The Potential Effect of Vitamin D Supplement on Selected Coagulability Predictors in Vape-Exposed Female Rats

1 The Potential Effect of Vitamin D Supplement on Selected 2 Coagulability Predictors in Vape-Exposed Female Rats 3 4 Aman M. Hammad1, Mahmoud Abu Samak1*, Rana Abu Farha1, Lujain F. Alzaghari 2, 5 Abdelrahim Alqudah3, Diana Malaeb4, Khaldoun Rasem Shnewer5, Souheil Hallit6, Muna 6 Barakat1* 7 1 Department of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Applied Science Private 8 University, Amman 11937, Jordan, [email protected], [email protected], 9 [email protected], [email protected] 10 2 Medab Pharmacy, Madaba, Jordan. [email protected] 11 3 Department of Clinical Pharmacy and Pharmacy Practice, Faculty of Pharmaceutical Sciences, The 12 Hashemite University, Zarqa 13133, Jordan. [email protected] 13 4 College of Pharmacy, Gulf Medical University, Ajman, UAE. [email protected] 14 5 Smart Medical Lab, Amman, Jordan. [email protected] 15 6 School of Medicine and Medical Sciences, Holy Spirit University of Kaslik, Jounieh, Lebanon, 16 [email protected] 17 *Correspondence: 18 Muna Barakat, Department of clinical pharmacy and therapeutics, Faculty of Pharmacy, Applied Science 19 Private University, Amman, 11937, Jordan, Email [email protected] 20 Mahmoud Abu Samak, Department of clinical pharmacy and therapeutics, Faculty of Pharmacy, 21 Applied Science Private University, Amman, 11937, Jordan, Email: [email protected] 22 23 24 25 26 27 28 29 30 31 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 32 Abstract 33 Background: Vaping and vitamin D deficiency impact blood coagulation and health. This study 34 aimed to investigate the effects of vitamin D supplementation on coagulation predictors in female 35 rats exposed to E-cigarette vaping. 36 Objective: To examine the effect of vaping alone and vaping with different VD doses on some 37 coagulation predictors, lungs, liver, and kidney functions 38 Methods: Forty-two female Wistar rats were divided into six groups, including vaping and non- 39 vaping with high (50,000 IU) and low (1000 IU) vitamin D doses. Blood samples and 40 histopathological analyses were conducted after one and three months. Nicotine, cotinine, 41 Interleukin-6 (IL-6), D-dimer, coagulation factor X (FX), thrombomodulin (TM), Alanine 42 Transaminase (ALT), and Creatinine levels were analyzed. Additionally, histopathological 43 analyses were conducted on the rats' liver, kidney, and lung. 44 Results: Exposing rats to vaping for one month caused a significant acute increase in D-dimer, 45 FX, and TM levels to 4402.05 ng/mL ± 785.15, 1.8687 μg/mL ± 0.3132, and 34.71 ng/mL ± 8.42, 46 respectively. However, after three months of exposure, those levels decreased significantly 47 compared to the one-month levels. Supplementation of the vape-exposed rats with a high vitamin 48 D dose reduced levels of IL-6, D-dimer, FX, and TM levels to become 93.285 pg/mL ± 12.715, 49 439.95 ng/mL ± 294.05, 0.647 μg/mL, and 17.375 ng/mL ± 3.895, respectively, at the end of the 50 three months. Moreover, vaping rats supplemented with the low and high doses of vitamin D had 51 significantly lower nicotine and cotinine levels than the EC group, with a p-value of <0.0001. The 52 histopathological examination revealed that the rat’s lung had necrotic pneumonia when exposed 53 to vaping without vitamin D treatment. Moreover, all vaping groups had an alveolar hemorrhage. 54 Bacterial pneumonia was seen in the high-dose vitamin D vape-exposed group. However, the 55 histopathological examination of the liver indicated no major differences between the groups. 56 One month of vaping raised D-dimer, FX, and TM levels, which decreased after three months. 57 High-dose vitamin D supplementation reduced IL-6, D-dimer, and FX levels while increasing TM 58 levels after three months. Vaping rats receiving vitamin D had lower nicotine and cotinine levels. 59 Histopathological findings showed necrotic pneumonia and alveolar hemorrhage in vaping rats, 60 with bacterial pneumonia in the high-dose group. 61 Conclusion: Vaping activates inflammatory and coagulation pathways, while high-dose vitamin 62 D appears to mitigate inflammation and blood coagulation issues associated with vaping, 63 potentially aiding in reducing nicotine dependence. 64 Keywords: Vitamin D, E-cigarette, D-dimer, Coagulation factor X, Creatinine. 65 66 67 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 68 Introduction 69 Smoking significantly contributes to early mortality and public health issues globally. E-cigarettes 70 (E-cigs), emerging around 2006-2009, have gained popularity, particularly among young adults, 71 with over 683,300 users in Jordan as of 2020 [1-4]. 72 E-cigs are devices that heat a solution usually composed of propylene glycol or glycerin, nicotine, 73 and flavoring ingredients to produce an aerosol, also known as vapor [5]. While traditional 74 smokers are switching to vaping as a quitting method, concerns arise regarding their role in 75 cardiovascular diseases. Yet, some recent studies concluded that e-cigs are not blameless for 76 causing cardiovascular diseases, including blood coagulation problems. A recent study has shown 77 that the thermal decomposition of vape components generates harmful compounds, which are 78 associated with a significant enhancement in platelet aggregation in vape-exposed mice, 79 suggesting an elevated risk of thrombosis-related cardiovascular disorders[6-8]. Concurrently, 80 vitamin D (VD) is highlighted for its health-modulating effects, including its anticoagulant 81 properties and association with reduced thrombosis risk [9]. Supporting this, a five-year cross- 82 sectional study reported that higher serum levels of VD were significantly associated with a 83 reduced risk of deep vein thrombosis and other serious health outcomes [10]. 