1,25(OH)2D3 effectively prevents the occurrence of high-altitude illness in rapid ascent to high altitude individuals
1,25(OH)2D3 improves adverse reactions to high altitude in the human body
Figure 1A shows the process of recruiting two groups of volunteers to conduct the experiment in this study. Comparison of baseline data between the two groups of healthy young volunteers showed no significant differences, and both groups had no underlying diseases (Table.1). According to the Lake Louise Score (LLS) screening for acute mountain sickness (AMS), diagnostic criteria include rapidly ascending to high-altitude areas (>2500m) , Lake Louise AMS score total of 3 or higher indicates AMS, including at least one point from headache in the setting of a recent ascentor gain in altitude. As shown in Fig. 1B, on the first day in the high-altitude area, the Lake Louise Score for the H5D group volunteers was 2.72±0.94, significantly higher than the VDH5D group's score of 1.74±0.72 (P<0.05), but neither group's score exceeded 3 points, so it could not be diagnosed as AMS. On the fifth day in the high-altitude area, the H5D group volunteers had a score of 3.40±1.08, exceeding 3, and 21 people had headache reactions, which could be diagnosed as AMS. This score was significantly higher than the VDH5D group's score of 2.09±0.1 (P<0.05), with 5 people having headache reactions(Figure 1 B).
Table 1
Variables
|
Group 1 (n=23)
|
Group 2 (n=25)
|
P
|
Gender (male/female, n)
|
14/9
|
13/12
|
-
|
Age (years)
|
30.00 ± 8.60
|
30.28 ± 5.21
|
0.3526
|
Body mass index (kg/m2)
|
22.28 ±1.81
|
23.26 ± 2.59
|
0.5912
|
Heart rate
|
71.26 ± 3.18
|
72.00 ± 3.89
|
0.4488
|
Systolic blood pressure (mmHg)
|
120.40 ± 4.07
|
119.80 ± 4.91
|
0.9491
|
Diastolic blood pressure (mmHg)
|
74.00 ± 3.67
|
74.88 ± 2.09
|
01952
|
Pulse blood oxygen Saturation (%)
|
94.52 ± 0.85
|
94.40 ± 0.87
|
0.6286
|
Underlying disease
|
No
|
No
|
-
|
Compared with groups G1 and G2, both the VDH5D and H5D groups showed a significant decrease in blood oxygen saturation (P<0.05), and an increase in heart rate, systolic blood pressure, and diastolic blood pressure (P<0.05). However, the changes in these four indicators were more pronounced in the H5D group (Fig1.C). These results indicate that direct exposure to high-altitude low-oxygen environments can cause severe discomfort in the human body, and early treatment with 1,25(OH)2D3 can alleviate adverse reactions to high altitude, playing a role in inhibition and relief.
1,25(OH)2D3 inhibits the increase of inflammatory factors and inflammatory cells induced by hypoxia
Compared with groups G1 and G2, the VDH5D and H5D groups showed an increase in the levels of IL-6 and IL-1β in the serum, but the increase was more pronounced in the H5D group. The changes in the numbers of inflammatory cells in the blood routine were similar. Compared with groups G1 and G2, both the VDH5D and H5D groups showed an increase in the numbers of inflammatory cells, but the extent of increase was lower in the VDH5D group (Fig1.D). These results indicate that stimulation of the human body by high-altitude low-oxygen environments leads to the release of inflammatory factors and inflammatory reactions. Early treatment with 1,25(OH)2D3 can effectively reduce the expression of inflammatory factors in the body and improve the inflammatory damage caused by hypoxia.
1,25(OH)2D3 improves hypoxic liver injury in humans.
As shown in Figure 1E, after 5 days of exposure to high-altitude, low-oxygen environment, both the VDH5D and H5D groups showed an increase in serum ALT and AST levels. However, the extent of increase in the VDH5D group was significantly lower compared to the H5D group. As shown in Figure 1G, there was no significant difference in serum 25(OH)D levels between groups G1 and G2, and both were within the normal range (30-100 ng/mL). In contrast to groups G1 and G2, the VDH5D group showed a slight increase in serum 25(OH)D levels, while the H5D group exhibited a decreasing trend in serum 25(OH)D levels.
These results suggest that exposure to high-altitude hypoxia leads to liver injury in humans, causing the release of a large amount of ALT and AST into the bloodstream due to liver cell damage. The ineffective utilization of cholesterol results in its accumulation, and this impairment in liver function hinders the synthesis and utilization of 1,25(OH)2D3 in the body, leading to a decrease in serum 25(OH)D levels. However, 1,25(OH)2D3 effectively inhibits the increase in serum ALT, AST, and cholesterol levels, thereby increasing serum 25(OH)D levels and alleviating liver hypoxic injury.
1,25(OH)2D3 ameliorates hypoxic renal injury
As shown in Figure 1E, compared to Group G1 and G2, the levels of creatinine, BUN, and uric acid in the serum of volunteers in the VDH5D and H5D groups were significantly elevated (P<0.05). However, the elevation in the VDH5D group was significantly less than that in the H5D group, with the creatinine levels in the VDH5D group being significantly lower than in the H5D group (P<0.05). As depicted in Figure 1F, both Group G1 and G2 exhibited negative urine protein results. Following exposure to high-altitude hypoxia, 6 cases (24.0%) in the H5D group were positive for urine protein, 9 cases (36.0%) were weakly positive, and 10 cases (40.0%) were negative. In the VDH5D group, 1 case (4.4%) was positive, 3 cases (13.0%) were weakly positive, and 19 cases (82.6%) were negative. The positive rate of urine protein in the H5D group after exposure to high-altitude hypoxia was significantly higher than in the VDH5D group. This suggests that exposure to high-altitude hypoxia causes renal damage leading to glomerular insufficiency, resulting in elevated levels of serum creatinine, BUN, and uric acid, and a significantly increased positive rate of urine protein. However, 1,25(OH)2D3 can effectively inhibit the elevated trends of serum creatinine, BUN, and uric acid, reduce the positive rate of urine protein, and ameliorate the renal damage caused by hypoxia.
In summary, rapid ascent to high-altitude hypoxic regions can lead to numerous adverse reactions in humans. Hypoxia results in a reduction of oxygen inhaled into the lungs, decreased saturation of blood oxygen, reduced oxygen utilization efficiency in blood circulation, and elevated heart rate and blood pressure, which further cause hypoxic injuries to multiple organs such as the lungs, liver, and kidneys. Observation of the aforementioned phenotypes indicates that 1,25(OH)2D3 effectively ameliorates human adverse reactions to high-altitude hypoxia and hypoxic injuries; however, its mechanism of action still requires further exploration. Constructing acute hypoxic injury rat models to conduct animal experiments combined with metabolomic analysis is particularly important for elucidating the mechanism of action of 1,25(OH)2D3 in preventing and treating high-altitude diseases.
1,25(OH)2D3 ameliorates hypoxic injury in SD Rats
1,25(OH)2D3 ameliorates hypoxia-induced injury in the lung, liver, and kidney tissues of SD rats
Figure 2A is the flowchart of the animal experiment in this study. As shown in Fig. 2B, the histological structures of the pulmonary alveoli, liver lobules, and renal glomeruli in the CV and NV groups were normal; in the HV group, the pulmonary alveolar septa widened, the pulmonary alveoli enlarged, the liver cellular structure was disrupted, with cells arranged in a disorganized manner, showing signs of rupture, necrosis, and vacuolation. The renal tissue exhibited abnormalities in the renal tubular structure, with tubular epithelial cell shedding, unclear nuclear outlines, and substantial infiltration of red blood cells and inflammatory cells in the lung, liver, and kidney tissues. The MV group rats showed reduced tissue damage, with structures tending to be more intact, approaching those of the CV and NV groups. This suggests that acute hypoxic stress can induce damage to the pulmonary, hepatic, and renal tissues of rats, leading to the infiltration of red blood cells and inflammatory cells, resulting in multi-organ hypoxic injury in rats. Conversely, administration of 1,25(OH)2D3 prophylaxis can effectively mitigate the damage caused by hypoxia to various organ tissues, reduce tissue inflammation, and facilitate tissue repair.
1,25(OH)2D3 attenuates oxidative stress in SD rats
As shown in Figure 2C, there were no significant differences in oxidative stress indicators in the lung tissues of rats in the CV, NV, and MV groups, and these indicators were comparable. In comparison with the CV group, the activities of SOD and GSH-Px in the lung tissues of the HV group were significantly decreased (P<0.05), while MDA levels were significantly increased (P<0.05). Acute hypoxic stress can induce a strong oxidative stress response in the lung tissues of rats. In contrast to the HV group, the MV group exhibited an increasing trend in the activities of SOD and GSH-Px, and a significant decreasing trend in MDA levels (P<0.05), indicating that 1,25(OH)2D3 can reverse the oxidative stress state in rats under acute hypoxic stress and enhance their antioxidant stress resistance.
1,25(OH)2D3 inhibits the elevation of inflammatory cell numbers in SD rats subjected to hypoxic injury
As shown in Figure 2D, there were no significant differences in the numbers of inflammatory cells in the serum of rats in the CV, NV, and MV groups, which approached normal levels. In comparison with the CV and NV groups, the HV group exhibited a significant increase in the numbers of white blood cells, lymphocytes, neutrophils, monocytes, and eosinophils (P<0.05). Conversely, when compared to the HV group, the MV group showed a significant decrease in the numbers of inflammatory cells (P<0.05). The results suggest that acute hypoxic stress leads to a significant increase in the numbers of inflammatory cells within the rat body, resulting in an inflammatory response. However, the administration of 1,25(OH)2D3 in advance can significantly reduce the numbers of inflammatory cells, thereby mitigating the onset and progression of inflammation.
1,25(OH)2D3 ameliorates hypoxic liver injury in SD rats
As shown in Figure 2E, compared to the CV group, the HV group exhibited a significant elevation in both ALB and TBIL levels (P<0.05). In contrast to the HV group, the MV group demonstrated a decreasing trend in TBIL levels and a significant decrease in ALB levels (P<0.05). As depicted in Figure 2H, no significant differences were observed in the serum levels of 25(OH)D among the CV, NV, and MV groups, which were found to be comparable. In comparison to the CV group, the HV group showed a significant reduction in serum 25(OH)D levels (P<0.05). Conversely, the MV group demonstrated a significant elevation in serum 25(OH)D levels compared to the HV group. These findings suggest that acute hypoxic stress induces pathological changes in liver tissue, releasing ALB and TBIL into the bloodstream, leading to elevated serum levels. This process impairs the liver's capacity to synthesize and utilize 25(OH)D, resulting in decreased serum 25(OH)D levels. 1,25(OH)2D3 may modify this trend, mitigating the hypoxic injury to the rat liver and exerting a protective effect.
1,25(OH)2D3 improves hypoxic kidney injury in SD rats
As shown in Fig. 2E, compared to the CV group, the serum levels of CRE, BUN, and UA in the HV group significantly increased (P < 0.05). Compared to the HV group, the serum levels of CRE, BUN, and UA in the MV group significantly decreased (P < 0.05).
As shown in Fig. 2F, compared to the CV group, the HV group had 10 cases (100%) of positive urine protein, indicating a significant increase in the urine protein positivity rate. Compared to the HV group, the MV group had 6 negative cases (60%) and 4 positive cases (40%), indicating a decrease in the urine protein positivity rate. These results suggest that acute hypoxic stress leads to hypoxic damage to the rat kidney tissue, increasing the serum levels of CRE, BUN, and UA and causing kidney dysfunction, which results in urine protein in rats. Pre-treatment with 1,25(OH)2D3 can mitigate hypoxic kidney damage, reducing the serum levels of CRE, BUN, and UA and the urine protein positivity rate in rats.
1,25(OH)2D3 induces increased Th2 cell count in hypoxic SD rats
Flow cytometry was used to observe changes in Th1/Th2 cells in rats. As shown in Fig. 2G, the numbers of Th1 and Th2 cells in the Control group and the Hypoxia+VD3 group were similar without significant differences. Compared to the Control group, the number of Th2 cells in the Hypoxia group significantly increased (P < 0.05), and the number of Th1 cells decreased. Compared to the Hypoxia group, the number of Th2 cells in the Hypoxia+VD3 group significantly decreased (P < 0.05), and the number of Th1 cells significantly increased (P < 0.05). This indicates that hypoxic stress induces an inflammatory response in rats, polarizing Th1 to Th2 cells, which secrete anti-inflammatory factors to counteract hypoxia-induced inflammation. Conversely, in the high-altitude hypoxic environment, the number of Th1 cells decreased, and pre-treatment with 1,25(OH)2D3 resulted in a significant increase in Th1 cell count, suggesting that 1,25(OH)2D3 can balance the body's immune response, inhibiting the polarization of Th1 to Th2 cells and exerting an anti-inflammatory effect.
