Knockdown of lncRNA MEG3 promotes damage of vascular endothelial cells induced by vibration

Knockdown of lncRNA MEG3 promotes damage of vascular endothelial cells induced by vibration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Knockdown of lncRNA MEG3 promotes damage of vascular endothelial cells induced by vibration Xiuwen Hu, Fanfei Zeng, Ziyu Chen, Kuan Hu, Qingsong Chen, Yun Xia, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4454801/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hand–arm vibration syndrome (HAVS) is caused by long-term exposure to hand-transmitted vibration (HTV), and its pathogenesis has not been elucidated fully. We explored the molecular mechanism of HAVS and provided clues and a theoretical basis for the early prevention and treatment of HAVS. After vibration, samples were collected from the plasma of human workers, plasma of rat tails, and human umbilical vein endothelial cells (HUVECs). ELISAs were used to measure the expression of vasoactive factors. Cell Counting Kit-8 and electron microscopy were used to detect cell damage. Flow cytometry was employed to detect apoptosis. Real-time reverse transcription-polymerase chain reaction was used to measure the expression of long non-coding RNAs (lncRNAs). Western blotting was used to measure the expression of apoptosis-related proteins. Vibration could cause cell damage, apoptosis, and changes in the expression vasoactive factors and lncRNAs. The lncRNA maternally expressed gene 3 ( MEG3 ) had a significant regulatory effect on cell damage, apoptotic proteins, and vascular regulatory factors in the HUVEC damage induced by vibration, as shown by the further decrease in viability and aggravation of injury after knockdown of MEG3 expression in HUVECs treated with vibration. Expression of vasoactive factors and apoptosis-related proteins was changed after interfering with MEG3 expression. In conclusion, vibration can affect the expression of vasoactive factors and lncRNA, and cause damage to vascular endothelial cells. MEG3 may be involved in the inflammatory damage to vascular endothelial cells induced by vibration. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction About 25% of the 3 billion workers worldwide are exposed to vibration in the workplace (International Institute for Labour Studies, 2013 ). Currently, at least 2-million workers in China are exposed to hand-transmitted vibration (HTV) tools, and the main types of work are polishing, grinding, and sawing (Lin et al. 2005 ). Long-term exposure to HTV can lead to spontaneous hand symptoms and even lesions in the nervous, circulatory, muscular, or skeletal systems. Serious cases can further develop into occupational hand–arm vibration disease (HAVS) (Mahmood et al. 2017 ; Shen et al. 2017). HAVS can lead to the loss of the ability to work and live, bringing a huge economic burden to employers and the country. Vibration-induced white finger (VWF) is a typical manifestation of HAVS. HAVS development is irreversible. The pathogenesis of HAVS and influence on the vascular function of fingers have not been elucidated fully (Nilsson et al. 2017 ). Studies have shown that vibration-induced vascular endothelial dysfunction help to cause HAVS. Destruction of the structure and function of vascular endothelial cells as well as dysregulation of the production and secretion of vasoactive factors may be key factors in vibration-induced vascular injury (Herrick et al. 2005; Herrick et al. 2012). Long non-coding RNAs (lncRNAs) are located in the nucleus and are considered to have crucial regulatory roles in the proliferation (Kitagawa et al. 2013 ; Sirey et al. 2019 ) and apoptosis of cells, and invasion by cells (Evans et al. 2016 ; Tomita et al. 2017 ). Wang et al. showed that a lncRNA, maternally expressed gene 3 ( MEG3 ), may have an important role in the progression of endothelial dysfunction induced by hyperglycemia, and that knockout of MEG3 expression could aggravate inflammatory damage to endothelial cells. Knockout of MEG3 expression in human umbilical vein endothelial cells (HUVECs) has been shown to regulate the proliferation and apoptosis of endothelial cells by upregulating expression of B-cell lymphoma (Bcl)-2 and downregulating expression of Bax, caspase-3, and P53 (Wang et al. 2018 ). In the present study, plasma samples from humans and rats and HUVECs were selected to study changes in levels of vasoactive factors induced by vibration. We detected cellular damage in vitro under vibration. We interfered with MEG3 expression in HUVECs in vitro to further explore the effects of vibration-induced damage and the role of MEG3 in vibration-induced injury to vascular endothelial cells. Our data could provide: (i) a theoretical basis for revealing the epigenetic mechanism of HAVS; (ii) clues for exploring HAVS pathogenesis and finding early biomarkers. In these ways, early prevention, diagnosis, and treatment of HAVS could be achieved. Methods and materials Cell culture and treatments HUVECs were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. HUVEC culture medium (10% fetal bovine serum, penicillin, 0.25% streptomycin) were purchased from Thermo Fisher (MA, USA) and Bioshark (Beijing, China). Before the start of the experiment, cells were kept in an incubator at 37℃, 5% CO 2 . After observing cell morphology and growth, aspirate the old culture using a sterile Pasteur pipette, wash the culture flask with PBS. The cells were completely suspended by digestion and washing using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Then transfer the cells to a new flask, replenish with the appropriate amount of new medium, and place them in a 37°C, 5% CO 2 incubator for culture, and the experiment was started when HUVEC were cultured to 70–80% abundance. The experimental group was a vibration group with multiple vibrational frequency gradients of 10–100 Hz, 100–200 Hz, and 500 Hz. The vibration frequencies and accelerations are shown in Table S1 , the schematic diagram of the vibration experiment is shown in Figure S1 . The acceleration values corresponding to the vibration exposure at each frequency were obtained by instrumental measurements. The control group had the same conditions as the experimental group except that they were not exposed to vibration. Cell ultrastructure detection At the end of the vibration, cells were collected for fixation, embedding, then cutting (ultrathin sections) and staining. Changes in the internal structure of the cells, such as cell shape, mitochondria and endoplasmic reticulum, were observed under an electron microscope. Cell viability assay Cell viability was assayed using the Cell Counting Kit CCK-8. At the end of vibration, 10 µl CCK-8 reagent (UE Landi, Suzhou, China) was added and incubated for 2.5 h. The absorbance value was measured at 450 nm (Swiss Sunrise Automatic Enzyme Labeler). Flow cytometry (FCM) assay After the vibration treatment, the cells were harvested and stained with FITC-Annexin V and PI according to the FITC-Annexin V apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Flow cytometry (FACScan, BD Biosciences) was performed. Molecular epidemiological survey of occupational population Judgmental sampling was used to divide 150 employees of a factory in Zhongshan City (Guangdong, China) into three groups, of which 50 patients with a chief complaint hospital diagnosis of HAVS were the vibration white finger group, 50 hand-transmitted vibration workers without HAVS in the same period as the vibration non-white finger group, and another 50 non-hand-transmitted vibration workers were selected as the control group. In the early morning, 4 ml/person of median venous blood from the elbow of the research subjects was uniformly collected, placed in an anticoagulation tube (see supplementary materials for population inclusion criteria). All methods were performed in accordance with the relevant guidelines and regulations. The survey was approved by the ethics board of the first affiliated hospital of Guangdong Pharmaceutical University (batch number: (2021) No. [116]). Rat tail vibration exposure model Twenty-five SPF-rated male SD rats, weighing 178.3 ± 6.1 g and aged 7–8 weeks, were randomly divided into five groups: the control group, 1-day vibration exposure group, 3-day vibration exposure group, 7-day vibration group, and 14-day vibration group, with five rats in each group. The vibration exposure conditions were as follows: The tail of each rat was exposed to vibration for 4 h/day at a vibration frequency of 125 Hz and an acceleration of 6.4 m/s 2 . The exposure conditions were chosen from the information provided by most studies on the frequency-related effects of changes in vibration injury (Wang et al. 2018 ). After anesthesia, blood was collected from the abdominal aorta of the rats and euthanized by air embolization. All methods were performed in accordance with the relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. Detection of vasoactive factors by ELISA The detection of the vasoactive factors leukotriene B4 (LTB4), 5-hydroxy tryptamine (5-HT), VEGF, interleukin β (IL-1β), Myosin light chain-2 (MLC2), Calcitonin gene-related peptide (CGRP) and Endothelin-1(ET-1) in human and rat plasma after thawing at room temperature, in cells, and supernatants was performed according to the ELISA kit procedure produced by MEIMIAN (Jiangsu China). The optical density values of each well were measured at 450 nm(Swiss Sunrise Automatic Enzyme Labeler), the standard curve was plotted, and the concentration levels of the above indicators were calculated. Real-time fluorescence quantitative reverse transcription PCR Total RNAs were extracted using Trizol reagent (Invitrogen), equal amounts of total RNA were reverse transcribed using a cDNA synthesis kit (TaKaRa, Japan), and real-time fluorescent quantitative PCR (qRT-PCR) was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference to detect the relative expression levels. The primers used were as follows supplementary Table S2. lncRNA MEG3 knock-down Before conducting lncRNA MEG3 knockdown experiments, the most suitable siRNA (small interfering RNA) transfection dose needs to be determined first. We selected FMA-modified siRNA fragments and used universal liposomes Lipofectamine 2000 (ThermoFisher, USA) as transfectants. The fluorescence intensity of each well was observed under a fluorescence microscope, and the liposome concentration with the strongest fluorescence was recorded. MEG3 siRNA and siRNA were transfected into HUVECs at a concentration of 50 nmol/L using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. MEG3 -specific siRNA and siRNA were designed and synthesized from GenePharma (Shanghai, China). Cells were harvested after 48 h for qRT-PCR, western blot, cell proliferation and apoptosis analyses. Western blot Cellular protein content in lysates was assessed by the BCA protein assay (Thermo Fisher, USA). Cell protein lysates were separated in 10%SDS-PAGE and transferred to a 0.22µm polyvinylidene difluoride membrane. Membranes were subsequently incubated with specific antibodies (ABCAM, UK) overnight at 4°C. Secondary antibodies were added and incubated for 1 h at 37°C. The immunoreactive bands were visualized using the ECL kit (Thermo Fisher, USA). Statistical analysis All statistical analyses were performed using the SPSS 21.0. The measured data were tested for normality, and data conforming to a normal distribution were described by ( \(\stackrel{-}{X}\) ± S). Median (M) and interquartile spacing ( P 25 to P 75) were used to describe the number of disobedience to normal distribution. One-way ANOVA or two-way ANOVA were used for comparison of means between multiple groups. Before comparing between groups, the sameness test of variance was performed on the data of each index, and the interaction between the treatment factors was analyzed. If the variance was equal, the least significant difference (LSD) rank test was used for pairwise comparison. Otherwise, use Dunnet's T3 for pairwise comparison. Rates of enumeration data were compared using the Pearson χ² test or Fisher's exact test. All tests were two-tailed and P < 0.05 was defined as statistically significant. GraphPad was used for plotting. Results Vibration alters the viability and subcellular structure to induce HUVEC apoptosis Compared with the control group, HUVEC viability increased at 1, 1.5, 2, and 4 h, and HUVEC viability in the vibration group at 2.5 h and 3 h decreased (Fig. 1 A). These data indicated that this vibration condition elicited the greatest effect upon HUVECs. Exposure to 125-Hz vibration could increase HUVEC viability for 3 h and 4 h. These results suggested that longer exposure could increase HUVEC viability, and could not be considered a beneficial effect of vibration at this frequency. Based on this result, we investigated whether there were other frequencies that reduced HUVEC viability. HUVEC viability decreased at 20 and 200 Hz, and increased at 10, 40, 80, and 130 Hz, compared with the control group, for 2.5 h. After exposure to vibration for 3 h, HUVEC viability decreased at 125 and 130 Hz and increased at 205 Hz (Fig. 1 B). Due to the survival status of HUVECs, only a 2-day experiment could be conducted. Compared with the control group, HUVEC viability increased at 10 Hz, and decreased at 20 and 125 Hz for 2.5 h of vibration. HUVEC viability increased at 20 and 125 Hz for 3 h and 4 h of vibration (Fig. 1 C). Therefore, we selected a vibration time of 2.5 h and frequencies of 20 and 125 Hz as criteria for subsequent experiments. Transmission electron microscopy revealed that the control group had normal nuclei, endoplasmic reticuli (ER), and mitochondria, but the structure was faint. The cytoplasm was abundant and dense, and a small amount of vacuolization was visible. In the 20-Hz, 2-day group, pyknosis, chromatin margination, and swollen ER were observed. The cytoplasm was abundant and dense, cell outgrowth was reduced, and some of the cytoplasm was vacuolated. In the 125-Hz, 2-day group, pyknosis, chromatin margination, nuclear-membrane irregularities, and considerable cytoplasm vacuolization were observed. ER were swollen and cell outgrowth was reduced (Fig. 1 D). Compared with the control group, 125-Hz vibration for 2.5 h caused an increase in apoptosis in the 1-day group and 2-day group (Fig. 1 I). These results suggested that vibration induced cell damage and promoted HUVEC apoptosis. (A) Effect of vibration at 125 Hz for 1–4 h on HUVEC viability. N = 3 experimental replicates per group. (B) Effect of vibration at different frequencies for 2.5 h (left) and 3 h (right) on one-day group HUVEC viability. N = 3 experimental replicates per