84 Based on published literature, this study will focus on female rats due to the higher reported risk 85 of thrombosis in females and the increasing use of e-cigs among women, especially during 86 pregnancy [11]. This research aims to investigate the effects of vape exposure on blood coagulation 87 and inflammation, as previous studies have reported conflicting results. In addition, the study will 88 evaluate the potential protective effect of vitamin D against vaping-induced changes in coagulation 89 and inflammatory markers. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 90 Materials and methods 91 Study design 92 VOOPOO DRAG M100S vape device with PnP-TM2 0.8 Ohms coil resistance, VGOD berry 93 bomb (sour strawberry belt) flavor, 50% propylene glycol, 50% vegetable glycerin, and 18mg/ml 94 nicotine. Hi Dee® (2000 IU VD/5 drops) and tera D ® (400 IU VD/ml) were used. The study was 95 conducted in two phases as follows: 96 Phase one: 97 Three groups of female rats were exposed to vaping for one month, and the other three groups 98 were not exposed to vaping nor received any treatment. 99 Phase two: 100 After the first month of exposure, VD was given to two vaping and two non-vaping groups for 2 101 months. The two vaping groups received VD at different dosages—one with a low dose (1000) 102 and the other with a high dose (50,000 IU weekly). Additionally, two other rat groups will receive 103 vitamin D without vape exposure, one with a high dose (50,000) and the other with a low dose 104 (1000 IU daily). 105 Animal management 106 This study was an in vivo study which used 42 Wistar rats, aged 12 weeks, housed at Applied 107 Science Private University in Amman, Jordan, for acclimatization over one week under standard 108 laboratory conditions, including a temperature of 21–23 °C, relative humidity of 35–70%, and a 109 12-hour light/dark cycle, with free access to food and water. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 110 All experimental procedures involving animals were conducted in accordance with institutional 111 and international guidelines for the care and use of laboratory animals. Ethical approval was 112 granted by the Research and Ethics Committee of the Faculty of Pharmacy, ASU (Approval No.: 113 2024-PHA-40). 114 Rats were randomly assigned to six experimental groups (n = 7 per group) as follows: Control 115 group (C): This group was not exposed to vaping nor VD treatment for the entire 12-week period 116 (negative control). Vaping group (EC): This group was exposed to vaping only for the entire 12 117 weeks, 2 hours per day, 5 days a week, without receiving VD treatment (positive control). Vaping 118 + low dose VD (ECD1000): This group was exposed to vaping for 4 weeks. Then, 1000 IU of VD 119 was administered daily for 8 weeks while continuing vaping exposure. Vaping + high dose VD 120 (ECD50,000): This group was exposed to vaping for 4 weeks. Then, 50,000 IU of VD was 121 administered once weekly for 8 weeks while continuing vaping exposure. Low-dose VD (D1000): 122 This group took 1000 IU of VD daily for 8 weeks without exposure to vaping. High dose VD 123 (D50,000): This group took 50,000 IU of VD weekly for 8 weeks without exposure to vaping. 124 The exposure settings 125 The vaping groups were removed from their cages to a 50 cm (length) x 50 cm (width) smoking 126 chamber shown in Figure.1, and back again to their cages after the end of the exposure. Rats were 127 exposed to the vape twice daily in two separate sessions, one hour in the morning and one hour in 128 the evening. After modification, an air pump was used to pull the vapor from the mouthpiece 129 through plastic oxygen tubes directly connected to the smoking chamber. At the beginning, the 130 chamber was filled with vapor (saturation phase). In the saturation phase, the pump was activated 131 for 20 seconds. During this time, the fire button was pressed for 5 seconds, followed by 2-3 seconds .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 132 of rest to avoid coil and mouthpiece overheating. The second exposure phase began after the 133 chamber was filled with vapor. During this phase, the pump was activated for 5 seconds, followed 134 by 20 seconds of rest, mimicking real situations of typical user behavior. 135 After the first hour of exposure, the pump was stopped, and the chamber remained closed until the 136 rats inhaled the remaining vapor inside. Then, the chamber was opened and supplied with fresh air 137 by opening a window directly beside the chamber, and the rats remained inside until the next hour 138 of exposure. The exact process was repeated in the second hour of exposure. 139 VD treatment 140 VD was given orally by directly dropping the dose into the rat’s mouth after one month of vape 141 exposure for 4 groups. It was administered in two doses: 1,000 IU daily and 50,000 IU weekly. 142 The equivalent doses were calculated using this equation: animal equation dose = Human dose/60 143 × Km ratio, Km = 6.2 [12]. The equivalent dose of 50,000 IU was 5 drops from the commercially 144 available Hi Dee® (2000 IU/5 drops) vial and 3 drops from the commercially available tera D® 145 (400 IU/ml) vial for a 1000 IU dose. The vitamin was given in the morning. 146 Blood sampling 147 Blood samples were collected in ethylenediamine tetraacetic acid (EDTA) tubes. The blood plasma 148 was obtained and stored in Eppendorf tubes at -80 °C. 149 Measurement of nicotine and cotinine levels 150 Nicotine and cotinine concentrations were measured using LC-MS-8030 As LIQUID 151 CHROMATOGRAPH MASS SPECTROMETER-Triple Quad MS (Shimadzu Corp.Japan). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 152 Briefly, a C18 4.5 mmx15cm x 0.2um column was used as a stationary phase, with a mobile phase 153 of 75% acetonitrile mixed with 0.05% formic acid and 25% distilled water, eluted isocratically at 154 a flow rate of 0.3 ml/min for 4 minutes. The MS interface was electrospray ionization (ESI) 155 running in positive ionization mode to generate [M+H]+ ions at m/z 162.23, 176.21 for nicotine 156 and cotinine, respectively. 157 Measurement of IL-6, D-dimer, TM, and FX levels 158 Commercially available rat-specific ELISA kits (ELK Biotechnology, Wuhan, China) were used 159 to detect IL-6, D-dimer, and TM, according to the manufacturer's instructions. Briefly, Plasma 160 samples, standards, and controls were placed in wells pre-coated with particular capture antibodies, 161 then incubated and washed. The detection antibodies were then coupled with horseradish 162 peroxidase (HRP), and colorimetric detection was performed on a TMB substrate. The reaction 163 ended with a sulfuric acid solution, and the absorbance was measured at 450 nm using a microplate 164 reader. The concentrations were determined using standard curves developed from the established 165 values included in each kit. Each sample was examined in duplicate. 166 FX levels were evaluated using a mouse-specific sandwich ELISA kit (Mouse F10 ELISA Kit, 167 Reed Biotech Ltd., Hubei, China), following the manufacturer's protocol, which was similar to the 168 previously used method. Although the kit was designed for mouse samples, it was used due to 169 documented cross-reactivity with rat plasma. Each sample was examined in duplicate. 170 Measurement of ALT and Creatinine levels 171 Serum alanine aminotransferase (ALT) and creatinine levels were measured using the BioSystems 172 ALT and Creatinine kits (BioSystems S.A., Barcelona, Spain). ALT activity was assessed by .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 173 monitoring NADH oxidation at 340 nm via spectrophotometry, while creatinine levels were 174 determined through the Jaffé reaction, which forms a colored complex measured at 500 nm. 175 Histopathological analysis 176 Lung, kidneys, and liver were harvested from three rats from each group after sacrifice. Organs 177 were fixed in 10% formalin for 1 week. Then, the organs were transferred to Smart Lab to make 178 the histopathological analysis. The organs were trimmed into cassettes and then a high 179 concentration of alcohol was added to dehydrate the tissues. A clearing agent (xylene) was added 180 to replace the alcohol. Then, melted paraffin wax was added to support the tissues. Using a 181 microtome, the paraffin block was cut into 4-µm-thick sections and placed on a glass slide to be 182 stained with hematoxylin and eosin for examination under the light microscope. 183 Statistical analysis 184 All results were expressed as mean ± standard deviation. Data were analyzed using the two-factor 185 analysis of variance (ANOVA) in GraphPad PRISM, the tenth version of statistical software to 186 calculate the statistical significance between the groups at different times. Tukey’s post hoc test 187 was then used to compare the differences between the groups, considering a P value of <0.05 a 188 statistically significant value. In addition, the Pearson test was used to detect the correlation 189 between nicotine concentration and other parameters in the vaping groups. 190 191 Results 192 Nicotine and Cotinine .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 193 As shown in Fig. 2, after the first month of exposure, the nicotine and cotinine levels increased 194 significantly from 0 ng/mL to 5.5 ng/mL ± 0.1 and 103 ng/mL ± 5, respectively. After 3 months, 195 a significant increase occurred in both compounds’ mean levels to 12.45 ng/mL ± 0.55 in nicotine 196 and 283.5 ng/mL ± 3.5 in cotinine, with a p-value of <0.0001 in the EC group. 197 However, the ECD1000 group had nicotine concentration close to the one-month level, with a 198 slight increase of 7.27%. Consequently, the cotinine levels increased significantly in three months 199 to 120 ng/mL ± 9.5, with a p-value of 0.0010. 200 Regarding the ECD50,000 group, the nicotine mean level was 8.25 ng/mL ± 0.25, and the cotinine 201 mean level was 174 ng/mL ± 6, representing a statistically significant increase in nicotine and 202 cotinine levels after three months of exposure to a p-value of <0.0001. Pearson test results 203 indicated that there was no correlation between nicotine level and other parameters. 204 Interleukin-6 (IL-6) 205 At baseline, the mean IL-6 level was 71.843 pg/mL ±10.414. After 1 month, the IL-6 level was 206 97.19 pg/mL ± 14.4294, representing a slight, non-significant increase of approximately 35%. 207 After 3 months, no statistically significant difference was observed in the EC group. The mean IL- 208 6 level was 115.1 pg/mL ± 10.6, representing 60% and 18% higher levels than the baseline and 209 one-month levels, respectively. 210 For the VD-treated groups, the mean value for the D1000 group was 108.175 pg/mL ± 28.325, 211 which was 1.5-fold higher than the baseline level. The mean value for the D50,000 group was 212 235.9 pg/mL ± 109.1, which was significantly higher than both the D1000 group and the C group, 213 with a p-value of <0.0001. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 214 Among the vape-exposed groups, the lowest IL-6 levels were in the ECD50,000 group, with a 215 mean of 93.285 pg/mL ± 12.715, and the highest levels were in the ECD1000 group, with a mean 216 of 122.23 pg/mL ± 46.67. Group ECD1000 had approximately 31% higher, non-significant IL-6 217 levels compared to group ECD50,000. 218 As Figure 3 summarizes, group D50,000 had significantly higher IL-6 mean levels than the EC, 219 ECD1000, ECD50,000, and C groups, with p-values of 0.0002, 0.0004, <0.0001, and <0.0001, 220 respectively. Group ECD1000 had approximately 13% higher IL-6 than the D1000 group. 