Ozone therapy ameliorates tubulointerstitial inflammation by regulating TLR4 in adenine-induced CKD rats

Figures & data

Table 1. Primer list.

Gene	Sequence
TLR4	Forward: 5′-CCAGAGCCGTTGGTGTATCT-3′	239 bp
	Reverse: 5′-TCAAGGCTTTTCCATCCAAC-3′
MCP-1	Forward: 5′-GATGCAGTTAATGCCCCACT-3′	168 bp
	Reverse: 5′-TTCCTTATTGGGGTCAGCAC-3′
TNF-α	Forward: 5′-TGCCTCAGCCTCTTCTCATT-3′	180 bp
	Reverse: 5′-GGGCTTGTCACTCGAGTTTT-3′
IL-1β	Forward: 5′-CTGTGACTCGTGGGATGATG-3′	211 bp
	Reverse: 5′-AGGGATTTTGTCGTTGCTTG-3′
IL-6	Forward: 5′-AGTTGCCTTCTTGGGACTGA-3′	218 bp
	Reverse: 5′-ACAGTGCATCATCGCTGTTC-3′
GAPDH	Forward: 5′-ACAGCAACAGGGTGGTGGAC-3′	253 bp
	Reverse: 5′-TTTGAGGGTGCAGCGAACTT-3′

Table 2. Serum biochemical parameters in different groups at week 4.

Serum biochemical indexes	Sham group	OT group	ADE group
Cr (μm/L)	28 ± 3.58	131.17 ± 8.78**	219.33 ± 14.33^#
BUN (μm/L)	3.82 ± 0.02	36.05 ± 1.72**	66.81 ± 3.93^#
K (mg/day)	4.68 ± 0.17	5.23 ± 0.28**	6.16 ± 0.25^#
Ca (mg/day)	2.34 ± 0.13	2.61 ± 0.12*	2.74 ± 0.13^#
IP (mg/day)	2.56 ± 0.33	4.43 ± 0.23*	4.74 ± 0.28^#

* p < 0.05 versus ADE group.

** p < 0.01 versus ADE group.

# p < 0.01 versus Sham group.

Figure 1. Effect of ozone therapy on tubulointerstitium morphology at week 4. H&E staining was performed in the kidneys of different groups, original magnification ×400 (A); MT staining was performed in the kidneys of different groups, original magnification ×200 (B). Bar graphs depict the statistical analyses of relative percentages of tubulointerstitial fibrosis in the kidneys of different groups after MT staining (C). Bars represent means ± SEM. *p < 0.05 versus ADE group; #p < 0.01 versus Sham group. H&E, hematoxylin and eosin; MT, Masson trichrome.

Figure 2. Effect of ozone therapy on inflammation-related cytokines in kidney at week 4. MCP-1 and TNF-α infiltration in the renal of different groups was detected by immunohistochemical staining (A), original magnification ×400. The protein level and mRNA level of MCP-1, TNF-α, IL-1β and IL-6 were respectively detected by Western blot analysis (B) and Real-time PCR analysis (C). Data were represented by mean ± SEM. *p < 0.05 versus ADE group; **p < 0.01 versus ADE group; ^#p < 0.01 versus Sham group.

Figure 3. Effect of ozone therapy on TLR4 and p-NF-KB P65 in kidney at week 4. TLR4 and p-NF-KB P65 infiltration were detected by immunohistochemical staining (A). The protein level and mRNA level of TLR4 and the protein level of p-NF-KB P65 were detected by Western blot analysis (B) and Real-time PCR analysis (C). Data were represented by mean ± SEM. **p < 0.01 versus ADE group; ^#p < 0.01 versus Sham group.

Figure 4. The relationship between the protein level of TLR4 and the protein levels of MCP-1 (A) and TNF-α (B) in the kidney of experiment rats.