group. (C) Effect of vibration at different frequencies for 2.5 h (left), 3 h (middle) and 4 h (right) on two-day group HUVEC viability. N = 3 experimental replicates per group. (D) Intracellular structural hanges were observed at different magnifications of 1 µm-10 kx, 2 µm-5 kx, 2 µm-4 kx, and 2 µm-5 kx. Nucleus pyknosis, chromatin margination, swollen endoplasmic reticulum and mitochondria, cell budding and vacuolization are indicated with red arrows. (E to F) Effect of vibration at 125 Hz (E) and 20 Hz (F) for 2.5 h on one-day group HUVEC apoptosis. N = 3 experimental replicates per group. (G to H) Effect of vibration at 125 Hz (G) and 20 Hz (H) for 2.5 h on two-day group HUVEC apoptosis. N = 3 experimental replicates per group. (I) Flow cytometry was used to measure % of apoptotic cells. N = 3 experimental replicates per group. *P < 0.05, **P < 0.01, compared with the control group. Vibration induces changes in the expression of vasoactive factors in the plasma of workers and rats, HUVECs, and HUVEC supernatants All worker groups comprised 50 males. The mean age of the vibration-white-finger group was 39.86 ± 6.70 years and they had 11.92 ± 5.39 years of service. The mean age of the vibration-non-white-finger group was 35.92 ± 6.61 years, and they had 6.98 ± 6.84 years of service. The mean age of the control group was 34.50 ± 5.75 years and they had 10.70 ± 6.21 years of service. There were significant differences in age, duration of service, and hand symptoms among these three groups (Table 1 ). Expression of LTB4, IL-1β, CGRP, and ET-1 in the vibration-white-finger group was higher than that in the vibration-non-white-finger group and control group. The distribution of MLC2 was reversed in these three groups, and differences in VEGF expression among these three populations was not significant (Fig. 2 A). Changes in expression of vasoactive factors in rat plasma were measured (Fig. 2 B). Expression of LTB4 in 3-, 7-, and 14-day groups was higher than that in the control group and 1-day group. VEGF expression in 7- and 14-day groups was higher than that in control, 1-, and 3-day groups. IL-1β expression in 3-, 7-, and 14-day groups was higher than that in control and 1-day groups. IL-1β expression in the 14-day group was higher than that in 3- and 7-day groups. MLC2 expression in 3-, 7-, and 14-day groups was lower than that in control and 1-day groups. CGRP expression in 7- and 14-day groups was lower than that in control, 1- and 3-day groups. ET-1 expression in 3-, 7-, and 14-day groups was higher than that in control and 1-day groups. ET-1 expression in the 14-day group was higher than that in 3- and 7-day groups. We also detected the changes in expression of vasoactive factors in HUVECs and HUVEC supernatants (Fig. 2 C). Expression of LTB4, VEGF, IL-1β, CGRP, and ET-1 was increased in HUVECs and/or HUVEC supernatants in 20- and 125-Hz groups compared with that in the control group. MLC2 expression was reduced in HUVECs and HUVEC supernatants in the 125-Hz, 2-day group compared with that in the control group. LTB4 expression was decreased in HUVECs and HUVEC supernatants in the 125-Hz group compared with that in the 20-Hz group. Expression of VEGF and ET-1 was increased in HUVECs in the 125-Hz, 1-day group compared with that in the 20-Hz, 1-day group. MLC2 expression was decreased in HUVECs in the 125-Hz, 2-day group compared with that in the 20-Hz, 2-day group. CGRP expression was increased in the HUVEC supernatants of the 125-Hz, 1-day group and HUVECs in the 125-Hz, 2-day group, compared with that in the 20-Hz group with the same number of days of exposure. Thus, vibration could induce changes in the expression and secretion of the vasoactive factors LTB4, IL-1β, MLC2, CGRP, and ET-1 in HUVECs and HUVEC supernatants. Table 1 Comparison of the basic situation of the three groups of workers Variables Control (n = 50) non-VWF (n = 50) VWF (n = 50) F/χ2 P Age, y 34.50 ± 5.75 35.92 ± 6.16 39.86 ± 6.70 9.979 a <0.001 Length of service, y 10.70 ± 6.21 6.98 ± 6.84 11.92 ± 5.39 16.949 a <0.001 Smoking, n (%) 21(29.6) 24(33.8) 26(36.6) 1.016 b 0.048 Drinking, n (%) 32(36.0) 26(29.2) 31(34.8) 1.713 b 0.079 Hand symptoms, n (%) 3(4.1) 23(31.1) 48(64.9) 81.348 b <0.001 [Note]: a is F value; b is χ2 value; compared with the control group, P < 0.05. (A) Comparison of plasma concentrations of six vasoactive factors among three groups of workers. ** P < 0.01, compared with the control group (n = 50); ## P < 0.01, compared between the vibration white finger (VWF) group (n = 50) and the vibration non-white finger (non-VWF) group (n = 50). (B) Comparison of plasma concentrations of six vasoactive factors among five groups of rats. * P < 0.05, ** P < 0.01, compared with the control group (n = 5); # P < 0.05, ## P < 0.01, compared between 1 day (n = 5) and 3 days (n = 5), 7 days (n = 5), 14 days (n = 5); N P < 0.05, NN P < 0.01, compared between 3 days and 7 days, 14 days; X P < 0.05, XX P < 0.01, compared between 7 days and 14 days. (C) Cells and supernatants in one-day group and two-day group were collected after vibration for 2.5 h. N = 3 experimental replicates per group. * P < 0.05, ** P < 0.01, compared with the control group; # P < 0.05, ## P < 0.01, compared with the 20 Hz group. Changes in lncRNA expression induced by vibration in HUVECs In 1-day groups, expression of the lncRNAs MEG3 , nuclear-enriched abundant transcript 1 ( NEAT1 ), taurine-upregulated gene 1 ( TUG1 ), and metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) decreased in the 20-Hz group and 125-Hz group compared with that in the control group. In 2-day groups, expression of MEG3 , TUG1 , and MALAT1 decreased in the 20-Hz group, but NEAT1 expression increased; MEG3 had increased expression in the 125-Hz group, but TUG1 expression decreased (Fig. 3 ). These data suggested that vibration induced changes in the expression of MEG3 , NEAT1 , TUG1 and MALAT1 in HUVECs. Use qPCR to analyse the expression of lncRNA in HUVEC after vibration for 2.5 h. N = 3 experimental replicates per group. * P < 0.05, ** P < 0.01, compared with the control group. Vibration induces changes in MEG3 expression in HUVECs In 1-day groups, MEG3 expression decreased in the 20-Hz group and 125-Hz group compared with that in the control group. In 2-day groups, MEG3 expression decreased in the 20-Hz group (Fig. 4 B). These results indicated that vibration could cause changes in MEG3 expression. Fluorescence microscopy revealed that intracellular fluorescence could be observed in transfected HUVECs in each group. An appropriate fluorescence effect and transfection efficiency could be observed at the dose of 5 µL of liposome (Fig. 4 C). Compared with the control group, expression of small interfering (si)RNA MEG3-3#5 mRNA was decreased (Fig. 4 D–F), indicating that knockdown of MEG3 expression had been achieved. In the 125-Hz, 1- and 2-day groups, compared with the negative control group, expression in the siRNA-control group and negative control-vibration group decreased; compared with the negative control-vibration group, MEG3 expression was decreased in the siRNA-vibration group (Fig. 4 G–J). In the 20-Hz, 1-day group, expression increased in the negative control-vibration group compared with that in the negative control group; in the 2-day group, expression in the siRNA-control group decreased, and expression in the negative control-vibration group increased. Compared with the negative control-vibration group, MEG3 expression in the siRNA-vibration group decreased. These results indicated that vibration could affect MEG3 expression in HUVECs, and that MEG3 had an important role in HUVEC damage induced by vibration. (A) Diagram of the vibration instrument used in the experiment. (B) Effect of vibration on the expression of lncRNA MEG3 . (C) Expression of FAM-modified siRNA with different doses of Lipofectamine 2000. (D-F) Transfection efficiency of three different doses of siRNA. (G-J) Changes of lncRNA MEG3 expression under different vibration frequencies. * P < 0.05, ** P < 0.01, compared with negative control group (n = 3); # P < 0.05, ## P < 0.01, compared between negative control-vibration group (n = 3) and siRNA-vibration group (n = 3). Effect of MEG3 on the internal structure in vibration-induced HUVECs injury Electron microscopy showed that in the 20-Hz, 2-day group, the nucleus, ER, and mitochondria of the negative control group were normal, but the structure was fuzzy. The cytoplasm was abundant and dense, with a small amount of vacuolization. In the siRNA-control group, nuclear pyknosis, malformation, slightly irregular nuclear membrane, inconspicuous chromatin, relatively swollen ER and mitochondria, and some cytoplasmic vacuolization were observed. In the negative control-vibration group, nuclear pyknosis, chromatin margination, no swelling or expansion of ER, no obvious swelling of mitochondria, and some cytoplasmic vacuolization were observed. In the siRNA-vibration group, nuclear pyknosis and malformation, a relatively regular nuclear membrane, and chromatin margination were observed. ER and mitochondria were swollen, and vacuolization was observed in the cytoplasm (Fig. 5 A). In the 125-Hz, 2-day group, the nucleus, ER, and mitochondria were normal in the negative control group, chromatin margination was absent, the cytoplasm was dense, and some vacuolization was observed. In the siRNA-control group, we observed nuclear pyknosis and malformation, slightly irregular nuclear membranes, and inconspicuous chromatin. Rough endoplasmic reticuli (RER) were slightly dilated, ER ribosomes had been shed, mitochondria were swollen and ruptured, and some cytoplasm was vacuolated. In the negative control-vibration group, we observed nuclear pyknosis and chromatin margination. RER were arranged in a lamellar pattern, ribosomes were near ER membranes, mitochondria were swollen and ruptured, and a small part of the cytoplasm was vacuolated. In the siRNA-vibrative group, nuclear pyknosis, malformation, and chromatin margination were observed. Expanded RER were seen in the cytoplasm. Mitochondria were swollen and morphologically abnormal. Extensive vacuolization was observed in the cytoplasm (Fig. 5 B). In the 20- and 125-Hz groups at 1 day, compared with the negative control group, HUVEC viability decreased in the siRNA-control group. In the 125-Hz, 2-day group, compared with the negative control group, HUVEC viability decreased in the siRNA-control group and negative control-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, HUVEC viability increased in the siRNA-vibration group (Fig. 5 C–F). Compared with the negative vibration group, cell damage in the MEG3 knockdown vibration group was more obvious and severe. These data suggested that MEG3 may be involved in vibration-induced injury and may inhibit HUVEC damage. (A and B) Intracellular structural changes of cells in 20 Hz, two-day group (A) and 125 Hz, two-day group (B) were respectively observed at different magnifications of 1 µm-10 kx, 0.5 µm-25 kx, 1 µm-10 kx, 0.5 µm-30 kx, and 2 µm-5 kx. Nucleus pyknosis, abnormal chromatin or chromatin margination, swollen endoplasmic reticulum and mitochondria and vacuolization are indicated with red arrows. (C to F) Effect of vibration on cell viability in each group. N = 5 experimental replicates per group. * P < 0.05, ** P < 0.01, compared with the negative control group. MEG3 regulates the expression of Bax, caspase-3, and Bcl-2 during vibration-induced damage to HUVECs In the 20-Hz, 1-day group, compared with the negative control group, Bax expression was decreased in the negative control-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, Bax expression was increased in the siRNA-control group and negative control-vibration group, and Bax expression in the siRNA-vibration group was higher than that in the siRNA-control group. In the 20-Hz, 1-day group, caspase-3 expression was decreased in the negative siRNA-control group and increased in the siRNA-vibration group compared with that in the negative control group; compared with the negative control-vibration group, caspase-3 expression was increased in the siRNA-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, caspase-3 expression was decreased in the negative control-vibration group and siRNA-vibration group; compared with the negative control-vibration group, caspase-3 expression was decreased in the siRNA-vibration group. In the 20-Hz, 1-day group, compared with the negative control group, Bcl-2 expression was decreased in the siRNA-control group and negative control-vibration group; compared with the negative control-vibration group, Bcl-2 expression was increased in the siRNA-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, Bcl-2 expression was reduced in the negative control-vibration group and siRNA-vibration group; compared with the negative control-vibration group, Bcl-2 expression was reduced in the siRNA-vibration group (Fig. 6 ). In the 125-Hz, 1-day group, compared with the negative control group, Bax expression was decreased in the siRNA-control group and siRNA-vibration group, and Bax expression in the siRNA-vibration group was lower than that in the siRNA-control group. In the 2-day group, compared with the negative control group, Bax expression was decreased in the negative control-vibration group and siRNA-vibration group. In the 125-Hz, 2-day group, caspase-3 expression was decreased in the negative siRNA-control group compared with that in the negative control group. In the 125-Hz, 2-day group, compared with the negative control group, Bcl-2 expression was decreased in the siRNA-control group and siRNA-vibration group, and Bcl-2 expression was increased in the negative control-vibration group; compared with the negative control-vibration group, Bcl-2 expression was decreased in the siRNA-vibration group (Fig. 6 ). These results showed that vibration-induced expression of apoptotic proteins (Bax and caspase-3) decreased, whereas that of an anti-apoptotic protein (Bcl-2) increased. After interfering with MEG3 , the expression of Bax, caspase-3, and Bcl2 decreased. This finding suggested that MEG3 may be involved in vibration-induced HUVEC injury by regulating the expression of Bax, caspase-3, and Bcl-2. (A and B) The effect of MEG3 on the expression levels of Bax, Caspase3 and Bcl2 in HUVECs injury induced by vibration. (C and D) The effect of MEG3 on the expression levels of Bax in HUVECs injury induced by vibration. (E and F) The effect of MEG3 on the expression levels of Caspase3 in HUVECs injury induced by vibration. (G and H) The effect of MEG3 on the expression levels of Bcl2 in HUVECs injury induced by vibration. N = 3 experimental replicates per group. * P < 0.05, ** P < 