221 D-dimer, FX, and TM 222 At baseline, the mean D-dimer, FX, and TM levels were 1412.13 ng/mL ± 1072.1288, 0.8027 223 μg/mL ± 0.0945, and 7.606 ng/mL ± 1.6511, respectively. After 1 month, all the parameters 224 increased significantly after the first month of exposure. The D-dimer levels increased to 4402.05 225 ng/mL ± 785.15, with a p-value of 0.0019. Moreover, the FX level increased to 1.8687 μg/mL ± 226 0.3132 with a p-value of < 0.0001, and the TM level increased to 34.71 ng/mL ± 8.42 with a p- 227 value of <0.0001. After 3 months, in the EC group, the mean D-dimer level decreased significantly 228 with a p-value of 0.0016 from the one-month level to become higher by only 3% than the C group 229 reading, with mean values of 1370.65 ng/mL ± 853.05 and 1332.25 ng/mL ± 376.85, respectively. 230 The FX level was 0.904 µg/ml ±0.233, which was lower by 106.71% than the one-month level. 231 The mean level did not differ significantly from the C group, which had a mean value of 0.9975 232 µg/ml ± 0.3965. The mean level of TM was 13.475 ng/mL ± 2.125, which represented a 233 statistically significant decrease with a p-value of <0.0001. This TM mean value is lower than in 234 the C group by 5.79%, indicating a non-significant difference. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 235 After VD treatment, D-dimer levels did not differ significantly between the VD-supplemented 236 groups. Group D1000 had the highest D-dimer level compared to group D50,000 and the C group, 237 with mean values of 2345.95 ng/ml ± 1585.65, 1785.05 ng/ml ± 1420.05, and 1332.25 ng/ml ± 238 376.85, respectively. Group D1000 had approximately 31.42% higher levels than group D50,000 239 and 76.07% than the C group. In group D50,000, the d-dimer level was higher by 34% than in the 240 C group. 241 The mean D-dimer level in the ECD1000 group was 956.9 ng/mL ± 589.6, which decreased 242 significantly from the one-month mean level with a p-value of 0.0003. The ECD50,000 group had 243 a D-dimer mean level of 439.95 ng/mL ± 294.05, representing the lowest D-dimer level compared 244 with the EC and ECD1000 groups. Although the difference between the group’s levels was not 245 statistically significant, group ECD50,000 had 117.5% and 211.6% lower levels than the ECD1000 246 and EC groups, respectively. Both VD supplemented with vape exposure groups had lower D- 247 dimer levels than the C group. The C group had approximately 39.23% higher levels than group 248 ECD1000 and 202.8% than group ECD50,000. 249 The analysis showed no statistically significant differences between the groups, as shown in 250 Figure S4A. However, the D-dimer levels in the D1000 and D50,000 groups were higher than 251 those in the ECD1000 and ECD50,000 groups by 145.17% and 305.78%, respectively. The lowest 252 D-dimer levels were found in group ECD50,000 among all other groups. 253 The highest increase in FX mean level among all the groups was found in group D50,000, which 254 had a significant elevation from the baseline level to 1.5575 μg/mL ± 0.2335 and a p-value of 255 <0.0001. A statistically significant difference between group D50,000 from group D1000 mean 256 value of 0.9275 μg/mL ± 0.0235, and the C group mean value of 0.9975 μg/mL ± 0.3965, appeared 257 after analysis with p-values of 0.0015 and 0.0062, respectively. In contrast, group D1000 had a .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 258 higher FX mean level of 15.56% from the baseline and a lower mean level of 7.55% from the C 259 group. 260 The FX mean level decreased significantly from the one-month mean value to 0.752 μg/mL ± 261 0.245 with a p-value of <0.0001 in the ECD1000 group. a non-statistically significant decrease in 262 FX mean level by 20.21% in the ECD1000 group compared to the EC group. The lowest FX mean 263 level was found in group ECD50,000, which was equal to 0.647 μg/mL. Even not statistically 264 significant, the ECD50,000 group had a 16.23% lower mean FX level than the ECD1000 and 265 39.72% than the EC groups. Both the ECD1000 and the ECD50,000 groups had lower FX levels 266 than the C group by 32.64% and 54.17% respectively. 267 According to Figure S4C, group D50,000 had a significantly higher mean FX level than all the 268 groups. When group D50,000 was compared with vaping groups, the results were different p- 269 values of 0.0009 against the EC group, <0.0001 against group ECD1000, and <0.0001 against 270 group ECD50,000. The C group had the second-highest mean FX level, followed by group D1000 271 and the EC group, which had close mean values. Group D1000 had a higher mean FX level of 272 43.36% compared to group ECD50,000 and 23.34% compared to group ECD1000. However, 273 group D1000 had a lower FX mean value than the EC group by 2.60%. 274 Regarding TM, a slight, non-significant decrease of 23.95% compared to the baseline level was 275 detected in group D1000, which had a mean value of 6.1365 ng/ml ± 2.4315. In contrast, group 276 D50,000 showed an increase in TM level by 5.47% from baseline with a mean value of 8.022 277 ng/ml ± 1.251. Group D1000 had a lower TM mean value of 30.73% than group D50,000. The 278 D1000 and D50,000 groups had no statistically significantly lower TM mean values than the C 279 group, which had a mean level of 14.255 ng/ml ± 3.17002, by 132.32% and 77.68%, respectively. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 280 In addition, the TM mean level decreased significantly in the ECD1000 and ECD50,000 IU VD 281 groups, with mean values of 20.41 ng/mL ± 8.35 and 17.375 ng/mL ± 3.895, respectively, and 282 corresponding p-values of 0.0002 and <0.0001. The highest TM mean level was in group 283 ECD1000, which was higher than the EC group by 51% and by 17.47% compared to group 284 ECD50,000. Both the ECD1000 and the ECD50,000 groups had higher, but non-significant, TM 285 mean values compared to the C group, by 43.18% and 21.89%, respectively. 