1,25(OH)2D3 downregulates serum short-chain fatty acids in SD rats to exert synergistic anti-iInflammatory effects
A metabolomic analysis of rat serum was conducted to further explore the protective mechanism of 1,25(OH)2D3 against hypoxic injury. The TIC overlay analysis showed good technical reproducibility of metabolites (Supplementary Fig. S1). The Pearson correlation coefficient of QC samples ranged from 0.989 to 1.000 (Supplementary Fig. S1), demonstrating good detection stability and high data quality, laying a foundation for subsequent studies.
Pairwise comparisons between the CV, HV, and MV groups were performed with PLS-DA statistical analysis (Figs. 3A-B), showing clear separation between different groups, indicating good grouping. The model evaluation parameters R2 and Q2 obtained through orthogonal partial least squares discriminant analysis were close to 1 (Figs. 3C-D), indicating significant differences between the models and strong predictive power, allowing for further analysis. After refining the targeted metabolomic data analysis with VIP > 1.0 and P < 0.05 and fold change ≥ 2 as criteria, we found significant differences in metabolites between groups, detecting a total of 664 metabolites (Supplementary Fig. S2). There were 217 differential metabolites between HV and CV, with 155 metabolites showing an upward trend and 62 a downward trend (Fig. 3E). There were 88 differential metabolites between MV and HV, with 14 showing an upward trend and 74 a downward trend (Fig. 3F). The intergroup comparison further elucidated the differences in metabolites between groups.
After performing KEGG enrichment analysis on the differential metabolites (Figs. 3G-H), the differential metabolites in the HV vs. CV comparison group were mainly enriched in thyroid hormone synthesis, fructose, and mannose metabolism (P < 0.05). The differential metabolites in the MV vs. HV comparison group were mainly enriched in starch and sucrose metabolism (P < 0.05). Based on the KEGG enrichment analysis, we screened key differential metabolic pathways of organic acids and selected key metabolites in these pathways for further analysis (Supplementary Fig. S3). The results showed that both analyses annotated the butyrate metabolism pathway (Figs. 3I-J). Using the criteria of VIP > 1.0 and P < 0.05 and fold change ≥ 2, we further analyzed key metabolites in the butyrate metabolism pathway. Among them, we identified four differential metabolites regulated by 1,25(OH)2D3: (R)-3-hydroxyisobutyric acid, (S)-2-hydroxybutyric acid, 2-hydroxybutyric acid, and β-hydroxyisobutyric acid (Fig. 3K).
Compared to the CV group, the HV rats experienced oxidative stress and inflammatory responses due to hypoxia. To cope with acute hypoxia, the body produced a large amount of short-chain fatty acids, resulting in increased levels of the four butyrate salts (R)-3-hydroxyisobutyric acid, (S)-2-hydroxybutyric acid, 2-hydroxybutyric acid, and β-hydroxyisobutyric acid to adapt to the hypoxic environment and mitigate hypoxic damage. Conversely, compared to the HV group, the levels of these four butyrate salts in the MV rats showed a decreasing trend after treatment with 1,25(OH)2D3, approaching the levels seen in the normal group.
Safety of 1,25(OH)2D3 administration
To investigate the safety of 1,25(OH)2D3 administration, normal rats were given 1,25(OH)2D3 intervention and designated as the NV group. Comparisons were made with the CV group to observe whether 1,25(OH)2D3 had any toxic side effects on the rats. HE staining of lung, liver, and kidney tissue sections showed no differences between the NV and CV groups, with both displaying normal tissue structures and no pathological damage (Fig. 2B).Comparing the NV group with the CV group, the levels of ALB and TBIL were similar and showed no significant differences, both remaining within the normal reference range25 (Fig. 2E). The serum levels of 25(OH)D in both groups were also similar, showing no significant differences and remaining within the normal range (36-53 ng/mL) (Fig. 2H). 1,25(OH)2D3 did not cause a significant increase in ALB and TBIL levels in normal rats, indicating no toxic effects on the liver.Comparing the NV group with the CV group, the serum levels of CRE, BUN, and UA were similar and showed no significant differences, all remaining within the normal reference range25 (Fig. 2H). No individuals in either group tested positive for urine protein (Fig. 2F), indicating that 1,25(OH)2D3 did not exert toxic side effects on the kidneys.
In summary, acute hypoxic stress causes hypoxic damage to the liver and kidneys in rats, but 1,25(OH)2D3 can alleviate hypoxia-induced liver and kidney damage, providing a protective effect. Moreover, no toxic side effects of 1,25(OH)2D3 on the liver and kidneys of rats were observed.