0.01, compared with the negative control group; # P < 0.05, ## P < 0.01, compared between negative control-vibrative group and siRNA-vibrative group. Effect of MEG3 on vasoactive factors in vibration-induced damage to HUVECs In the 20-Hz, 1-day group, compared with the negative control group, ET-1 expression was increased and VEGF expression was decreased in HUVECs; after knockdown of MEG3 expression, compared with the negative control-vibration group, VEGF expression was increased. Expression of IL-1β, CGRP, and ET-1 was increased in HUVEC supernatants; after knockdown of MEG3 expression, compared with the negative control-vibration group, VEGF expression was increased but expression of ET-1 and IL-1β decreased (Fig. 7 ). In the 20-Hz, 2-day group, compared with the negative control group, expression of IL-1β, 5-HT, CGRP, and ET-1 was increased and VEGF expression was decreased in HUVECs; after knockdown of MEG3 expression, compared with the negative control-vibrative group, CGRP expression was increased and expression of LTB4, IL-1β, 5-HT, and ET-1 was decreased. Expression of LTB4, IL-1β, 5-HT, CGRP, and ET-1 was increased and VEGF expression was decreased in HUVEC supernatants; after knockdown of MEG3 expression, compared with the negative control-vibration group, CGRP expression was increased and expression of LTB4, VEGF, IL-1β, 5-HT, and ET-1 was decreased (Fig. 7 ). In the 125-Hz, 1-day group, compared with the negative control group, expression of LTB4, IL-1β, 5-HT, and ET-1 in HUVECs was increased; after knockdown of MEG3 expression, compared with the negative control-vibration group, expression of IL-1β, 5-HT, and ET-1 was decreased. Expression of 5-HT and CGRP was increased in HUVEC supernatants; after knockdown of MEG3 expression, compared with the negative control-vibration group, VEGF expression was increased and expression of ET-1 and 5-HT was decreased (Fig. 7 ). In the 125-Hz, 2-day group, compared with the negative control group, expression of LTB4, IL-1β, and ET-1 was increased and VEGF expression was decreased in HUVECs; after knockdown of MEG3 expression, compared with the negative control-vibration group, VEGF expression was increased and expression of LTB4 and IL-1β was decreased. Expression of LTB4, IL-1β, 5-HT, and ET-1 was increased in HUVEC supernatants; after knockdown of MEG3 expression, compared with the negative control-vibration group, expression of LTB4 and 5-HT was decreased (Fig. 7 ). These results indicated that MEG3 regulated the expression of vasoactive factors in vibration-induced inflammation. Hence, MEG3 may be involved in vibration-induced damage to HUVECs by regulating the expression of vasoactive factors. Cells and supernatants were collected after vibration for 2.5 h, and the expression level of vasoactive factors were measured by ELISA. N = 3 experimental replicates per group. N-C: negative control, siRNA-C: siRNA-control, N-V: negative control-vibrative, siRNA-V: siRNA-vibrative. * P < 0.05, ** P < 0.01, compared with the negative control group; # P < 0.05, ## P < 0.01, compared between negative control-vibrative group and siRNA-vibrative group. Discussion Studies on the pathophysiology of HAVS have focused mainly on its clinical manifestations and diagnosis. Studies on the mechanism of injury to vascular endothelial cells caused by vibration are scarce (Budd et al. 2018). Previously, we found that Chinese workers were exposed to high intensity and long duration of vibration without effective protective measures, and that the hazards of vibration were seriously underestimated or ignored (Chen et al. 2012 ). The effect of vibration on peripheral vascular function has been the focus of HAVS research, and vasoactive factors help to regulate vascular function. We measured expression of the vasoactive factors LTB4, IL-1β, CGRP, MLC2, and ET-1 using in vivo and in vitro models of vibration exposure. This approach provided a theoretical basis for future studies on the pathogenesis of HTV-induced HAVS. In addition, few studies have investigated the effect of vibration on cells in vitro . White et al. proposed that the absolute size of the temporal gradient and the frequency/duration of each pulse cycle had roles in the biomechanical transduction of fluid vibration in endothelial cells (2004). Therefore, we also employed a cellular model to investigate the effects of, and damage of vibration to, cells. By ultrastructural observation we found, for the first time, that vibration could induce changes in the viability of, and damage to, HUVECs. LTB4 is an important metabolite of arachidonic acid. It is produced by the action of 5-lipoxygenase and other enzymes. It has a powerful activating effect on leukocytes, stimulating their chemotaxis and aggregation, releasing oxygen radicals and lysosomal enzymes, and increasing vascular permeability and vascular-wall constriction (Rosenblum et al. 1985; Woodward et al. 1983 ). Our in vivo experimental results showed that, in workers exposed to HTV, those with a higher plasma level of LTB4 were more likely to suffer from VWF. In rats, the longer the duration of vibration, the higher was the LTB4 expression in plasma, suggesting that there may be a dose–response relationship between LTB4 expression and the duration of vibration exposure. The in vitro HUVEC model revealed LTB4 expression in vibration groups to be higher than that in the control group. VEGF acts specifically and multilaterally on vascular endothelial cells, causing their proliferation, thereby promoting neovascularization. However, in the present study, VEGF expression was not associated with VWF. This finding may have been due to the small sample size of our study. The increased VEGF expression in plasma in the 7- and 14-day groups of rats may have been because repair of vascular endothelial cells was at a compensatory stage. The increased VEGF expression in HUVECs in the 2-day vibration group and no change in VEGF expression in HUVEC supernatants may have been due to impaired secretion of VEGF in HUVECs as a result of prolonged vibration. IL-1β is a key mediator of intercellular communication in the immune system. It is also a key driver of local and systemic immune responses in atherosclerotic cardiovascular disease (Viana-Huete et al. 2019). Our in vivo results showed that the higher the IL-1β expression in a population exposed to HTV, the greater was the chance of developing occupational HAVS. In rats, the longer the duration of vibration, the higher was the IL-1β expression in plasma, suggesting that there may be a dose–response relationship between IL-1β expression and duration of exposure to vibration. Myosin consists of one pair of heavy chains and two pairs of light chains. Phosphorylation of myosin light chains is the basis for increasing vascular endothelial permeability (Hu et al. 2018 ). We showed that vibration decreased MLC2 expression in rat plasma and HUVECs in vitro . Also, if the vibration lasted longer, considerable necrosis of HUVEC occurred, and then expression in vibration groups was lower than that in the control group. We speculated that humans have a regulatory function in which short-term vibration increases MLC2 secretion. CGRP is the main transmitter of sensory nerves. It is distributed widely in central and peripheral tissues, and produces biological effects by interacting with specific receptors. CGRP can elicit vasodilatation, protect vascular endothelial cells, and inhibit the proliferation of smooth muscle cells (Rosenfeld et al. 1983 ; Brain et al. 1985 ). We observed that vibration decreased CGRP expression rat tails. Vibration increased CGRP expression significantly in human plasma and HUVECs. The different results between humans and rats may have been because stress in humans can be regulated by the immune system, whereas rats exhibit a short-term stress response. The pathology associated with sensory-nerve deficits in patients with HAVS has been described, but the pathogenesis of these deficits has not, which merits requires further investigation. ET-1 is a potent vasoconstrictor secreted by vascular endothelial cells that mediates vasoconstriction, proinflammatory effects, and mitogenesis. ET-1 overproduction may be important in cardiovascular diseases (Davie et al. 2009 ). Our in vivo and in vitro models showed that vibration increased ET-1 expression, which led to HUVEC injury. We elucidated that, under different frequencies and times of damage to HUVECs, changes in the expression of LTB4, VEGF, IL-1β, MLC2, CGRP, and ET-1 in vibration-induced in vivo and in vitro models may be involved in HAVS pathogenesis. However, quantifying the effects of vibration exposure accurately is challenging, and the number of validated biomarkers of exposure is limited. The association between lncRNAs and HAVS is not clear. We found that vibration induced altered expression of the lncRNAs MEG3 , NEAT1 , TUG1 , and MALAT1 in HUVECs. MEG3 expression has been shown to be significantly higher in the plasma of patients suffering from sepsis compared with that in healthy controls (Yuan et al. 2020). However, it has also been shown that MEG3 expression decreases in the retinal epithelial cells of diabetic mice under oxidative stress and high-glucose stimulation in vitro and in vivo (Wang et al. 2018 ). MEG3 expression may differ in different conditions or diseases. We suggest that lncRNAs may be affected by the frequency and duration of vibration, which may lead to a different extent of expression. lncRNAs have a very important regulatory role in vascular endothelial inflammation and may be involved in vibration-induced endothelial injury. The specific role of lncRNAs in HAVS progression will be explored further in cellular experiments and animals by our research team, and may reveal HAVS pathogenesis. We found that MEG3 was involved in the regulation of damage, apoptotic proteins, and vasoactive factors in HUVECs induced by vibration. Vibration treatment of HUVECs with knockdown of MEG3 expression caused a further reduction in cell viability and aggravation of injury. Bax expression decreased in the 1- and 2-day groups for 20-Hz vibration. In the 1-day group, caspase-3 expression increased and decreased in the 2-day group, data which align with results from work by Song and colleagues. Vibration induced a decrease in the expression of the apoptotic proteins Bax and caspase-3. After knockdown of MEG3 expression, the expression of Bax and caspase-3 decreased more significantly, and Bcl-2 expression increased. These findings suggest that MEG3 may be involved in the vibration-induced injury to vascular endothelial cells by regulating the expression of Bax, caspase-3, and Bcl-2. Vibration increased the expression of LTB4, 5-HT, IL-1β, CGRP, and ET-1, and decreased the expression of VEGF when MEG3 was intact. After knockdown of MEG3 expression, expression of IL-1β, 5-TH, CGRP, and VEGF was increased. Expression of LTB4, IL-1β, ET-1, and CGRP in the siRNA-vibration group was lower than that in the negative control-vibration group. These data suggested that, under the influence of vibration and siRNA, expression of LTB4, IL-1β, ET-1, CGRP, and VEGF was reversed. After knockdown of MEG3 expression, VEGF expression was increased in the 1-day group, but decreased in the 2-day group, which may be a way that cells protect themselves from stress. Studies have shown that MEG3 affects the expression of inflammatory factors. Wang et al. found that knockout of MEG3 expression increased the expression of VEGF, TNF-α, and IL-6, significantly in HUVECs, suggesting that MEG3 may inhibit the secretion of inflammatory factors (2018). Conversely, Liu et al. showed that downregulation of MEG3 expression inhibited the secretion of TNF-α, IL-1β, and IL-6 in lipopolysaccharide-treated human dental pulp cells (2021). However, our data suggest that downregulation of MEG3 expression has different effects on the expression of vasoactive factors under different vibration conditions. There were differences between the in vitro cell model and actual human exposure to vibration, which merit further study. Knockdown of MEG3 expression in HUVECs can regulate the proliferation and apoptosis of endothelial cells by upregulating expression of Bcl-2 and downregulating expression of Bax, caspase-3, and P53 (Wang et al. 2018 ). In contrast, Liu and colleagues found that downregulation of MEG3 expression in HUVECs induced apoptosis and inhibited cell proliferation (2021). In addition, knockdown of MEG3 expression reduced neovascularization and VEGF-induced angiogenesis in alkali-burned corneas through stromal cell-derived factor-1/chemokine receptor-4 (SDF-1/CXCR4) and Smad2/3 signaling pathways (Shen et al. 2022 ). MEG3 may have different roles in the proliferation and apoptosis of endothelial cells and inflammatory injury due to different conditions for cell growth. Therefore, more studies are needed to understand the role and mechanism of action of MEG3 . MEG3 is involved in the physiological and pathologic processes of several vascular diseases. MEG3 is an effective indicator of changes in vascular structure and function. Increasing numbers of studies have shown that lncRNAs are regulators of protein-coding genes, and that their dysfunction affects the occurrence and development of diseases. We discovered that, according to electron microscopy, downregulation of MEG3 expression aggravated cell damage. We conclude that MEG3 has a protective role in the cell damage induced by vibration. Conclusions MEG3 may be involved or have a regulatory role in the inflammatory damage experienced by vascular endothelial cells induced by vibration. We used a strategy of gene-expression inhibition to explore the relationship between MEG3 and vibration-induced damage to vascular endothelial cells. We clarified the expression pattern of MEG3 and its role in vibration-induced inflammatory damage and disorders of secretion of vasoactive substances. Thus, our data could reveal the molecular mechanism of HAVS, and provide clues and a theoretical basis for the early prevention and treatment of HAVS. Declarations Ethics approval and consent to participate The blood samples of this study population were approved by the Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University, Guangdong Province, batch number: (2021) No. [116], and the written informed consent of the research subjects was obtained. All methods were performed in accordance with the relevant guidelines and regulations. The animal experiments in this study have been approved by the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University (acceptance number: gdpulac2021212). All methods were performed in accordance with the relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. Consent for publication Consent obtained from individuals participating in stakeholder interviews. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by National Natural Science Foundation of China(82204007); the Natural Science Foundation of Guangdong Province-General Project (2022A1515012412); the Natural Science Foundation of Guangdong Province-General Project (2022A1515011357); Major basic and applied basic research projects of Guangdong Province of China (2019A1515110492); Guangzhou Science and Technology Planning Project(201904010222), Guangdong Medical Science and Technology Research Fund (A2022210). Authors' contributions Hongyu Yang contributed to the overall planning of the experiment, the technical guidance of the experiment, and the guidance of the writing of the thesis. Fanfei Zeng contributed to the thesis writing. Ziyu Chen contributed to the questionnaire survey and experimental operation. Kuan Hu contributed to the data analysis. Qingsong Chen contributed to the questionnaire design and experimental design. Yun Xia contributed to the experimental planning and thesis guidance. Xiuwen Hu contributed to the questionnaire survey, experimental operation and data analysis. All authors have read and approved the final manuscript. Acknowledgements The authors acknowledge the Medical Laboratory Animal Center provided by the Guangdong Provincial Occupational Disease Prevention and Control Institute. The authors thank workers at the Guangsheng factory in Zhongshan City, Guangdong Province for providing blood samples. 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Chin J Clin Lab Sci, 2020; https://doi.org/10.13602/j.cnki.jcls.2020.09.09 Additional Declarations No competing interests reported. Supplementary Files sup.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4454801","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308508323,"identity":"93f85f8e-e2d3-4bd9-91f4-77a81e0942ca","order_by":0,"name":"Xiuwen Hu","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Xiuwen","middleName":"","lastName":"Hu","suffix":""},{"id":308508324,"identity":"b1559fe9-110c-4946-9d83-9f007a37c315","order_by":1,"name":"Fanfei Zeng","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Fanfei","middleName":"","lastName":"Zeng","suffix":""},{"id":308508325,"identity":"b713f42a-a4ea-48bf-9488-3f7559fcd953","order_by":2,"name":"Ziyu Chen","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Ziyu","middleName":"","lastName":"Chen","suffix":""},{"id":308508326,"identity":"decc80a1-7626-4949-a164-e73318fe0c6d","order_by":3,"name":"Kuan Hu","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Kuan","middleName":"","lastName":"Hu","suffix":""},{"id":308508327,"identity":"c5de808b-070e-4dca-8a45-7e40e121f770","order_by":4,"name":"Qingsong Chen","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Qingsong","middleName":"","lastName":"Chen","suffix":""},{"id":308508328,"identity":"c37e3cb3-09e8-440c-bffa-cfef661812de","order_by":5,"name":"Yun Xia","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Xia","suffix":""},{"id":308508329,"identity":"e6e16910-3297-4c24-991e-0e211bfb4de0","order_by":6,"name":"Hongyu Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYFCCBAYGxgYGOTb29gOkaTHm4zmTQJqWxHkSDgbEaZB3zz0mzbvjcHqbBFDzj4pthLUYnnmXJs175nBum3TjAcaeM7eJ0DIjx0yatw2oReZAAjNjGwla0tkkEgyI0yIvAdGSQLwWA553yZZz29IN24CBfJAov8i35x688bbNWl6+vf3ggx8VxNhygIeBiYehGcw5QFg9yJYGHgbGHwx1RCkeBaNgFIyCEQoA+QE9HyKdl/MAAAAASUVORK5CYII=","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-05-21 12:25:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4454801/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4454801/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57643424,"identity":"68a2ff9e-d963-4287-9651-cce9ef6e756e","added_by":"auto","created_at":"2024-06-03 18:19:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2665788,"visible":true,"origin":"","legend":"\u003cp\u003eVibration-induced changes in viability, subcellular structure and apoptosis in HUVECs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Effect of vibration at 125 Hz for 1-4 h on HUVEC viability. N = 3 experimental replicates per group. \u003cstrong\u003e(B)\u003c/strong\u003e Effect of vibration at different frequencies for 2.5 h (left) and 3 h (right) on one-day group HUVEC viability. N = 3 experimental replicates per group. \u003cstrong\u003e(C)\u003c/strong\u003e Effect of vibration at different frequencies for 2.5 h (left), 3 h (middle) and 4 h (right) on two-day group HUVEC viability. N = 3 experimental replicates per group. \u003cstrong\u003e(D)\u003c/strong\u003e Intracellular structural hanges were observed at different magnifications of 1 μm-10 kx, 2 μm-5 kx, 2 μm-4 kx, and 2 μm-5 kx. Nucleus pyknosis, chromatin margination, swollen endoplasmic reticulum and mitochondria, cell budding and vacuolization are indicated with red arrows. \u003cstrong\u003e(E \u003c/strong\u003eto\u003cstrong\u003e F)\u003c/strong\u003e Effect of vibration at 125 Hz (E) and 20 Hz (F) for 2.5 h on one-day group HUVEC apoptosis. N = 3 experimental replicates per group. \u003cstrong\u003e(G \u003c/strong\u003eto\u003cstrong\u003e H)\u003c/strong\u003e Effect of vibration at 125 Hz (G) and 20 Hz (H) for 2.5 h on two-day group HUVEC apoptosis. N = 3 experimental replicates per group. \u003cstrong\u003e(I)\u003c/strong\u003e Flow cytometry was used to measure % of apoptotic cells. N = 3 experimental replicates per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, compared with the control group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/f3b5193aaaa7742f38fbf4c7.png"},{"id":57643427,"identity":"58ddee55-2027-4115-baad-c4216cfa005a","added_by":"auto","created_at":"2024-06-03 18:19:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":545604,"visible":true,"origin":"","legend":"\u003cp\u003eVibration-induced changes in the expression of vasoactive factors in plasma of workers, plasma of rats, HUVEC and supernatant\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Comparison of plasma concentrations of six vasoactive factors among three groups of workers. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the control group (n=50); \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, compared between the vibration white finger (VWF) group (n=50) and the vibration non-white finger (non-VWF) group (n=50). \u003cstrong\u003e(B)\u003c/strong\u003e Comparison of plasma concentrations of six vasoactive factors among five groups of rats. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the control group (n=5); \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between 1 day (n=5) and 3 days (n=5), 7 days (n=5) , 14 days (n=5); \u003csup\u003eN\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003eNN\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between 3 days and 7 days, 14 days; \u003csup\u003eX\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003eXX\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between 7 days and 14 days. \u003cstrong\u003e(C)\u003c/strong\u003e Cells and supernatants in one-day group and two-day group were collected after vibration for 2.5 h. N = 3 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the 20 Hz group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/eb15f4ff135302b9dd500411.png"},{"id":57643429,"identity":"ce20298a-8b81-4830-845a-c4816c3a3784","added_by":"auto","created_at":"2024-06-03 18:19:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73684,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in lncRNA expression induced by vibration in HUVEC\u003c/p\u003e\n\u003cp\u003eUse qPCR to analyse the expression of lncRNA in HUVEC after vibration for 2.5 h. N = 3 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the control group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/d291cede2125b36fedc5b4ee.png"},{"id":57643430,"identity":"b48906d0-1446-490f-ae41-bd0666447b40","added_by":"auto","created_at":"2024-06-03 18:19:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":506658,"visible":true,"origin":"","legend":"\u003cp\u003eRole of knockdown of lncRNA MEG3 in vibration-induced HUVEC injury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Diagram of the vibration instrument used in the experiment. \u003cstrong\u003e(B)\u003c/strong\u003e Effect of vibration on the expression of lncRNA \u003cem\u003eMEG3\u003c/em\u003e. \u003cstrong\u003e(C)\u003c/strong\u003e Expression of FAM-modified siRNA with different doses of Lipofectamine 2000. \u003cstrong\u003e(D-F)\u003c/strong\u003e Transfection efficiency of three different doses of siRNA. \u003cstrong\u003e(G-J)\u003c/strong\u003e Changes of lncRNA \u003cem\u003eMEG3\u003c/em\u003e expression under different vibration frequencies.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with negative control group (n=3); \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between negative control-vibration group (n=3) and siRNA-vibration group (n=3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/2bb6226ffbc211315af4c3f5.png"},{"id":57643426,"identity":"a0ec7c66-af8d-4720-9c09-88103c10e540","added_by":"auto","created_at":"2024-06-03 18:19:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1339694,"visible":true,"origin":"","legend":"\u003cp\u003eVibration treatment of \u003cem\u003eMEG3\u003c/em\u003e knockdown HUVECs induces cells injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A \u003c/strong\u003eand\u003cstrong\u003e B)\u003c/strong\u003e Intracellular structural changes of cells in 20 Hz, two-day group (A) and 125 Hz, two-day group (B) were respectively observed at different magnifications of 1 μm-10 kx, 0.5 μm-25 kx, 1 μm-10 kx, 0.5 μm-30 kx, and 2 μm-5 kx. Nucleus pyknosis, abnormal chromatin or chromatin margination, swollen endoplasmic reticulum and mitochondria and vacuolization are indicated with red arrows. \u003cstrong\u003e(C\u003c/strong\u003e to\u003cstrong\u003e F)\u003c/strong\u003e Effect of vibration on cell viability in each group. N = 5 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the negative control group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/bdd5f6192490b028726dd51a.png"},{"id":57643431,"identity":"9d5e22ee-3cf4-4319-a31e-2f0a1f223b56","added_by":"auto","created_at":"2024-06-03 18:19:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":454561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMEG3\u003c/em\u003e regulates the expression of Bax, Caspase-3 and Bcl2 in vibration-induced damage in HUVECs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A \u003c/strong\u003eand\u003cstrong\u003e B)\u003c/strong\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Bax, Caspase3 and Bcl2 in HUVECs injury induced by vibration. \u003cstrong\u003e(C\u003c/strong\u003e and\u003cstrong\u003e D)\u003c/strong\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Bax in HUVECs injury induced by vibration. \u003cstrong\u003e(E \u003c/strong\u003eand\u003cstrong\u003e F)\u003c/strong\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Caspase3 in HUVECs injury induced by vibration. \u003cstrong\u003e(G \u003c/strong\u003eand\u003cstrong\u003e H)\u003c/strong\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Bcl2 in HUVECs injury induced by vibration. N = 3 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the negative control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between negative control-vibrative group and siRNA-vibrative group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/9c88b93ace587b4f396a8a4b.png"},{"id":57643806,"identity":"72b151df-d71c-459c-b7a1-37ca97e1f00c","added_by":"auto","created_at":"2024-06-03 18:27:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":529975,"visible":true,"origin":"","legend":"\u003cp\u003eCells and supernatants were collected after vibration for 2.5 h, and the expression level of vasoactive factors were measured by ELISA. N = 3 experimental replicates per group. N-C: negative control, siRNA-C: siRNA-control, N-V: negative control-vibrative, siRNA-V: siRNA-vibrative. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared with the negative control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between negative control-vibrative group and siRNA-vibrative group.\u003c/p\u003e\n\u003cp\u003eKnockdown of \u003cem\u003eMEG3\u003c/em\u003e affects the expression and secretion of vasoactive factors in vibration-induced HUVEC injury\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/dd8e8989fed527742002e5ce.png"},{"id":59644567,"identity":"64fd5ee2-4c13-45d6-a590-6567f4c1e2cb","added_by":"auto","created_at":"2024-07-04 08:30:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7508075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/cd050269-29ef-4435-9e3c-0e2c76414b91.pdf"},{"id":57643425,"identity":"e54cc2bf-a831-4e2c-9f41-6f7ba8194cf9","added_by":"auto","created_at":"2024-06-03 18:19:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":78774,"visible":true,"origin":"","legend":"","description":"","filename":"sup.docx","url":"https://assets-eu.researchsquare.com/files/rs-4454801/v1/0a560442ee3cd6006e61b575.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Knockdown of lncRNA MEG3 promotes damage of vascular endothelial cells induced by vibration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAbout 25% of the 3\u0026nbsp;billion workers worldwide are exposed to vibration in the workplace (International Institute for Labour Studies, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Currently, at least 2-million workers in China are exposed to hand-transmitted vibration (HTV) tools, and the main types of work are polishing, grinding, and sawing (Lin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Long-term exposure to HTV can lead to spontaneous hand symptoms and even lesions in the nervous, circulatory, muscular, or skeletal systems. Serious cases can further develop into occupational hand\u0026ndash;arm vibration disease (HAVS) (Mahmood et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shen \u003cem\u003eet al.\u003c/em\u003e 2017). HAVS can lead to the loss of the ability to work and live, bringing a huge economic burden to employers and the country. Vibration-induced white finger (VWF) is a typical manifestation of HAVS. HAVS development is irreversible. The pathogenesis of HAVS and influence on the vascular function of fingers have not been elucidated fully (Nilsson et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStudies have shown that vibration-induced vascular endothelial dysfunction help to cause HAVS. Destruction of the structure and function of vascular endothelial cells as well as dysregulation of the production and secretion of vasoactive factors may be key factors in vibration-induced vascular injury (Herrick \u003cem\u003eet al.