286 When comparing all the groups, the group receiving D1000 had a significantly lower TM mean 287 level than both the ECD1000 and ECD50,000 groups, with p-values of 0.0006 and 0.0106, 288 respectively. Regarding group D1000, a non-statistically significant difference of 119.59% lower 289 TM mean level than the EC group was found after analysis. The D50,000 group had a significantly 290 lower mean value than group ECD1000 IU VD, with a p-value of 0.0037 and a non-significant 291 lower mean level than group ECD50,000 IU VD by 116.64%. In addition, group D50,000 had a 292 lower TM level of 67.99% than the EC group. Figure S4B visually summarizes those findings. 293 294 ALT and creatinine 295 At baseline, the ALT and creatinine mean levels were 60.1 U/L ± 4.1605 and 0.645 µmol/L ± 296 0.015, respectively. After 1 month, ALT level decreased significantly after one month of exposure 297 to 42.7 U/L ± 3.8105, with a p-value of 0.0028. However, the Creatinine level was 0.69 µmol/L ± 298 0.09, which was considered a non-significant increase by 6.98% from baseline levels. After 3 299 months, in the EC group, the ALT mean level increased significantly to 57.5 U/L ± 3.5 and a p- 300 value of 0.0136, less by 4.33% than the baseline and 15.38% than the C group, which had a mean 301 value of 67.95 U/L ± 1.65. A 3% decrease in creatinine levels occurred compared to the first .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 302 month’s levels to be 0.67 µmol/L. However, compared to the control’s mean level of 0.61 µmol/L 303 ± 0.03, the EC group had 9.84% higher creatinine levels. 304 After VD treatment in both groups, an increase in ALT levels appeared. Group D1000 had 15.35%, 305 and group D50,000 had 13.71% higher values than the baseline, with mean values of 71 U/L ± 8.5 306 and 69.65 U/L ± 7.85, respectively. Compared with the C group, group D1000 and group D50,000 307 had higher mean ALT levels by 4.30% and 2.44%, respectively. Both groups, D1000 and D50,000 308 had very close mean values, with group D1000 having approximately 1.9% higher values. 309 Regarding creatinine, group D1000 had the highest creatinine level of 0.665 µmol/L ± 0.005 by 310 3.10% and 9.02%, respectively, compared with group D50,000 and the C group. When compared 311 with the C group, group D50,000 had a mean level of 0.645 µmol/L ± 0.015, which was 5.74% 312 higher. 313 Both the ECD1000 and the ECD50,000 group's ALT mean levels increased compared to one 314 month after VD treatment, with mean values of 75.25 U/L ± 16.15 and 51.9 U/L ± 5.2, respectively. 315 This increase was significant in the ECD1000 group with a p-value of <0.0001, and non- 316 statistically significant in group ECD50,000, with a 21.54% increase only. The ECD1000 group 317 had the highest ALT mean level among the EC group and group ECD50,000, with p-values of 318 0.0015 and 0.0001, respectively. Group ECD50,000 had a 9.74% lower ALT mean value than the 319 EC group. 320 Figure S5A showed that the highest ALT mean level was in group ECD1000 compared to all other 321 groups. It had a significantly higher mean level of 75.25 U/L ± 16.15 than the EC group with a p- 322 value of 0.0051. Both groups D1000 and D50,000 had lower ALT mean values of 5.65% and .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 323 7.44% than the ECD1000, respectively. In addition, group ECD1000 had a 9.70% higher mean 324 ALT level than the C group. 325 However, the group ECD50,000 mean ALT level was 9.74% lower than the EC group. Upon 326 analysis, the ECD50,000 group had a significantly lower ALT mean value than the D1000, 327 D50,000, and the C groups, with p-values of 0.0021, 0.0051, and 0.0145, respectively. 328 The creatinine in the ECD1000 group was significantly higher than the EC group and the C groups, 329 with a mean value of 0.78 μmol/L ± 0.01 and p-values of 0.0120 and <0.0001, respectively. In 330 contrast, the ECD50,000 group had a mean creatinine level of 0.72 μmol/L ± 0.06, which was 331 7.69% less than the group ECD1000 IU VD mean level and higher than the EC group by 7.46%. 332 A significantly higher mean creatinine level with a p-value of 0.0120 was found between the 333 ECD50,000 and the C group. 334 A significantly higher mean creatinine level was observed in group ECD1000 than in the D1000 335 and D50,000 groups, with p-values of 0.0076 and 0.0011, respectively. Regarding group 336 ECD50,000, it had an 8.27% higher mean creatinine level than group D1000 and 11.635% higher 337 than group D50,000. There were very close mean values in the EC, D1000, and D50,000 groups. 338 The EC group had 0.75% higher creatinine levels than group D1000 and 3.88% higher than group 339 D50,000, as illustrated in Figure S5B. 340 Histopathological examination 341 Liver 342 The EC and the C groups had a resemble hepato-microscopic examination with moderate 343 lymphocytic infiltration in the portal tracts, and no significant fibrosis, steatosis, or cirrhosis was 344 observed, as shown in Figure S6A and S6B. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 345 The hepatic tissue in group D1000 resembled the C group by having mild chronic inflammation in 346 the portal tracts, with a predominantly lymphocytic infiltrate without significant hepatocellular 347 damage or fibrosis. As shown in Figure S6C. In contrast, group D50,000 had normal liver tissue 348 without significant fibrosis or inflammation. As shown in Figure S6D. 349 Histopathological examination reveals chronic inflammation with lymphocytic infiltration and 350 occasional macrophages in the portal tracts in the ECD1000 group, without signs of fibrosis, 351 necrosis, or steatosis, see Figure S6E. Moreover, in the ECD50,000 group, the liver showed 352 evidence of chronic inflammation with mild lymphocytic infiltration around the portal tracts 353 without significant hepatocellular damage or fibrosis, see Figure S6F. 354 355 Kidneys 356 In the C group, the glomeruli demonstrate cellular expansion in both mesangial and endocapillary 357 areas, with reduced capillary lumen size. Tubules show preserved morphology or mild nonspecific 358 changes, as illustrated in Figure S7A. 359 The glomeruli in the EC group exhibited diffuse mesangial hypercellularity and mild endocapillary 360 proliferation, with some narrowing of capillary lumina. There was no significant 361 glomerulosclerosis or crescent formation, and the tubules appeared largely preserved, as shown in 362 Figure S7B. In group D1000, the glomeruli demonstrate mild mesangial expansion with focal 363 endocapillary hypercellularity. Tubules are largely preserved, with minimal signs of atrophy and 364 rare protein casts, as Figure S7C provides. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 365 In group D50,000, Glomeruli showed mesangial and endocapillary hypercellularity with capillary 366 lumen narrowing. There was no evidence of crescent formation or significant glomerular scarring. 367 Tubules are unremarkable or show mild reactive changes, as Figure S7D provides. 368 In the ECD1000 group, as shown in Figure S7E, glomeruli showed mesangial and endocapillary 369 hypercellularity with capillary lumen narrowing. Tubules were unremarkable or showed mild 370 reactive changes. Blood vessels were within normal limits, without evidence of vasculitis or 371 thrombosis, which indicates glomerular inflammation or injury. 372 The glomerular inflammation and injury were more severe in the ECD50,000 group, as shown in 373 Figure S7F, which was characterized by the expansion of the mesangial matrix with focal crescent 374 formation and endocapillary hypercellularity. 375 Lungs 376 In the C group, the lung tissues appeared with no significant inflammatory infiltrates, numerous 377 blood cells, and preserved alveolar walls, as represented in Figure S8A. However, in the EC group, 378 the alveolar spaces were filled with abundant neutrophils, and areas of necrosis were evident, 379 consistent with necrotizing pneumonia. The alveolar walls exhibit marked disruption and extensive 380 hemorrhage in the affected regions, as shown in Figure S8B. 381 Diffuse interalveolar hemorrhage was evident in group D1000, with blood in the alveolar spaces 382 and mild edema in the interstitium without any alveolar wall necrosis or significant inflammation 383 observation, as evidenced by Figure S8C. In contrast, group D50,000 had normal lung tissue 384 without hemorrhage, fibrosis, or inflammation, as evidenced by Figure S8D. 385 Extensive red blood cells were present in the alveolar spaces in the ECD1000 group, indicating 386 hemorrhage. The alveolar walls were intact, but blood-filled spaces were noted, with occasional .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 387 hemosiderin-laden macrophages. Mild interstitial edema was present without significant 388 inflammation, as illustrated in Figure S8E. 389 In the ECD50,000 group, the lung was more injured, as evidenced in Figure S8F, by having the 390 alveolar spaces filled with neutrophils, indicating acute bacterial pneumonia. The alveolar walls 391 showed mild inflammation, and a few alveolar septa were thickened due to edema. The 392 mucopurulent exudate was present within the bronchioles, and the surrounding alveoli were 393 congested. 394 Discussion 395 The extensive advertising of e-cigs as a safer and healthier alternative to traditional tobacco 396 smoking has considerably boosted their popularity, particularly among teenagers and young adults. 397 The appealing tastes and generally moderate odor of vaping devices have particularly attracted 398 female consumers, emphasizing the importance of researching any potential risks linked with their 399 usage. According to a global survey conducted in 2020, an estimated 68 million people are active 400 e-cig users globally [13]. A survey conducted by a team of British scientists found that the majority 401 of vape shop consumers are between the ages of 18 and 25, with females accounting for around 402 41% of this population. Moreover, information received from vape shop workers suggested that 403 fruit-flavored e-liquids were the most popular option among their customers [14]. According to 404 emerging data from several research, tobacco smoking has a deleterious impact on VD status by 405 interfering with key enzymes involved in its production and metabolism. Furthermore, smoking 406 has been linked to increased liver damage indicators, which may inhibit VD synthesis and lead to 407 a greater risk of deficiencies among tobacco users [15]. This study is particularly significant .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 408 because there is little information on the consequences of VD on e-cig users, particularly in terms 409 of blood coagulability parameters and inflammatory markers. 410 The current study's principal findings show that one month of exposure to e-cig vapor led in a 411 substantial increase in blood levels of nicotine, TM, FX, D-dimer, and IL-6. These findings are 412 consistent with previous studies that found similar short-term effects of vaping on coagulation 413 indicators and inflammatory markers [16, 17]. The significant rise in certain clinical markers might 414 be attributed to nicotine or the vape juice ingredients themselves. Many in vivo and in vitro 415 investigations revealed that nicotine, flavoring ingredients, hygroscopic carriers, and metals 416 emitted by the heated coil might cause cardiac toxicity and increase IL-6 and other inflammatory 417 markers. likewise, previous research found that acute vape exposure raises the risk of 418 cardiovascular disease via worsening endothelial dysfunction [18, 19]. However, during the third 419 month of exposure, the levels of these biomarkers were decreased, except for nicotine in the vape- 420 only group and IL-6, which remained high. Notably, nicotine concentrations were lower in both 421 the vape + 1000 IU VD and vape + 50,000 IU VD groups, indicating a possible modulatory impact 422 of VD supplementation. In support of this, Knihtilä et al. found that appropriate maternal VD levels 423 during pregnancy were related with reduced cotinine concentrations in tobacco-exposed mothers, 424 which contributed to better respiratory outcomes in their children [20]. The study also identified a 425 significant interaction between cotinine and VD levels, though the underlying mechanism remains 426 unclear. This interaction may be attributed to the anti-inflammatory and antioxidant properties of 427 VD [15]. 