). TheIL-1β levels in high-dose groups of Nano-Si60and Nano-Si30groups weresignificantly up-regulated compared with Si600groups (P＜0.05). The IL-6levels inall experimental groups of Nano-Si30groups, the high-and low-dose groups ofNano-Si60groups and the middle-dose groups of Nano-Si90groups weresignificantly up-regulated when compared with Si600groups (P＜0.05). 7Effects of four sizes particles after exposure on blood coagulation andendothelial dysfunction in blood in ratsThere was a significant increase in the levels of D-dimer, ET-1and P-selectin inall experimental groups of four sizes silica particles compared with the control group(P＜0.05). At the same size particle groups, the levels of D-dimer and ET-1in thehigh-dose groups of four sizes silica particles were higher than those in the middle-and low-dose groups. The levels of P-selectin in the high-dose groups of Nano-Si90,Nano-Si60, and Nano-Si30groups were higher than those in the middle-and low-dosegroups (P＜0.05). There was a significant increase in the levels of P-selectin in thehigh-dose groups of Si600compared with the low-dose groups (P＜0.05).Furthermore, the levels of D-dimer, ET-1and P-selectin of the middle-dose groups ofNano-Si90, Nano-Si60and Nano-Si30groups significantly elevated when comparedwith the low-dose groups (P＜0.05). At the same dose groups, the levels of D-dimerand ET-1in the high-dose groups of Nano-Si30groups, the middle and high-dosegroups of Nano-Si90and Nano-Si60groups and the levels of P-selectin in thehigh-dose groups of Nano-Si30groups significantly increased when compared withthe Si600groups (P＜0.05).Except in low-dose of Si600groups, there was a significant increase in the levelsof sICAM-1and sVCAM-1in all expression groups of four sizes silica particlescompared with the control groups (P＜0.05). At the same size particle group, thelevels of sICAM-1in the high-dose group of four sizes silica particles significantlyincreased (P＜0.05). There was a significant increase in the levels of sICAM-1in themiddle-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groups compared withthe low-dose groups (P＜0.05). Further, the levels of sICAM-1in the middle andhigh-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groups significantlyelevated when compared with Si600groups (P＜0.05). Compared with themiddle-dose groups, there was a significant increase in the levels of sVCAM-1in thehigh-dose groups of Nano-Si60and Nano-Si30groups (P＜0.05). However, only the levels of sVCAM-1in the high-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groups were significantly elevated when compared with the Si600groups (P＜0.05).There was a significant decrease in the levels of NO and the activity of NOS andeNOS in all experimental groups of four sizes silica particles compared with thecontrol groups (P＜0.05). But except the low-dose of four sizes particles and themiddle-dose of Si600, the activity of iNOS in the other groups were significantlyup-regulated when compared with the control groups (P＜0.05). In the same sizeparticle groups, the levels of NO in the high-dose group of four sizes silica particlesand middle-dose of Nano-Si90, Nano-Si60and Nano-Si30groups were significantlydown-regulated, when compared with the low-dose groups (P＜0.05). In the samedose groups, the levels of NO in the high and middle-dose group of Nano-Si90,Nano-Si60and Nano-Si30groups were lower than that in the Si600groups, and therewas a significant decrease in the levels of NO in the high-dose group of Nano-Si30groups compared with Nano-Si90and Nano-Si60groups (P＜0.05). In the same sizegroup, there was a significant decrease in the activity of NOS and eNOS in thehigh-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groups (P＜0.05). In thesame dose, compared with Si600groups, only the activity of NOS and eNOS in thehigh-dose groups of Nano-Si60and Nano-Si30groups and the activity of NOS in thehigh-dose groups of Nano-Si90groups were significantly down-regulated (P＜0.05).However, in the same size groups, there was a significant increase in the activity ofiNOS in the high-dose groups of Nano-Si90, Nano-Si60and Nano-Si30groupscompared with the low and middle-dose groups (P＜0.05).After intratracheal instillation, each exposure group myocardial expression ofTGF-β1protein were increased, in addition to other exposure groups the Si600exposed group low dose group, myocardial cytoplasm were higher than the degree ofyellow dye control group, and with the increased exposure doses and the particle sizedecreases, the degree of staining deep, in addition to the low-dose group of the Si600exposure group, the myocardial TGF-beta1the gray values in the other experimental groups of the four scales silica particles exposed groups were significantly lower thanthe control group (P＜0.05).8Myocardial apoptosis induced by silica nanoparticles exposureThere was a significant increase in the activity of CK-MB and LDH in allexperimental groups compared with the control groups after four sizes silica particlesinstillation (P＜0.05). In the same size group, there was a significant increase in theactivity of CK-MB and LDH in the high-dose groups of four sizes particles groups (P＜0.05). The activity of LDH in the middle-dose groups of four sizes particle groupswas higher than that in the low-dose groups (P＜0.05). In the same dose groups, onlyin the high-dose groups of Nano-Si30, the activities of CK-MB and LDH weresignificantly up-regulated when compared with the Si600groups (P＜0.05).The nucleus of the normal control group rat cardiomyocytes is brown stainingafter TUNEL determination of myocardial apoptosis. In addition to the low-dosegroup in the Si600-exposed group, the number of myocardial cell nucleus brownstaining in the other exposed groups is higher than that in the control group, and withelevated exposure dose and the particle size decreases, the number increases, Inaddition to the low-dose group in the Si600-exposed group, four scales silica particlesexposed group gray value of each experimental groups were significantly lower thanthe control group (P＜0.05).After intratracheal instillation four scales silica particles, each exposure groupmyocardial Bax protein expression were increased, but bcl-2protein expression wasdecreased. In addition to the low dose group of the Si600-exposed groups, eachexperimental group myocardial Bax protein expression gray values were significantlylower than those in the control group (P＜0.05), while myocardial gray value of Bcl-2protein expression were significantly higher (P＜0.05). Myocardial Caspase-3proteinexpression was increased in each exposure group. In addition to the low dose group ofthe Si600-exposed groups, Caspase-3protein gray value of the other groups in thefour-scale silica particles exposed group was significantly lower those in the control group (P＜0.05).Based on the present experiment, we found an interesting event that four sizessilica particles in low dosage (2mg/Kg·bw) could induce the toxic effects ofcardiovascular system, however, the toxic effect was no significant difference amongthe low dosage of particles. Intratracheal instillation of silica nanoparticles can passthrough the alveolar-capillary barrier into systemic circulation. There were dose-andsize-dependent cardiovascular toxicity in male Wistar Rats after exposure to differentsizes of silica nanoparticles. Heart is a target organ of silica nanoparticles.Intratracheal instillation of silica nanoparticles can cause inflammatory reaction,oxidative stress and endothelial dysfunction. ROS generation in the cardiovascularcould be one of the mechanisms responsible for the cardiovascular toxic effects ofsilica nanoparticles. In addition, we firstly found endothelial NO/NOS systemdisorder caused by nanoparticles could be one of the mechanisms for endothelialdysfunction. The results will provide a scientific basis to evaluate the risk of silicaparticles in the ecosystem and to human health.