\u003c/em\u003e 2005; Herrick \u003cem\u003eet al.\u003c/em\u003e 2012).\u003c/p\u003e \u003cp\u003eLong non-coding RNAs (lncRNAs) are located in the nucleus and are considered to have crucial regulatory roles in the proliferation (Kitagawa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sirey et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and apoptosis of cells, and invasion by cells (Evans et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tomita et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Wang \u003cem\u003eet al.\u003c/em\u003e showed that a lncRNA, maternally expressed gene 3 (\u003cem\u003eMEG3\u003c/em\u003e), may have an important role in the progression of endothelial dysfunction induced by hyperglycemia, and that knockout of \u003cem\u003eMEG3\u003c/em\u003e expression could aggravate inflammatory damage to endothelial cells. Knockout of \u003cem\u003eMEG3\u003c/em\u003e expression in human umbilical vein endothelial cells (HUVECs) has been shown to regulate the proliferation and apoptosis of endothelial cells by upregulating expression of B-cell lymphoma (Bcl)-2 and downregulating expression of Bax, caspase-3, and P53 (Wang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, plasma samples from humans and rats and HUVECs were selected to study changes in levels of vasoactive factors induced by vibration. We detected cellular damage \u003cem\u003ein vitro\u003c/em\u003e under vibration. We interfered with \u003cem\u003eMEG3\u003c/em\u003e expression in HUVECs \u003cem\u003ein vitro\u003c/em\u003e to further explore the effects of vibration-induced damage and the role of \u003cem\u003eMEG3\u003c/em\u003e in vibration-induced injury to vascular endothelial cells. Our data could provide: (i) a theoretical basis for revealing the epigenetic mechanism of HAVS; (ii) clues for exploring HAVS pathogenesis and finding early biomarkers. In these ways, early prevention, diagnosis, and treatment of HAVS could be achieved.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatments\u003c/h2\u003e \u003cp\u003eHUVECs were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. HUVEC culture medium (10% fetal bovine serum, penicillin, 0.25% streptomycin) were purchased from Thermo Fisher (MA, USA) and Bioshark (Beijing, China). Before the start of the experiment, cells were kept in an incubator at 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e. After observing cell morphology and growth, aspirate the old culture using a sterile Pasteur pipette, wash the culture flask with PBS. The cells were completely suspended by digestion and washing using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Then transfer the cells to a new flask, replenish with the appropriate amount of new medium, and place them in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for culture, and the experiment was started when HUVEC were cultured to 70\u0026ndash;80% abundance. The experimental group was a vibration group with multiple vibrational frequency gradients of 10\u0026ndash;100 Hz, 100\u0026ndash;200 Hz, and 500 Hz. The vibration frequencies and accelerations are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the schematic diagram of the vibration experiment is shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The acceleration values corresponding to the vibration exposure at each frequency were obtained by instrumental measurements. The control group had the same conditions as the experimental group except that they were not exposed to vibration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell ultrastructure detection\u003c/h2\u003e \u003cp\u003eAt the end of the vibration, cells were collected for fixation, embedding, then cutting (ultrathin sections) and staining. Changes in the internal structure of the cells, such as cell shape, mitochondria and endoplasmic reticulum, were observed under an electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eCell viability was assayed using the Cell Counting Kit CCK-8. At the end of vibration, 10 \u0026micro;l CCK-8 reagent (UE Landi, Suzhou, China) was added and incubated for 2.5 h. The absorbance value was measured at 450 nm (Swiss Sunrise Automatic Enzyme Labeler).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry (FCM) assay\u003c/h2\u003e \u003cp\u003eAfter the vibration treatment, the cells were harvested and stained with FITC-Annexin V and PI according to the FITC-Annexin V apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Flow cytometry (FACScan, BD Biosciences) was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular epidemiological survey of occupational population\u003c/h2\u003e \u003cp\u003eJudgmental sampling was used to divide 150 employees of a factory in Zhongshan City (Guangdong, China) into three groups, of which 50 patients with a chief complaint hospital diagnosis of HAVS were the vibration white finger group, 50 hand-transmitted vibration workers without HAVS in the same period as the vibration non-white finger group, and another 50 non-hand-transmitted vibration workers were selected as the control group. In the early morning, 4 ml/person of median venous blood from the elbow of the research subjects was uniformly collected, placed in an anticoagulation tube (see supplementary materials for population inclusion criteria). All methods were performed in accordance with the relevant guidelines and regulations. The survey was approved by the ethics board of the first affiliated hospital of Guangdong Pharmaceutical University (batch number: (2021) No. [116]).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRat tail vibration exposure model\u003c/h2\u003e \u003cp\u003eTwenty-five SPF-rated male SD rats, weighing 178.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 g and aged 7\u0026ndash;8 weeks, were randomly divided into five groups: the control group, 1-day vibration exposure group, 3-day vibration exposure group, 7-day vibration group, and 14-day vibration group, with five rats in each group. The vibration exposure conditions were as follows: The tail of each rat was exposed to vibration for 4 h/day at a vibration frequency of 125 Hz and an acceleration of 6.4 m/s\u003csup\u003e2\u003c/sup\u003e. The exposure conditions were chosen from the information provided by most studies on the frequency-related effects of changes in vibration injury (Wang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). After anesthesia, blood was collected from the abdominal aorta of the rats and euthanized by air embolization. All methods were performed in accordance with the relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetection of vasoactive factors by ELISA\u003c/h2\u003e \u003cp\u003eThe detection of the vasoactive factors leukotriene B4 (LTB4), 5-hydroxy tryptamine (5-HT), VEGF, interleukin β (IL-1β), Myosin light chain-2 (MLC2), Calcitonin gene-related peptide (CGRP) and Endothelin-1(ET-1) in human and rat plasma after thawing at room temperature, in cells, and supernatants was performed according to the ELISA kit procedure produced by MEIMIAN (Jiangsu China). The optical density values of each well were measured at 450 nm(Swiss Sunrise Automatic Enzyme Labeler), the standard curve was plotted, and the concentration levels of the above indicators were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eReal-time fluorescence quantitative reverse transcription PCR\u003c/h2\u003e \u003cp\u003eTotal RNAs were extracted using Trizol reagent (Invitrogen), equal amounts of total RNA were reverse transcribed using a cDNA synthesis kit (TaKaRa, Japan), and real-time fluorescent quantitative PCR (qRT-PCR) was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference to detect the relative expression levels. The primers used were as follows supplementary Table S2.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003elncRNA\u003c/b\u003e \u003cb\u003eMEG3\u003c/b\u003e \u003cb\u003eknock-down\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eBefore conducting lncRNA \u003cem\u003eMEG3\u003c/em\u003e knockdown experiments, the most suitable siRNA (small interfering RNA) transfection dose needs to be determined first. We selected FMA-modified siRNA fragments and used universal liposomes Lipofectamine 2000 (ThermoFisher, USA) as transfectants. The fluorescence intensity of each well was observed under a fluorescence microscope, and the liposome concentration with the strongest fluorescence was recorded. \u003cem\u003eMEG3\u003c/em\u003e siRNA and siRNA were transfected into HUVECs at a concentration of 50 nmol/L using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u0026rsquo;s instruction. \u003cem\u003eMEG3\u003c/em\u003e-specific siRNA and siRNA were designed and synthesized from GenePharma (Shanghai, China). Cells were harvested after 48 h for qRT-PCR, western blot, cell proliferation and apoptosis analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCellular protein content in lysates was assessed by the BCA protein assay (Thermo Fisher, USA). Cell protein lysates were separated in 10%SDS-PAGE and transferred to a 0.22\u0026micro;m polyvinylidene difluoride membrane. Membranes were subsequently incubated with specific antibodies (ABCAM, UK) overnight at 4\u0026deg;C. Secondary antibodies were added and incubated for 1 h at 37\u0026deg;C. The immunoreactive bands were visualized using the ECL kit (Thermo Fisher, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using the SPSS 21.0. The measured data were tested for normality, and data conforming to a normal distribution were described by (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{-}{X}\\)\u003c/span\u003e\u003c/span\u003e\u0026plusmn; S). Median (M) and interquartile spacing (\u003cem\u003eP\u003c/em\u003e25 to \u003cem\u003eP\u003c/em\u003e75) were used to describe the number of disobedience to normal distribution. One-way ANOVA or two-way ANOVA were used for comparison of means between multiple groups. Before comparing between groups, the sameness test of variance was performed on the data of each index, and the interaction between the treatment factors was analyzed. If the variance was equal, the least significant difference (LSD) rank test was used for pairwise comparison. Otherwise, use Dunnet's T3 for pairwise comparison. Rates of enumeration data were compared using the Pearson χ\u0026sup2; test or Fisher's exact test. All tests were two-tailed and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was defined as statistically significant. GraphPad was used for plotting.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVibration alters the viability and subcellular structure to induce HUVEC apoptosis\u003c/h2\u003e \u003cp\u003eCompared with the control group, HUVEC viability increased at 1, 1.5, 2, and 4 h, and HUVEC viability in the vibration group at 2.5 h and 3 h decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These data indicated that this vibration condition elicited the greatest effect upon HUVECs. Exposure to 125-Hz vibration could increase HUVEC viability for 3 h and 4 h. These results suggested that longer exposure could increase HUVEC viability, and could not be considered a beneficial effect of vibration at this frequency. Based on this result, we investigated whether there were other frequencies that reduced HUVEC viability. HUVEC viability decreased at 20 and 200 Hz, and increased at 10, 40, 80, and 130 Hz, compared with the control group, for 2.5 h. After exposure to vibration for 3 h, HUVEC viability decreased at 125 and 130 Hz and increased at 205 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Due to the survival status of HUVECs, only a 2-day experiment could be conducted. Compared with the control group, HUVEC viability increased at 10 Hz, and decreased at 20 and 125 Hz for 2.5 h of vibration. HUVEC viability increased at 20 and 125 Hz for 3 h and 4 h of vibration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Therefore, we selected a vibration time of 2.5 h and frequencies of 20 and 125 Hz as criteria for subsequent experiments.\u003c/p\u003e \u003cp\u003eTransmission electron microscopy revealed that the control group had normal nuclei, endoplasmic reticuli (ER), and mitochondria, but the structure was faint. The cytoplasm was abundant and dense, and a small amount of vacuolization was visible. In the 20-Hz, 2-day group, pyknosis, chromatin margination, and swollen ER were observed. The cytoplasm was abundant and dense, cell outgrowth was reduced, and some of the cytoplasm was vacuolated. In the 125-Hz, 2-day group, pyknosis, chromatin margination, nuclear-membrane irregularities, and considerable cytoplasm vacuolization were observed. ER were swollen and cell outgrowth was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eCompared with the control group, 125-Hz vibration for 2.5 h caused an increase in apoptosis in the 1-day group and 2-day group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These results suggested that vibration induced cell damage and promoted HUVEC apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Effect of vibration at 125 Hz for 1\u0026ndash;4 h on HUVEC viability. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003cb\u003e(B)\u003c/b\u003e Effect of vibration at different frequencies for 2.5 h (left) and 3 h (right) on one-day group HUVEC viability. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003cb\u003e(C)\u003c/b\u003e Effect of vibration at different frequencies for 2.5 h (left), 3 h (middle) and 4 h (right) on two-day group HUVEC viability. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003cb\u003e(D)\u003c/b\u003e Intracellular structural hanges were observed at different magnifications of 1 \u0026micro;m-10 kx, 2 \u0026micro;m-5 kx, 2 \u0026micro;m-4 kx, and 2 \u0026micro;m-5 kx. Nucleus pyknosis, chromatin margination, swollen endoplasmic reticulum and mitochondria, cell budding and vacuolization are indicated with red arrows. \u003cb\u003e(E\u003c/b\u003e to \u003cb\u003eF)\u003c/b\u003e Effect of vibration at 125 Hz (E) and 20 Hz (F) for 2.5 h on one-day group HUVEC apoptosis. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003cb\u003e(G\u003c/b\u003e to \u003cb\u003eH)\u003c/b\u003e Effect of vibration at 125 Hz (G) and 20 Hz (H) for 2.5 h on two-day group HUVEC apoptosis. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003cb\u003e(I)\u003c/b\u003e Flow cytometry was used to measure % of apoptotic cells. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the control group.