428 The observed increases in IL-6 might be attributed to the increase in toxic substances generated 429 by vaping and their buildup in the lungs. These irritating substances produce oxidative stress, 430 which increases the production of white blood cells and cytokines. The observed decrease in blood .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 431 coagulation indicators after one month of exposure might be due to the rats' physiological ability 432 to adapt and maintain homeostasis over time [21]. This conclusion is supported by Garrett's study, 433 which examined the long-term effects of cigarette smoke exposure on coagulation in rats. In this 434 study, rats exposed to tobacco smoke for 22 weeks showed no significant variations in plasma 435 clotting times compared to the control group, indicating an adaptive response. Furthermore, the 436 study revealed an age-dependent effect, with the prothrombotic effect of cigarette smoke being 437 more prominent in older rats (24 months) than in younger rats (about 3 months), indicating that 438 age may influence susceptibility to smoking-induced coagulopathy [22]. In research on the 439 cardiovascular impacts of vaping, Dai et al. exposed 6-week-old rats to e-cig aerosol for 5 hours 440 per day, 4 days per week, for 3 months. Their findings showed that this exposure regimen did not 441 cause substantial changes in blood pressure or heart rate at the end of the research [23]. To further 442 explore the temporal dynamics of cardiovascular adaptation to vaping, El-Mahdy et al. conducted 443 a prolonged exposure study in which rats were subjected to e-cig vapor for 60 weeks, with pulse 444 measurements recorded at multiple intervals. During the initial 8 weeks, pulse rates were elevated 445 relative to baseline values, suggesting an acute physiological response. However, from weeks 8 to 446 16, pulse rates declined, indicative of adaptive mechanisms. Following this period, from week 16 447 onward, pulse rates increased progressively and significantly, persisting through the remainder of 448 the 60-week exposure period [24]. Additionally, Rafiq et al. confirmed an inverse connection 449 between IL-6 and TM. In individuals with coronary artery disease, NF-kB was down-regulated 450 whereas TM expression was up-regulated. In vitro and in mouse lung injury models, inhibiting 451 NF-kB activity reduced cytokine-induced TM downregulation [25]. These prior findings are 452 consistent with the findings of this investigation, which showed that TM and IL-6 levels were 453 conflicting [26]. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 454 Recently, in a vitro study conducted by Cirillo et al, tissue factor expression at gene and protein 455 levels and the pro-coagulation activity were increased after incubating human umbilical vein 456 endothelial cells with vape containing 18 mg/mL nicotine [27]. The inflammation that occurred in 457 vaping groups increased the levels of IL-6, which consequently elevated platelet count and 458 increased the tendency to blood coagulation and thrombosis. In addition, the presence of tissue 459 factor leads to the acceleration in factor VIII conversion to its active form which will convert FX 460 to its active form sequentially [28-30]. However, it has been approved by Cimmino et al, that VD 461 can decrease tissue factor expression and atherosclerotic risk by modulating the nuclear factor 462 kappa B in pre-incubated cells with VD. Although not all VD doses have the ability to decrease 463 the activity of nuclear factor kappa B, the low dose VD of 1000 IU caused a reverse effect by 464 increasing its activity in ulcerative colitis patients [31, 32]. That result clearly elucidates that 1000 465 IU of VD daily will cause an elevation in inflammatory biomarkers and increase the risk of 466 thrombogenesis during stress conditions and inflammation. As the dose of VD increases, the 467 activity of nuclear factor kappa B will be diminished in a dose-dependent manner during 468 inflammation or stress conditions [33]. Those results explain the effect of 1000 IU and 50,000 IU 469 on increasing and decreasing the inflammatory marker IL-6 and blood coagulation predictor levels 470 in vape+50,000 IU VD and vape+1000 IU VD groups, respectively. In addition, the vape+1000 471 IU VD group had the highest creatine levels among all the groups with evidence of 472 glomerulonephritis, which affected IL-6 clearance and led to its accumulation in the body. 473 The immunomodulatory action of VD is dose-dependent and changes according to physiological 474 circumstances. According to Bock et al., giving healthy people 140,000 IU VD once a month 475 markedly increased regulatory T cell activity, suggesting that large dosages of VD stimulate the 476 immune system [34]. In a similar vein, Bader et al. showed that high-dose VD (50,000 IU) caused .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 477 a cytokine storm and increased IL-6 levels in healthy individuals [35]. In contrast, under 478 inflammatory or stress conditions, VD has been shown to reduce IL-6 levels in a dose-dependent 479 manner [36]. 