Exposure to DEP results in increased ACE2 protein expression in the bronchioles of C57BL/6 mice
To investigate whether subchronic inhalational exposure to DEP and consumption of a HF diet alter ACE2 expression, we measured ACE2 protein expression by immunofluorescent histology (Fig. 1A-M) and mRNA transcript expression by RT-qPCR (Fig. 1N). We observed a significant increase in ACE2 protein expression in experimental groups that were exposed to DEP, regardless of diet. Compared with that in the LF + CON group, ACE2 protein expression was significantly greater in the LF + DEP (p < 0.0001) and HF + DEP groups (p < 0.0001). Compared with that in the HF + CON group, there was a significant increase in ACE2 protein expression in the LF + DEP (p < 0.0001) and HF + DEP groups (p < 0.0001). There was no significant difference in protein expression observed among the exposure groups. Exposure F = 71.07, diet F = 2.494, and exposure x diet F = 0.08277. There was no significant difference in ACE2 mRNA transcript expression between the groups.
Probiotic treatment mitigated the DEP-mediated increase in ACE2 receptor protein expression in the lungs of C57BL/6 mice
To determine whether probiotic treatment mitigates DEP- ± HF diet-mediated alterations in ACE2 expression, we measured ACE2 protein expression via immunofluorescent histology (Fig. 2A-M) and mRNA transcript expression by RT-qPCR (Fig. 2N). We observed a significant decrease in ACE2 protein expression in the HF + CON + PRO (p < 0.0001) and HF + DEP + PRO (p < 0.0001) groups compared with the HF + DEP group. There was no significant difference in ACE2 protein expression between the probiotic groups and the HF + CON group. Exposure F = 43.91, probiotics F = 38.18, exposure x probiotics F = 46.77.
We also observed a significant decrease in ACE2 mRNA expression in the HF + CON + PRO (p = 0.0012) and HF + DEP + PRO (p = 0.0017) groups compared to the HF + CON group. Similarly, ACE2 mRNA expression was significantly lower in the HF + CON + PRO (p = 0.0003) and HF + DEP + PRO (p = 0.0005) groups than in the HF + DEP group. There was no significant difference in ACE2 mRNA expression between the HF + CON and HF + DEP groups or between the probiotic treated groups. Exposure F = 32.10, probiotics F = 0.5418, exposure x probiotics F = 0.4563.
Exposure to diesel exhaust particulate matter in conjunction with consumption of a HF diet results in increased TMPRSS2 expression in the lungs of C57BL/6 mice
To assess whether subchronic inhalational exposure to DEP and consumption of a HF diet alter TMPRSS2 expression, we measured TMPRSS2 protein expression by immunofluorescent histology (Fig. 3A-M) and mRNA transcript expression by RT-qPCR (Fig. 3N). We observed a significant increase in TMPRSS2 protein expression in the HF + DEP group compared to the LF + CON (p = 0.0004), LF + DEP (p < 0.0001), and HF + CON (p < 0.0001) groups. Compared with the LF + CON group, the LF + DEP (p < 0.0001) and HF + CON (p = 0.0004) groups presented significantly lowerTMPRSS2 protein expression. Compared to the LF + DEP group, the HF + CON (p = 0.0273) group displayed significantly increased TMPRSS2 protein expression was significantly increased. Exposure F = 1.325, diet F = 16.98, exposure x diet F = 56.07.
TMPRSS2 mRNA expression was not significantly different between the LF + CON, LF + DEP, and HF + CON groups, but the expression of TMPRSS2 mRNA in the HF + DEP group was significantly greater than that in the LF + DEP (p = 0.0016) and HF + CON (p = 0.0067) groups. Exposure F = 0.7838, diet F = 2.562, exposure x diet F = 12.77.
Probiotic treatment mitigated the DEP- and HF diet-mediated increase of TMPRSS2 protein expression in the lungs of C57BL/6 mice
To establish whether probiotic treatment mitigates DEP- ± HF diet-mediated alterations in TMPRSS2 expression, we measured TMPRSS2 expression by immunofluorescent histology (Fig. 4A-M) and mRNA transcript expression by RT-qPCR (Fig. 4N). We observed a significant decrease in TMPRSS2 protein expression in groups treated with probiotics (HF + CON + PRO (< 0.0001), HF + DEP + PRO (< 0.0001)) compared to the HF + DEP group. Within the probiotic groups, TMPRSS2 protein expression was observed to be significantly decreased in the HF + DEP + PRO (p = 0.0049) group compared to the HF + CON + PRO group. Exposure F = 20.93, probiotics F = 3.547, exposure x probiotics F = 38.84.
TMPRSS2 mRNA expression was not found to be statistically different between the HF + CON, HF + DEP, and HF + DEP + PRO groups. TMPRSS2 mRNA expression in the HF + CON + PRO (p = 0.0275) group was observed to be significantly lower than that of the HF + DEP group. Exposure F = 2.137, probiotics F = 4.328, exposure x probiotics F = 0.1592.
Exposure to diesel exhaust particulate matter in conjunction with the consumption of a HF diet results in increased androgen receptor protein expression in the bronchioles of C57BL/6 mice
To determine if subchronic inhalational exposure to DEP and consumption of a HF diet alters AR expression, we measured AR protein expression by immunofluorescent histology (Fig. 5A-M) and mRNA transcript expression by RT-qPCR (Fig. 5N). We observed a significant increase in AR protein expression in the HF + DEP group compared with the LF + CON (p < 0.0001), LF + DEP (p < 0.0001), and HF + CON (p < 0.0001) groups. There was no significant difference observed between the LF + CON, LF + DEP, and HF + CON groups. Exposure F = 10.21, diet F = 17.17, exposure x diet F = 22.58.
AR mRNA expression was significantly greater in the LF + DEP (p = 0.0188) group than in the LF + CON group as well as decreased in the HF + DEP group (p = 0.0307) compared to the LF + DEP group. Exposure F = 1.669, diet F = 1.270, exposure x diet F = 5.069.
Probiotic treatment mitigated the DEP- and HF diet-mediated increase in androgen receptor protein expression in the bronchioles of C57BL/6 mice
To ascertain whether probiotic treatment mitigates DEP- ± HF diet-mediated alterations in AR expression, we measured AR expression via immunofluorescent histology (Fig. 6A-M) and mRNA transcript expression by RT-qPCR (Fig. 6N). We observed a significant decrease in AR protein expression in the groups treated with probiotics [HF + CON + PRO (p < 0.0001), HF + DEP + PRO (p < 0.0001)] compared with the HF + DEP group. There was no significant difference observed between the HF + CON and probiotic groups. Exposure F = 14.03, probiotics F = 5.946, exposure x probiotics F = 28.60. There was no significant difference in AR mRNA expression observed between groups.
Exposure to diesel exhaust particulate matter in conjunction with the consumption of a HF diet results in increased NRP1 protein expression in the bronchioles of C57BL/6 mice
To investigate whether subchronic inhalational exposure to DEP and consumption of a HF diet alter NRP1 expression, we measured NRP1 protein expression via immunofluorescent histology (Fig. 7A-M) and mRNA transcript expression by RT-qPCR (Fig. 7N). We observed a significant increase in NRP1 protein expression in the HF + DEP group compared with the LF + CON (p < 0.0001), LF + DEP (p < 0.0001), and HF + CON (p < 0.0001) groups. There was no significant difference observed between the two LF groups. NRP1 protein expression was decreased in the HF + CON group compared with the two LF groups [LF + CON (p = 0.0159), LF + DEP (p < 0.0001)]. Exposure F = 44.21, diet F = 2.039, exposure x diet F = 27.47.
NRP1 mRNA expression was observed to be significantly greater in the LF + DEP group than in the LF + CON (p = 0.0205), HF + CON (p = 0.0125), and HF + DEP (p = 0.0239) groups. There was no significant difference in NRP1 mRNA expression among the LF + CON, HF + CON, and HF + DEP groups. Exposure F = 3.732, diet F = 3.976, exposure x diet F = 2.236.
Probiotic treatment mitigated the DEP- and HF diet-mediated increase in NRP1 protein expression in the bronchioles of C57BL/6 mice
To determine whether probiotic treatment mitigates DEP- ± HF diet-mediated alterations in NRP1 expression, we measured NRP1 expression via immunofluorescent histology (Fig. 8A-M) and mRNA transcript expression by RT-qPCR (Fig. 8N). We observed a significant decrease in NRP1 protein expression in groups treated with probiotics [HF + CON + PRO (p < 0.0001), HF + DEP + PRO (p < 0.0001)] compared with the HF + DEP group. There was no significant difference observed between the HF + CON and probiotic groups. Exposure F = 38.85, probiotics F = 41.20, exposure x probiotics F = 27.18.
We also observed a significant increase in NRP1 mRNA expression in the HF + DEP + PRO group compared with the HF + CON (p = 0.0345) and HF + CON + PRO (p = 0.0161) groups. There was no significant difference in NRP1 mRNA expression among the HF + CON, HF + DEP, and HF + CON + PRO groups. Exposure F = 1.792, probiotics F = 3.385, exposure x probiotics F = 2.405.
Exposure to diesel exhaust particulate matter in conjunction with consumption of a HF diet results in increased furin protein expression in the bronchioles of C57BL/6 mice
To establish whether subchronic inhalational exposure to DEP and consumption of a HF diet alter furin expression, we measured furin expression via immunofluorescent histology (Fig. 9A-M) and mRNA transcript expression by RT-qPCR (Fig. 9N). We observed a significant increase in furin protein expression in the HF + DEP group compared with the LF + CON (p < 0.0001), LF + DEP (p < 0.0001), and HF + CON (p < 0.0001) groups. There was no significant difference observed between the LF + CON, LF + DEP, and HF + CON groups. Exposure F = 30.95, diet F = 28.62, and exposure x diet F = 45.02. We also observed no significant difference in furin mRNA expression among the four groups.
Probiotic treatment mitigated the DEP- and HF diet-mediated increase in furin protein expression in the bronchioles of C57BL/6 mice
To determine whether probiotic treatment mitigates DEP- ± HF diet-mediated alterations in furin expression, we measured furin expression by immunofluorescent histology (Fig. 10A-M) and mRNA transcript expression by RT-qPCR (Fig. 10N). We observed a significant decrease in furin protein expression in the groups treated with probiotics [HF + CON + PRO (p < 0.0001), HF + DEP + PRO (p < 0.0001)] compared with the HF + DEP group. Furin protein expression was significantly lower in the HF + CON + PRO group than in the HF + CON (0.0360) and HF + DEP + PRO (p = 0.0077) groups. Exposure F = 46.02, probiotics F = 60.13, exposure x probiotics F = 12.00.
Furin mRNA expression was decreased in the HF + CON + PRO group compared with the HF + CON (p = 0.0399) and HF + DEP + PRO (p = 0.0022) groups. There was no significant difference observed in furin mRNA expression among the HF + CON, HF + DEP, and HF + DEP + PRO groups. Exposure F = 0.04313, probiotics F = 3.122, exposure x probiotics F = 7.989.
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