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eVibration induces changes in the expression of vasoactive factors in the plasma of workers and rats, HUVECs, and HUVEC supernatants\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAll worker groups comprised 50 males. The mean age of the vibration-white-finger group was 39.86\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70 years and they had 11.92\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39 years of service. The mean age of the vibration-non-white-finger group was 35.92\u0026thinsp;\u0026plusmn;\u0026thinsp;6.61 years, and they had 6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;6.84 years of service. The mean age of the control group was 34.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75 years and they had 10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;6.21 years of service. There were significant differences in age, duration of service, and hand symptoms among these three groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Expression of LTB4, IL-1β, CGRP, and ET-1 in the vibration-white-finger group was higher than that in the vibration-non-white-finger group and control group. The distribution of MLC2 was reversed in these three groups, and differences in VEGF expression among these three populations was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eChanges in expression of vasoactive factors in rat plasma were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Expression of LTB4 in 3-, 7-, and 14-day groups was higher than that in the control group and 1-day group. VEGF expression in 7- and 14-day groups was higher than that in control, 1-, and 3-day groups. IL-1β expression in 3-, 7-, and 14-day groups was higher than that in control and 1-day groups. IL-1β expression in the 14-day group was higher than that in 3- and 7-day groups. MLC2 expression in 3-, 7-, and 14-day groups was lower than that in control and 1-day groups. CGRP expression in 7- and 14-day groups was lower than that in control, 1- and 3-day groups. ET-1 expression in 3-, 7-, and 14-day groups was higher than that in control and 1-day groups. ET-1 expression in the 14-day group was higher than that in 3- and 7-day groups.\u003c/p\u003e \u003cp\u003eWe also detected the changes in expression of vasoactive factors in HUVECs and HUVEC supernatants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Expression of LTB4, VEGF, IL-1β, CGRP, and ET-1 was increased in HUVECs and/or HUVEC supernatants in 20- and 125-Hz groups compared with that in the control group. MLC2 expression was reduced in HUVECs and HUVEC supernatants in the 125-Hz, 2-day group compared with that in the control group. LTB4 expression was decreased in HUVECs and HUVEC supernatants in the 125-Hz group compared with that in the 20-Hz group. Expression of VEGF and ET-1 was increased in HUVECs in the 125-Hz, 1-day group compared with that in the 20-Hz, 1-day group. MLC2 expression was decreased in HUVECs in the 125-Hz, 2-day group compared with that in the 20-Hz, 2-day group. CGRP expression was increased in the HUVEC supernatants of the 125-Hz, 1-day group and HUVECs in the 125-Hz, 2-day group, compared with that in the 20-Hz group with the same number of days of exposure. Thus, vibration could induce changes in the expression and secretion of the vasoactive factors LTB4, IL-1β, MLC2, CGRP, and ET-1 in HUVECs and HUVEC supernatants.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the basic situation of the three groups of workers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;50)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003enon-VWF (n\u0026thinsp;=\u0026thinsp;50)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVWF (n\u0026thinsp;=\u0026thinsp;50)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF/χ2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.92\u0026thinsp;\u0026plusmn;\u0026thinsp;6.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.86\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.979 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLength of service, y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;6.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.92\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.949 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21(29.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24(33.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26(36.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.016 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.048\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrinking, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32(36.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26(29.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31(34.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.713 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.079\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHand symptoms, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23(31.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48(64.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.348 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e[Note]: a is \u003cem\u003eF\u003c/em\u003e value; b is \u003cem\u003eχ2\u003c/em\u003e value; compared with the control group, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Comparison of plasma concentrations of six vasoactive factors among three groups of workers. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the control group (n\u0026thinsp;=\u0026thinsp;50); \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared between the vibration white finger (VWF) group (n\u0026thinsp;=\u0026thinsp;50) and the vibration non-white finger (non-VWF) group (n\u0026thinsp;=\u0026thinsp;50). \u003cb\u003e(B)\u003c/b\u003e Comparison of plasma concentrations of six vasoactive factors among five groups of rats. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05,\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the control group (n\u0026thinsp;=\u0026thinsp;5); \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared between 1 day (n\u0026thinsp;=\u0026thinsp;5) and 3 days (n\u0026thinsp;=\u0026thinsp;5), 7 days (n\u0026thinsp;=\u0026thinsp;5), 14 days (n\u0026thinsp;=\u0026thinsp;5); \u003csup\u003eN\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003eNN\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between 3 days and 7 days, 14 days; \u003csup\u003eX\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003eXX\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between 7 days and 14 days. \u003cb\u003e(C)\u003c/b\u003e Cells and supernatants in one-day group and two-day group were collected after vibration for 2.5 h. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the 20 Hz group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChanges in lncRNA expression induced by vibration in HUVECs\u003c/h2\u003e \u003cp\u003eIn 1-day groups, expression of the lncRNAs \u003cem\u003eMEG3\u003c/em\u003e, nuclear-enriched abundant transcript 1 (\u003cem\u003eNEAT1\u003c/em\u003e), taurine-upregulated gene 1 (\u003cem\u003eTUG1\u003c/em\u003e), and metastasis-associated lung adenocarcinoma transcript 1 (\u003cem\u003eMALAT1\u003c/em\u003e) decreased in the 20-Hz group and 125-Hz group compared with that in the control group. In 2-day groups, expression of \u003cem\u003eMEG3\u003c/em\u003e, \u003cem\u003eTUG1\u003c/em\u003e, and \u003cem\u003eMALAT1\u003c/em\u003e decreased in the 20-Hz group, but \u003cem\u003eNEAT1\u003c/em\u003e expression increased; \u003cem\u003eMEG3\u003c/em\u003e had increased expression in the 125-Hz group, but \u003cem\u003eTUG1\u003c/em\u003e expression decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These data suggested that vibration induced changes in the expression of \u003cem\u003eMEG3\u003c/em\u003e, \u003cem\u003eNEAT1\u003c/em\u003e, \u003cem\u003eTUG1\u003c/em\u003e and \u003cem\u003eMALAT1\u003c/em\u003e in HUVECs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUse qPCR to analyse the expression of lncRNA in HUVEC after vibration for 2.5 h. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the control group.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eVibration induces changes in\u003c/b\u003e \u003cb\u003eMEG3\u003c/b\u003e \u003cb\u003eexpression in HUVECs\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn 1-day groups, \u003cem\u003eMEG3\u003c/em\u003e expression decreased in the 20-Hz group and 125-Hz group compared with that in the control group. In 2-day groups, \u003cem\u003eMEG3\u003c/em\u003e expression decreased in the 20-Hz group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results indicated that vibration could cause changes in \u003cem\u003eMEG3\u003c/em\u003e expression. Fluorescence microscopy revealed that intracellular fluorescence could be observed in transfected HUVECs in each group. An appropriate fluorescence effect and transfection efficiency could be observed at the dose of 5 \u0026micro;L of liposome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared with the control group, expression of small interfering (si)RNA MEG3-3#5 mRNA was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;F), indicating that knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression had been achieved. In the 125-Hz, 1- and 2-day groups, compared with the negative control group, expression in the siRNA-control group and negative control-vibration group decreased; compared with the negative control-vibration group, \u003cem\u003eMEG3\u003c/em\u003e expression was decreased in the siRNA-vibration group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u0026ndash;J). In the 20-Hz, 1-day group, expression increased in the negative control-vibration group compared with that in the negative control group; in the 2-day group, expression in the siRNA-control group decreased, and expression in the negative control-vibration group increased. Compared with the negative control-vibration group, \u003cem\u003eMEG3\u003c/em\u003e expression in the siRNA-vibration group decreased. These results indicated that vibration could affect \u003cem\u003eMEG3\u003c/em\u003e expression in HUVECs, and that \u003cem\u003eMEG3\u003c/em\u003e had an important role in HUVEC damage induced by vibration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Diagram of the vibration instrument used in the experiment. \u003cb\u003e(B)\u003c/b\u003e Effect of vibration on the expression of lncRNA \u003cem\u003eMEG3\u003c/em\u003e. \u003cb\u003e(C)\u003c/b\u003e Expression of FAM-modified siRNA with different doses of Lipofectamine 2000. \u003cb\u003e(D-F)\u003c/b\u003e Transfection efficiency of three different doses of siRNA. \u003cb\u003e(G-J)\u003c/b\u003e Changes of lncRNA \u003cem\u003eMEG3\u003c/em\u003e expression under different vibration frequencies. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05,\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with negative control group (n\u0026thinsp;=\u0026thinsp;3); \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared between negative control-vibration group (n\u0026thinsp;=\u0026thinsp;3) and siRNA-vibration group (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eMEG3\u003c/b\u003e \u003cb\u003eon the internal structure in vibration-induced HUVECs injury\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eElectron microscopy showed that in the 20-Hz, 2-day group, the nucleus, ER, and mitochondria of the negative control group were normal, but the structure was fuzzy. The cytoplasm was abundant and dense, with a small amount of vacuolization. In the siRNA-control group, nuclear pyknosis, malformation, slightly irregular nuclear membrane, inconspicuous chromatin, relatively swollen ER and mitochondria, and some cytoplasmic vacuolization were observed. In the negative control-vibration group, nuclear pyknosis, chromatin margination, no swelling or expansion of ER, no obvious swelling of mitochondria, and some cytoplasmic vacuolization were observed. In the siRNA-vibration group, nuclear pyknosis and malformation, a relatively regular nuclear membrane, and chromatin margination were observed. ER and mitochondria were swollen, and vacuolization was observed in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn the 125-Hz, 2-day group, the nucleus, ER, and mitochondria were normal in the negative control group, chromatin margination was absent, the cytoplasm was dense, and some vacuolization was observed. In the siRNA-control group, we observed nuclear pyknosis and malformation, slightly irregular nuclear membranes, and inconspicuous chromatin. Rough endoplasmic reticuli (RER) were slightly dilated, ER ribosomes had been shed, mitochondria were swollen and ruptured, and some cytoplasm was vacuolated. In the negative control-vibration group, we observed nuclear pyknosis and chromatin margination. RER were arranged in a lamellar pattern, ribosomes were near ER membranes, mitochondria were swollen and ruptured, and a small part of the cytoplasm was vacuolated. In the siRNA-vibrative group, nuclear pyknosis, malformation, and chromatin margination were observed. Expanded RER were seen in the cytoplasm. Mitochondria were swollen and morphologically abnormal. Extensive vacuolization was observed in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the 20- and 125-Hz groups at 1 day, compared with the negative control group, HUVEC viability decreased in the siRNA-control group. In the 125-Hz, 2-day group, compared with the negative control group, HUVEC viability decreased in the siRNA-control group and negative control-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, HUVEC viability increased in the siRNA-vibration group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;F).\u003c/p\u003e \u003cp\u003eCompared with the negative vibration group, cell damage in the \u003cem\u003eMEG3\u003c/em\u003e knockdown vibration group was more obvious and severe. These data suggested that \u003cem\u003eMEG3\u003c/em\u003e may be involved in vibration-induced injury and may inhibit HUVEC damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A\u003c/b\u003e and \u003cb\u003eB)\u003c/b\u003e Intracellular structural changes of cells in 20 Hz, two-day group (A) and 125 Hz, two-day group (B) were respectively observed at different magnifications of 1 \u0026micro;m-10 kx, 0.5 \u0026micro;m-25 kx, 1 \u0026micro;m-10 kx, 0.5 \u0026micro;m-30 kx, and 2 \u0026micro;m-5 kx. Nucleus pyknosis, abnormal chromatin or chromatin margination, swollen endoplasmic reticulum and mitochondria and vacuolization are indicated with red arrows. \u003cb\u003e(C\u003c/b\u003e to \u003cb\u003eF)\u003c/b\u003e Effect of vibration on cell viability in each group. N\u0026thinsp;=\u0026thinsp;5 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the negative control group.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMEG3\u003c/b\u003e \u003cb\u003eregulates the expression of Bax, caspase-3, and Bcl-2 during vibration-induced damage to HUVECs\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn the 20-Hz, 1-day group, compared with the negative control group, Bax expression was decreased in the negative control-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, Bax expression was increased in the siRNA-control group and negative control-vibration group, and Bax expression in the siRNA-vibration group was higher than that in the siRNA-control group. In the 20-Hz, 1-day group, caspase-3 expression was decreased in the negative siRNA-control group and increased in the siRNA-vibration group compared with that in the negative control group; compared with the negative control-vibration group, caspase-3 expression was increased in the siRNA-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, caspase-3 expression was decreased in the negative control-vibration group and siRNA-vibration group; compared with the negative control-vibration group, caspase-3 expression was decreased in the siRNA-vibration group. In the 20-Hz, 1-day group, compared with the negative control group, Bcl-2 expression was decreased in the siRNA-control group and negative control-vibration group; compared with the negative control-vibration group, Bcl-2 expression was increased in the siRNA-vibration group. In the 20-Hz, 2-day group, compared with the negative control group, Bcl-2 expression was reduced in the negative control-vibration group and siRNA-vibration group; compared with the negative control-vibration group, Bcl-2 expression was reduced in the siRNA-vibration group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the 125-Hz, 1-day group, compared with the negative control group, Bax expression was decreased in the siRNA-control group and siRNA-vibration group, and Bax expression in the siRNA-vibration group was lower than that in the siRNA-control group. In the 2-day group, compared with the negative control group, Bax expression was decreased in the negative control-vibration group and siRNA-vibration group. In the 125-Hz, 2-day group, caspase-3 expression was decreased in the negative siRNA-control group compared with that in the negative control group. In the 125-Hz, 2-day group, compared with the negative control group, Bcl-2 expression was decreased in the siRNA-control group and siRNA-vibration group, and Bcl-2 expression was increased in the negative control-vibration group; compared with the negative control-vibration group, Bcl-2 expression was decreased in the siRNA-vibration group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results showed that vibration-induced expression of apoptotic proteins (Bax and caspase-3) decreased, whereas that of an anti-apoptotic protein (Bcl-2) increased. After interfering with \u003cem\u003eMEG3\u003c/em\u003e, the expression of Bax, caspase-3, and Bcl2 decreased. This finding suggested that \u003cem\u003eMEG3\u003c/em\u003e may be involved in vibration-induced HUVEC injury by regulating the expression of Bax, caspase-3, and Bcl-2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A\u003c/b\u003e and \u003cb\u003eB)\u003c/b\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Bax, Caspase3 and Bcl2 in HUVECs injury induced by vibration. \u003cb\u003e(C\u003c/b\u003e and \u003cb\u003eD)\u003c/b\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Bax in HUVECs injury induced by vibration. \u003cb\u003e(E\u003c/b\u003e and \u003cb\u003eF)\u003c/b\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Caspase3 in HUVECs injury induced by vibration. \u003cb\u003e(G\u003c/b\u003e and \u003cb\u003eH)\u003c/b\u003e The effect of \u003cem\u003eMEG3\u003c/em\u003e on the expression levels of Bcl2 in HUVECs injury induced by vibration. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the negative control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared between negative control-vibrative group and siRNA-vibrative group.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eMEG3\u003c/b\u003e \u003cb\u003eon vasoactive factors in vibration-induced damage to HUVECs\u003c/b\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn the 20-Hz, 1-day group, compared with the negative control group, ET-1 expression was increased and VEGF expression was decreased in HUVECs; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, VEGF expression was increased. Expression of IL-1β, CGRP, and ET-1 was increased in HUVEC supernatants; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, VEGF expression was increased but expression of ET-1 and IL-1β decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the 20-Hz, 2-day group, compared with the negative control group, expression of IL-1β, 5-HT, CGRP, and ET-1 was increased and VEGF expression was decreased in HUVECs; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibrative group, CGRP expression was increased and expression of LTB4, IL-1β, 5-HT, and ET-1 was decreased. Expression of LTB4, IL-1β, 5-HT, CGRP, and ET-1 was increased and VEGF expression was decreased in HUVEC supernatants; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, CGRP expression was increased and expression of LTB4, VEGF, IL-1β, 5-HT, and ET-1 was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the 125-Hz, 1-day group, compared with the negative control group, expression of LTB4, IL-1β, 5-HT, and ET-1 in HUVECs was increased; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, expression of IL-1β, 5-HT, and ET-1 was decreased. Expression of 5-HT and CGRP was increased in HUVEC supernatants; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, VEGF expression was increased and expression of ET-1 and 5-HT was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the 125-Hz, 2-day group, compared with the negative control group, expression of LTB4, IL-1β, and ET-1 was increased and VEGF expression was decreased in HUVECs; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, VEGF expression was increased and expression of LTB4 and IL-1β was decreased. Expression of LTB4, IL-1β, 5-HT, and ET-1 was increased in HUVEC supernatants; after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, compared with the negative control-vibration group, expression of LTB4 and 5-HT was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results indicated that \u003cem\u003eMEG3\u003c/em\u003e regulated the expression of vasoactive factors in vibration-induced inflammation. Hence, \u003cem\u003eMEG3\u003c/em\u003e may be involved in vibration-induced damage to HUVECs by regulating the expression of vasoactive factors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCells and supernatants were collected after vibration for 2.5 h, and the expression level of vasoactive factors were measured by ELISA. N\u0026thinsp;=\u0026thinsp;3 experimental replicates per group. N-C: negative control, siRNA-C: siRNA-control, N-V: negative control-vibrative, siRNA-V: siRNA-vibrative. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared with the negative control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, compared between negative control-vibrative group and siRNA-vibrative group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies on the pathophysiology of HAVS have focused mainly on its clinical manifestations and diagnosis. Studies on the mechanism of injury to vascular endothelial cells caused by vibration are scarce (Budd \u003cem\u003eet al.\u003c/em\u003e 2018). Previously, we found that Chinese workers were exposed to high intensity and long duration of vibration without effective protective measures, and that the hazards of vibration were seriously underestimated or ignored (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The effect of vibration on peripheral vascular function has been the focus of HAVS research, and vasoactive factors help to regulate vascular function.\u003c/p\u003e \u003cp\u003eWe measured expression of the vasoactive factors LTB4, IL-1β, CGRP, MLC2, and ET-1 using \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models of vibration exposure. This approach provided a theoretical basis for future studies on the pathogenesis of HTV-induced HAVS. In addition, few studies have investigated the effect of vibration on cells \u003cem\u003ein vitro\u003c/em\u003e. White \u003cem\u003eet al.\u003c/em\u003e proposed that the absolute size of the temporal gradient and the frequency/duration of each pulse cycle had roles in the biomechanical transduction of fluid vibration in endothelial cells (2004). Therefore, we also employed a cellular model to investigate the effects of, and damage of vibration to, cells. By ultrastructural observation we found, for the first time, that vibration could induce changes in the viability of, and damage to, HUVECs.\u003c/p\u003e \u003cp\u003eLTB4 is an important metabolite of arachidonic acid. It is produced by the action of 5-lipoxygenase and other enzymes. It has a powerful activating effect on leukocytes, stimulating their chemotaxis and aggregation, releasing oxygen radicals and lysosomal enzymes, and increasing vascular permeability and vascular-wall constriction (Rosenblum \u003cem\u003eet al.\u003c/em\u003e 1985; Woodward et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Our \u003cem\u003ein vivo\u003c/em\u003e experimental results showed that, in workers exposed to HTV, those with a higher plasma level of LTB4 were more likely to suffer from VWF. In rats, the longer the duration of vibration, the higher was the LTB4 expression in plasma, suggesting that there may be a dose\u0026ndash;response relationship between LTB4 expression and the duration of vibration exposure. The \u003cem\u003ein vitro\u003c/em\u003e HUVEC model revealed LTB4 expression in vibration groups to be higher than that in the control group.\u003c/p\u003e \u003cp\u003eVEGF acts specifically and multilaterally on vascular endothelial cells, causing their proliferation, thereby promoting neovascularization. However, in the present study, VEGF expression was not associated with VWF. This finding may have been due to the small sample size of our study. The increased VEGF expression in plasma in the 7- and 14-day groups of rats may have been because repair of vascular endothelial cells was at a compensatory stage. The increased VEGF expression in HUVECs in the 2-day vibration group and no change in VEGF expression in HUVEC supernatants may have been due to impaired secretion of VEGF in HUVECs as a result of prolonged vibration.\u003c/p\u003e \u003cp\u003eIL-1β is a key mediator of intercellular communication in the immune system. It is also a key driver of local and systemic immune responses in atherosclerotic cardiovascular disease (Viana-Huete \u003cem\u003eet al.\u003c/em\u003e 2019). Our \u003cem\u003ein vivo\u003c/em\u003e results showed that the higher the IL-1β expression in a population exposed to HTV, the greater was the chance of developing occupational HAVS. In rats, the longer the duration of vibration, the higher was the IL-1β expression in plasma, suggesting that there may be a dose\u0026ndash;response relationship between IL-1β expression and duration of exposure to vibration.\u003c/p\u003e \u003cp\u003eMyosin consists of one pair of heavy chains and two pairs of light chains. Phosphorylation of myosin light chains is the basis for increasing vascular endothelial permeability (Hu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We showed that vibration decreased MLC2 expression in rat plasma and HUVECs \u003cem\u003ein vitro\u003c/em\u003e. Also, if the vibration lasted longer, considerable necrosis of HUVEC occurred, and then expression in vibration groups was lower than that in the control group. We speculated that humans have a regulatory function in which short-term vibration increases MLC2 secretion.\u003c/p\u003e \u003cp\u003eCGRP is the main transmitter of sensory nerves. It is distributed widely in central and peripheral tissues, and produces biological effects by interacting with specific receptors. CGRP can elicit vasodilatation, protect vascular endothelial cells, and inhibit the proliferation of smooth muscle cells (Rosenfeld et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Brain et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). We observed that vibration decreased CGRP expression rat tails. Vibration increased CGRP expression significantly in human plasma and HUVECs. The different results between humans and rats may have been because stress in humans can be regulated by the immune system, whereas rats exhibit a short-term stress response. The pathology associated with sensory-nerve deficits in patients with HAVS has been described, but the pathogenesis of these deficits has not, which merits requires further investigation.\u003c/p\u003e \u003cp\u003eET-1 is a potent vasoconstrictor secreted by vascular endothelial cells that mediates vasoconstriction, proinflammatory effects, and mitogenesis. ET-1 overproduction may be important in cardiovascular diseases (Davie et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models showed that vibration increased ET-1 expression, which led to HUVEC injury. We elucidated that, under different frequencies and times of damage to HUVECs, changes in the expression of LTB4, VEGF, IL-1β, MLC2, CGRP, and ET-1 in vibration-induced \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models may be involved in HAVS pathogenesis. However, quantifying the effects of vibration exposure accurately is challenging, and the number of validated biomarkers of exposure is limited.\u003c/p\u003e \u003cp\u003eThe association between lncRNAs and HAVS is not clear. We found that vibration induced altered expression of the lncRNAs \u003cem\u003eMEG3\u003c/em\u003e, \u003cem\u003eNEAT1\u003c/em\u003e, \u003cem\u003eTUG1\u003c/em\u003e, and \u003cem\u003eMALAT1\u003c/em\u003e in HUVECs. \u003cem\u003eMEG3\u003c/em\u003e expression has been shown to be significantly higher in the plasma of patients suffering from sepsis compared with that in healthy controls (Yuan \u003cem\u003eet al.\u003c/em\u003e 2020). However, it has also been shown that \u003cem\u003eMEG3\u003c/em\u003e expression decreases in the retinal epithelial cells of diabetic mice under oxidative stress and high-glucose stimulation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eMEG3\u003c/em\u003e expression may differ in different conditions or diseases. We suggest that lncRNAs may be affected by the frequency and duration of vibration, which may lead to a different extent of expression. lncRNAs have a very important regulatory role in vascular endothelial inflammation and may be involved in vibration-induced endothelial injury. The specific role of lncRNAs in HAVS progression will be explored further in cellular experiments and animals by our research team, and may reveal HAVS pathogenesis.