480 D-dimer and IL-6 levels were shown to positively correlate in vaping-exposed groups, which is in 481 line with research showing that fibrin breakdown products like D-dimer might increase vascular 482 inflammation and IL-6 release [37]. Additionally, it has been shown that during inflammatory 483 conditions, especially in lung infections as COVID-19, there is an inverse link between VD levels 484 and D-dimer concentration [38]. The differences in D-dimer levels across groups might be 485 explained by these interactions. The observed alterations in IL-6, D-dimer, TM, FX, ALT, and 486 creatinine were probably caused by other components of the vape aerosol rather than nicotine 487 alone, as evidenced by the notable lack of a significant link between nicotine and other measured 488 parameters according to Pearson correlation analysis. 489 490 Regarding the histopathological findings, the necrotic results reported in the vape group lung tissue 491 were consistent with an in vitro investigation conducted by Chastain Anderson et al, who 492 discovered that vaping caused cell necrosis. Since TM is found in the alveolar epithelial cells, the 493 drop in TM level that occurred after three months of exposure in the vape group was caused to 494 necrotic pneumonia, which emerged after the histological examination as authorized by Boehme 495 et al [39]. The Boehme et al. research discovered that TM levels increased dramatically before the 496 start of cell injury, but the release was lost once cell damage occurred. These findings explain why 497 the vape group had higher levels of IL-6 and lower levels of TM after one and three months, 498 respectively [40]. The bacterial pneumonia that emerged in the vape+50,000 IU VD group may be .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 499 attributed to the absence of a VD receptor, which impacts the innate defense against bacterial 500 infections in rodents and is confined to primates [41]. 501 As the hepatic regeneration capacity in Wistar rats reaches its maximum ability between 9 to 24 502 weeks of age [41]. This could strongly explain the histopathological examination of the liver, 503 which showed no significant changes in the hepatic tissues after three months of exposure. 504 The glomerular histopathology examination showed that there was immune-related kidney injury. 505 This damage was produced by inflammation and activation of the NF-kB pathway, which led to 506 an increase in IL-6 levels. This pathway activation increases the synthesis of proinflammatory 507 cytokines present in the nephritic glomeruli, resulting in glomerulonephritis in rats [42]. 508 Conclusion 509 In conclusion, our study shows that short-term nicotine vape exposure increases coagulability 510 indicators and inflammatory cytokines in female rats, but longer exposure may cause physiological 511 adaptation. High-dose VD supplementation (50,000 IU/week) protected vaping-exposed rats 512 against these changes, whereas low-dose VD (1,000 IU/day) showed moderate efficacy. Notably, 513 high-dose VD in non-vaping rats induced a proinflammatory response. These data indicate that 514 VD may reduce vaping-induced coagulopathy and inflammation while also potentially aiding in 515 nicotine detoxification. While based on an animal model, the findings emphasize the potential 516 therapeutic benefit of VD in vape users. 517 Declaration 518 Acknowledgment 519 The authors thank X-vape shop, Smart lab, and Al-Hikma lab. 520 Funding .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 521 The Deanship of Scientific Research at the Applied Science University, Amman, Jordan, funded 522 this research. 523 Data availability 524 All relevant data can be found within the manuscript and in the supporting file. 525 Author contributions 526 Conceptualization: Muna Barakat and Mahmoud Abu-Samak. 527 Methodology: Lujain F. Zghari, Aman M. Hammad. 528 Writing - original draft: Aman M. 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Bergman, P., et al., Vitamin D3 supplementation in patients with frequent respiratory tract 621 infections: a randomised and double-blind intervention study. BMJ Open, 2012. 2(6). 622 41. Vázquez, C., J. Buján, and D. Vallejo, Blood coagulation variations induced by carbon tetrachloride 623 inhalation in Wistar rats. Toxicol Appl Pharmacol, 1990. 103(2): p. 206-13. 624 42. Sakurai, H., et al., Activation of transcription factor NF-kappa B in experimental glomerulonephritis 625 in rats. Biochim Biophys Acta, 1996. 1316(2): p. 132-8. 626 627 Figure ligands 628 Figure 1. (A) The timeline of the experiment, and (B) the vaping chamber. 629 Figure 2. The (A) nicotine and (B) cotinine concentrations (ng/mL) among all vaping groups at 630 baseline, one month, and two months. 631 Figure 3. Summary of mean plasma IL-6 concentrations (pg/mL) among all the groups at baseline, 632 one month, and three months. 633 Figure S4. Summary of mean plasma (A) D-dimer concentrations (ng/mL), (B) TM concentrations 634 (ng/mL), and (C) FX concentrations (µg/mL) among all the groups at baseline, one month, and 635 three months. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint 636 Figure S5. (A) Summary of mean plasma ALT concentrations (U/L), and (B) Creatinine 637 concentrations (µmol/L) among all the groups at baseline, one month, and three months. 638 Figure S6. Histopathological appearance of liver tissue from all the groups under light 639 microscopy. 640 Figure S7. Histopathological appearance of kidney tissue from all the groups under light 641 microscopy. 642 Figure S8. Histopathological appearance of lung tissue from all the groups under light microscopy. 643 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint