25(OH)-Vitamin D alleviates neonatal infectious pneumonia via regulating TGFβ-mediated nuclear translocation mechanism of YAP/TAZ

Figures & data

Table 1. The sequences of VDR, CYP27B1, TGFβ and GAPDH

Primer name	Primer sequences
VDR	Forward: 5-TGCCTGACCCTGGAGACTTTGACC-3 Reverse: 5-CATCATGCCGATGTCCACACAGCG-3’
CYP27B1	Forward: 5-TGGCCCAGATCCTAACACATTT-3 Reverse: 5-GTCCGGGTCTTGGGTCTAACT-3
TGFβ	Forward: 5-TACCTGAACCCGTGTTGCTCTC-3 Reverse: 5-GTTGCTGAGGTATCGCCAGGAA-3
GAPDH (Reference)	Forward: 5-GGTATCGTGGAAGGACTCATGAC-3 Reverse: 5-ATGCCAGTGAGCTTCCCGTTCAGC-3

Table 2. Basic information in the control, mild infectious pneumonia and severe infectious pneumonia cases

Group	Cases	Gender (male/female)	Gestational age (x ± s, week)		Birth weight (x ± s, g)	Delivery mode (n (%))
						Cesarean delivery		Normal delivery
Control	60	29/31	38.2 ± 0.7		2963 ± 230	31 (51.7)		29 (48.3)
Mild pneumonia	34	16/18	38.2 ± 0.9		2861 ± 284	16 (47.1)		18 (52.9)
Severe pneumonia	31	15/16	38.3 ± 1.1		2863 ± 304	17 (54.8)		14 (45.2)
F value		0.0707	0.3012		2.356	0.1972
p value		0.9318	0.7404		0.0991	0.8213
Group	Season of birth (n (%))				Maternal illness during pregnancy (n (%))
	Spring	Summer	Autumn	Winter	Infection	Diabetes	Hypertension	Thyroid hypo-function
Control	13 (21.7)	15 (25.0)	14 (23.3)	18 (30.0)	0	10 (16.7)	8 (13.3)	5 (8.3)
Mild pneumonia	8 (23.5)	7 (20.6)	10 (29.4)	9 (26.5)	1 (2.9)	3 (8.8)	4 (11.8)	5 (14.7)
Severe pneumonia	6 (19.3)	8 (25.8)	7 (22.6)	10 (32.3)	2 (6.5)	4 (12.9)	7 (22.6)	3 (9.7)
F value	0.0525				0.5939
p value	0.9489				0.5538

p > 0.05 was performed to eliminate the effects caused by gender, gestational age, birth weight, delivery mode, season of birth and the maternal hypertension during pregnancy etc. on the results of 25-OH-VD.

Table 3. Comparison of 25-OH-VD deficiency rate among the controls, mild infectious pneumonia and severe infectious pneumonia cases

Group	Cases	Normal (n (%))	Insufficient (n (%))	Deficient (n (%))	Seriously deficient (n (%))
Control	60	12 (20.00)	27 (45.00)	21 (35.00)	0
Mild pneumonia	34	2 (5.88)	6 (17.65)	22 (64.71)	4 (11.76)
Severe pneumonia	31	0 (0.0)	2 (6.45)	20 (64.52)	9 (29.03)

According to the ‘Prevention and control proposal of the deficiency of micronutrients in children’ (prevention and control proposal), serum 25-OH-VD > 20.0 µg/L was considered as the normal level, 15.0 ~ 20.0 µg/L as the insufficient level, 5.1 ~ 14.9 µg/L as the deficient level, and ≤ 5.0 µg/L as the severely deficient level. In this study, serum 25-OH-VD ≤ 15.0 µg/L data were collected and calculated the rate of deficiency.