\u003c/p\u003e \u003cp\u003eWe found that \u003cem\u003eMEG3\u003c/em\u003e was involved in the regulation of damage, apoptotic proteins, and vasoactive factors in HUVECs induced by vibration. Vibration treatment of HUVECs with knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression caused a further reduction in cell viability and aggravation of injury. Bax expression decreased in the 1- and 2-day groups for 20-Hz vibration. In the 1-day group, caspase-3 expression increased and decreased in the 2-day group, data which align with results from work by Song and colleagues. Vibration induced a decrease in the expression of the apoptotic proteins Bax and caspase-3. After knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, the expression of Bax and caspase-3 decreased more significantly, and Bcl-2 expression increased. These findings suggest that \u003cem\u003eMEG3\u003c/em\u003e may be involved in the vibration-induced injury to vascular endothelial cells by regulating the expression of Bax, caspase-3, and Bcl-2.\u003c/p\u003e \u003cp\u003eVibration increased the expression of LTB4, 5-HT, IL-1β, CGRP, and ET-1, and decreased the expression of VEGF when \u003cem\u003eMEG3\u003c/em\u003e was intact. After knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, expression of IL-1β, 5-TH, CGRP, and VEGF was increased. Expression of LTB4, IL-1β, ET-1, and CGRP in the siRNA-vibration group was lower than that in the negative control-vibration group. These data suggested that, under the influence of vibration and siRNA, expression of LTB4, IL-1β, ET-1, CGRP, and VEGF was reversed.\u003c/p\u003e \u003cp\u003eAfter knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression, VEGF expression was increased in the 1-day group, but decreased in the 2-day group, which may be a way that cells protect themselves from stress. Studies have shown that \u003cem\u003eMEG3\u003c/em\u003e affects the expression of inflammatory factors. Wang \u003cem\u003eet al.\u003c/em\u003e found that knockout of \u003cem\u003eMEG3\u003c/em\u003e expression increased the expression of VEGF, TNF-α, and IL-6, significantly in HUVECs, suggesting that \u003cem\u003eMEG3\u003c/em\u003e may inhibit the secretion of inflammatory factors (2018). Conversely, Liu \u003cem\u003eet al.\u003c/em\u003e showed that downregulation of \u003cem\u003eMEG3\u003c/em\u003e expression inhibited the secretion of TNF-α, IL-1β, and IL-6 in lipopolysaccharide-treated human dental pulp cells (2021). However, our data suggest that downregulation of \u003cem\u003eMEG3\u003c/em\u003e expression has different effects on the expression of vasoactive factors under different vibration conditions. There were differences between the \u003cem\u003ein vitro\u003c/em\u003e cell model and actual human exposure to vibration, which merit further study.\u003c/p\u003e \u003cp\u003eKnockdown of \u003cem\u003eMEG3\u003c/em\u003e expression in HUVECs can regulate the proliferation and apoptosis of endothelial cells by upregulating expression of Bcl-2 and downregulating expression of Bax, caspase-3, and P53 (Wang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, Liu and colleagues found that downregulation of \u003cem\u003eMEG3\u003c/em\u003e expression in HUVECs induced apoptosis and inhibited cell proliferation (2021). In addition, knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression reduced neovascularization and VEGF-induced angiogenesis in alkali-burned corneas through stromal cell-derived factor-1/chemokine receptor-4 (SDF-1/CXCR4) and Smad2/3 signaling pathways (Shen et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eMEG3\u003c/em\u003e may have different roles in the proliferation and apoptosis of endothelial cells and inflammatory injury due to different conditions for cell growth. Therefore, more studies are needed to understand the role and mechanism of action of \u003cem\u003eMEG3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMEG3\u003c/em\u003e is involved in the physiological and pathologic processes of several vascular diseases. \u003cem\u003eMEG3\u003c/em\u003e is an effective indicator of changes in vascular structure and function. Increasing numbers of studies have shown that lncRNAs are regulators of protein-coding genes, and that their dysfunction affects the occurrence and development of diseases. We discovered that, according to electron microscopy, downregulation of \u003cem\u003eMEG3\u003c/em\u003e expression aggravated cell damage. We conclude that \u003cem\u003eMEG3\u003c/em\u003e has a protective role in the cell damage induced by vibration.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMEG3\u003c/em\u003e may be involved or have a regulatory role in the inflammatory damage experienced by vascular endothelial cells induced by vibration. We used a strategy of gene-expression inhibition to explore the relationship between \u003cem\u003eMEG3\u003c/em\u003e and vibration-induced damage to vascular endothelial cells. We clarified the expression pattern of \u003cem\u003eMEG3\u003c/em\u003e and its role in vibration-induced inflammatory damage and disorders of secretion of vasoactive substances. Thus, our data could reveal the molecular mechanism of HAVS, and provide clues and a theoretical basis for the early prevention and treatment of HAVS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood samples of this study population were approved by the Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University, Guangdong Province, batch number: (2021) No. [116], and the written informed consent of the research subjects was obtained. All methods were performed in accordance with the relevant guidelines and regulations. The animal experiments in this study have been approved by the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University (acceptance number: gdpulac2021212). All methods were performed in accordance with the relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent obtained from individuals participating in stakeholder interviews.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China(82204007); the Natural Science Foundation of Guangdong Province-General Project (2022A1515012412); the Natural Science Foundation of Guangdong Province-General Project (2022A1515011357); Major basic and applied basic research projects of Guangdong Province of China (2019A1515110492); Guangzhou Science and Technology Planning Project(201904010222), Guangdong Medical Science and Technology Research Fund (A2022210).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHongyu Yang contributed to the overall planning of the experiment, the technical guidance of the experiment, and the guidance of the writing of the thesis. Fanfei Zeng contributed to the thesis writing. Ziyu Chen contributed to the questionnaire survey and experimental operation. Kuan Hu contributed to the data analysis. Qingsong Chen contributed to the questionnaire design and experimental design. Yun Xia contributed to the experimental planning and thesis guidance. Xiuwen Hu contributed to the questionnaire survey, experimental operation and data analysis. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Medical Laboratory Animal Center provided by the Guangdong Provincial Occupational Disease Prevention and Control Institute. The authors thank workers at the Guangsheng factory in Zhongshan City, Guangdong Province for providing blood samples.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature. 1985; https://doi.org/10.1038/313054a0\u003c/li\u003e\n\u003cli\u003eBudd D, Holness DL. Raising awareness of hand-arm vibration syndrome (HAVS) using posters. Work. 2018; https://doi.org/10.3233/WOR-182775\u003c/li\u003e\n\u003cli\u003eChen Q, Xiao B, Chen J, Wen W, Xu G, Li W, Yan H. A cross-sectional study of hand-transmitted vibration hazards in six types of workers exposed to vibration. Chinese occupational medicine, 2012, 39(4): 298-299, 302.\u003c/li\u003e\n\u003cli\u003eDavie NJ, Schermuly RT, Weissmann N, Grimminger F, Ghofrani HA. The science of endothelin-1 and endothelin receptor antagonists in the management of pulmonary arterial hypertension: current understanding and future studies. Eur J Clin Invest. 2009; https://doi.org/10.1111/j.1365-2362.2009.02120.x\u003c/li\u003e\n\u003cli\u003eEvans JR, Feng FY, Chinnaiyan AM. The bright side of dark matter: lncRNAs in cancer. J Clin Invest. 2016; https://doi.org/10.1172/JCI84421\u003c/li\u003e\n\u003cli\u003eHerrick A L. Pathogenesis of Raynaud\u0026apos;s phenomenon. Rheumatology. Oxford; 2005.\u003c/li\u003e\n\u003cli\u003eHerrick AL. The pathogenesis, diagnosis and treatment of Raynaud phenomenon. Nat Rev Rheumatol. 2012; https://doi.org/10.1038/nrrheum.2012.96\u003c/li\u003e\n\u003cli\u003eHu S, Wu G, Wang S, Liu B. Effects and potential mechanism of myosin light chain kinase in cardiac hypertrophy induced by angiotensin Ⅱ. South China Journal of Cardiovascular Diseases. 2018; 24, 87-91.\u003c/li\u003e\n\u003cli\u003eInternational Institute for Labour Studies. World of Work Report 2013: Repairing the Economic and Social Fabric International Labour Organisation. 2013.\u003c/li\u003e\n\u003cli\u003eKitagawa M, Kitagawa K, Kotake Y, Niida H, Ohhata T. Cell cycle regulation by long non-coding RNAs. Cell Mol Life Sci. 2013; https://doi.org/10.1007/s00018-013-1423-0\u003c/li\u003e\n\u003cli\u003eLin W, Chunzhi Z, Qiang Z, Kai Z, Xiaoli Z. The study on hand-arm vibration syndrome in China. Ind Health. 2005; https://doi.org/10.2486/indhealth.43.480\u003c/li\u003e\n\u003cli\u003eLiu M, Chen L, Wu J, Lin Z, Huang S. Long noncoding RNA MEG3 expressed in human dental pulp regulates LPS-Induced inflammation and odontogenic differentiation in pulpitis. Exp Cell Res. 2021; https://doi.org/10.1016/j.yexcr.2021.112495\u003c/li\u003e\n\u003cli\u003eMahmood F, Ferguson KB, Clarke J, Hill K, Macdonald EB, Macdonald DJM. Hand-arm vibration in orthopaedic surgery: a neglected risk. Occup Med (Lond). 2017; https://doi.org/10.1093/occmed/kqx154\u003c/li\u003e\n\u003cli\u003eNilsson T, Wahlstr\u0026ouml;m J, Burstr\u0026ouml;m L. Hand-arm vibration and the risk of vascular and neurological diseases-A systematic review and meta-analysis. PLoS One. 2017; https://doi.org/journal.pone.0180795\u003c/li\u003e\n\u003cli\u003eRosenblum WI. Constricting effect of leukotrienes on cerebral arterioles of mice. Stroke. 1985; https://doi.org/10.1161/01.str.16.2.262\u003c/li\u003e\n\u003cli\u003eRosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WW, Evans RM. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature. 1983; https://doi.org/10.1038/304129a0\u003c/li\u003e\n\u003cli\u003eShen SC, House RA. Hand-arm vibration syndrome: What family physicians should know. Can Fam Physician. 2017 Mar;63(3):206-210.\u003c/li\u003e\n\u003cli\u003eShen T, Wu Y, Cai W, Jin H, Yu D, Yang Q, Zhu W, Yu J. 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Rev Esp Cardiol (Engl Ed). 2019; https://doi.org/10.1016/j.rec.2019.03.006\u003c/li\u003e\n\u003cli\u003eWang Z, Ding L, Zhu J, Su Y, Wang L, Liu L, Ma Q, Yao H. Long non-coding RNA MEG3 mediates high glucose-induced endothelial cell dysfunction. Int J Clin Exp Pathol. 2018 Mar 1;11(3):1088-1100.\u003c/li\u003e\n\u003cli\u003eWhite CR, Haidekker MA, Stevens HY, Frangos JA. Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration. J Physiol. 2004; https://doi.org/10.1113/jphysiol.2003.059899\u003c/li\u003e\n\u003cli\u003eWoodward DF, Weichman BM, Gill CA, Wasserman MA. The effect of synthetic leukotrienes on tracheal microvascular permeability. Prostaglandins. 1983; https://doi.org/10.1016/0090-6980(83)90142-9\u003c/li\u003e\n\u003cli\u003eYuan C, Jin N. Expression of LncRNA MEG3 in plasma of patients with sepsis and its clinical significance. Chin J Clin Lab Sci, 2020; https://doi.org/10.13602/j.cnki.jcls.2020.09.09\u003c/li\u003e\n\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":"","lastPublishedDoi":"10.21203/rs.3.rs-4454801/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4454801/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHand\u0026ndash;arm vibration syndrome (HAVS) is caused by long-term exposure to hand-transmitted vibration (HTV), and its pathogenesis has not been elucidated fully. We explored the molecular mechanism of HAVS and provided clues and a theoretical basis for the early prevention and treatment of HAVS. After vibration, samples were collected from the plasma of human workers, plasma of rat tails, and human umbilical vein endothelial cells (HUVECs). ELISAs were used to measure the expression of vasoactive factors. Cell Counting Kit-8 and electron microscopy were used to detect cell damage. Flow cytometry was employed to detect apoptosis. Real-time reverse transcription-polymerase chain reaction was used to measure the expression of long non-coding RNAs (lncRNAs). Western blotting was used to measure the expression of apoptosis-related proteins. Vibration could cause cell damage, apoptosis, and changes in the expression vasoactive factors and lncRNAs. The lncRNA maternally expressed gene 3 (\u003cem\u003eMEG3\u003c/em\u003e) had a significant regulatory effect on cell damage, apoptotic proteins, and vascular regulatory factors in the HUVEC damage induced by vibration, as shown by the further decrease in viability and aggravation of injury after knockdown of \u003cem\u003eMEG3\u003c/em\u003e expression in HUVECs treated with vibration. Expression of vasoactive factors and apoptosis-related proteins was changed after interfering with \u003cem\u003eMEG3\u003c/em\u003e expression. In conclusion, vibration can affect the expression of vasoactive factors and lncRNA, and cause damage to vascular endothelial cells. \u003cem\u003eMEG3\u003c/em\u003e may be involved in the inflammatory damage to vascular endothelial cells induced by vibration.\u003c/p\u003e","manuscriptTitle":"Knockdown of lncRNA MEG3 promotes damage of vascular endothelial cells induced by vibration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 18:19:02","doi":"10.21203/rs.3.rs-4454801/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":"d5cfb18c-8ab2-4a5c-9f31-c6f49c3bd515","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-04T08:22:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-03 18:19:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4454801","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4454801","identity":"rs-4454801","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}