Figure 1. The correlation between the levels of 25-OH-VD and Ca²⁺, PCT, CRP, and IL-6. (a) Statistical analysis of serum levels of 25-OH-VD, Ca²⁺, PCT, CRP, and IL-6 in the controls and mild and severe infectious pneumonia cases. (b) Correlations between the 25-OH-VD levels and four inflammatory indicators. ‘***’ p < 0.001 vs. control; ‘^###’ p < 0.001 vs. mild pneumonia cases

Table 4. Statistical analysis of serum levels of 25-OH-VD, Ca²⁺, PCT, CRP and IL-6 in the controls, mild infectious pneumonia and severe infectious pneumonia cases

Group	Cases (n)	25-OH-VD (µg/L)	Ca^2+ (mmol/L)	PCT (µg/L)	CRP (mg/L)	IL-6 (ng/L)
Control	60	16.28 ± 0.770	2.405 ± 0.042	0.060 ± 0.003	2.304 ± 0.032	4.305 ± 0.051
Mild pneumonia	34	11.13 ± 0.939	2.300 ± 0.048	0.210 ± 0.005	2.601 ± 0.040	28.80 ± 0.196
Severe pneumonia	31	8.433 ± 0.773	2.403 ± 0.046	0.559 ± 0.014	4.700 ± 0.048	43.40 ± 0.247
F value		23.63	1.536	1304	1011	19,278
p value		< 0.001	0.2195	< 0.001	< 0.001	< 0.001

p < 0.05 was considered to indicate a statistically significant result. The statistical differences were found in the serum levels of 25-OH-VD, PCT, CRP and IL-6 among the controls, mild pneumonia cases and severe pneumonia cases but not Ca²⁺.

Table 5. Correlations of 25-OH-VD with Ca²⁺, PCT, CRP and IL-6 among the neonates with infectious pneumonia

Indicators	rs value (95%CI)	p value
Ca^2+	0.042 (−0.121,0.210)	0.636
PCT	−0.340 (−0.463, −0.184)	<0.001
CRP	−0.135 (−0.298, 0.027)	0.113
IL-6	−0.345 (−0.481, −0.170)	<0.001

p < 0.05 was considered to indicate a statistically significant result. 25-OH-VD negatively correlate to serum levels of PCT and IL-6. 25-OH-VD did not correlate to serum levels of Ca²⁺ and CRP.

Figure 2. The analysis of TGFβ/YAP/TAZ pathway-related genes. (a) The mRNA expression levels of VDR and TGFβ in PBMCs, as determined using qRT-PCR. (b) Protein expression levels of VDR, TGFβ, n-YAP, and n-TAZ, as obtained using the western blot method. (c) Levels of inflammatory indicators IL-2, IFN-γ, TNFα and IL-1β, as determined using ELISA. NS, no significant difference with control; ‘*’ p < 0.05, ‘**’ p < 0.01, and ‘***’ p < 0.001 vs. control; ‘^#’ p < 0.05, ‘^##’ p < 0.01, and ‘^###’ p < 0.001 vs. mild pneumonia cases

Figure 3. 25-OH-VD inhibited the nuclear translocation of YAP/TAZ complex. (a) Cellular viability of BEAS-2B cells, as determined using CCK8 assay. (b) mRNA expression levels of CYP27B1, VDR, and TGFβ, as detected using qRT-PCR. (c) The positive expression of YAP in nuclei, as detected using ICC and concluded in a histogram; Bar: 50 μm. (d) IFA was applied to confirm the nuclear translocation of YAP/TAZ complex in BEAS-2B cells; Bar: 20 µm. (e) Expression levels of IL-2, IFN-γ, TNF-α, and IL-1β, as determined using the western blot method. NS, no significant difference with control; ‘*’ p < 0.05, ‘**’ p < 0.01, and ‘***’ p < 0.001 vs. control; ‘^@’ p < 0.05, ‘^@@’ p < 0.01, and ‘^@@@’ p < 0.001 vs. LPS group

Figure 4. The anti-inflammatory mechanism of 25-OH-VD in NIP. When 25-OH-VD diffused into BEAS-2B cells, it was secondarily hydroxylated to generate 1,25-OH-2D under the action of CYP27B1 and combined with VDR to inhibit the activation and expression of TGFβ, thus reducing the nuclear translocation of YAP/TAZ complex. Once the YAP/TAZ complex was translocated into the nucleus, as the promoter, it participated in the expression levels of inflammatory factors (IL-2, TNF-α, IL-1β, and IFN-γ)

Availability of data and material

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.