Vitamin D3 pretreatment regulates renal inflammatory responses during lipopolysaccharide-induced acute kidney injury

Vitamin D receptor (VDR) is highly expressed in human and mouse kidneys. Nevertheless, its functions remain obscure. This study investigated the effects of vitamin D3 (VitD3) pretreatment on renal inflammation during lipopolysaccharide (LPS)-induced acute kidney injury. Mice were intraperitoneally injected with LPS. In VitD3 + LPS group, mice were pretreated with VitD3 (25 μg/kg) at 48, 24 and 1 h before LPS injection. As expected, an obvious reduction of renal function and pathological damage was observed in LPS-treated mice. VitD3 pretreatment significantly alleviated LPS-induced reduction of renal function and pathological damage. Moreover, VitD3 pretreatment attenuated LPS-induced renal inflammatory cytokines, chemokines and adhesion molecules. In addition, pretreatment with 1,25(OH)2D3, the active form of VitD3, alleviated LPS-induced up-regulation of inflammatory cytokines and chemokines in human HK-2 cells, a renal tubular epithelial cell line, in a VDR-dependent manner. Further analysis showed that VitD3, which activated renal VDR, specifically repressed LPS-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65 subunit in the renal tubules. LPS, which activated renal NF-κB, reciprocally suppressed renal VDR and its target gene. Moreover, VitD3 reinforced the physical interaction between renal VDR and NF-κB p65 subunit. These results provide a mechanistic explanation for VitD3-mediated anti-inflammatory activity during LPS-induced acute kidney injury.

Vitamin D is known for its classical functions in calcium uptake and bone metabolism 16 . Recently, vitamin D is recognized for its non-classical actions including the modulation of innate immune and the regulation of cell proliferation 17,18 . Vitamin D itself is devoid of biological activity. Vitamin D3 (VitD3) is converted to 25(OH) D3 by cytochrome P450 (CYP)2R1 in the liver 19 . 25(OH)D3 is then converted into 1,25(OH)2D3 (also known as calcitriol), the active form of VitD3, by CYP27B1 in the kidney 20 . The actions of VitD3 are mediated by vitamin D receptor (VDR) that binds 1,25(OH)2D3 to induce both transcriptional and non-genomic responses 21 . Indeed, all components that mediate vitamin D activity, such as VDR and CYP27B1, are highly expressed in human and mouse kidneys 22,23 . Several studies demonstrated that VitD3 suppressed TGF-β -mediated tubular epithelial-to-mesenchymal transition and renal fibrosis in a VDR-dependent manner 24,25 . According to a recent report, the activated VDR physically interacts with Iκ B kinase β (IKKβ ) to block TNF-α -mediated translocation of nuclear factor kappa B (NF-κ B) p65 subunit from cytoplasm to nuclei in HEK293 cell 26 .
The aim of the present study was to investigate the effects of VitD3 pretreatment on early inflammatory response during LPS-induced acute kidney injury. We showed that VitD3 pretreatment alleviated early inflammatory response in LPS-induced acute kidney injury. We demonstrate that there is a mutual repression between VitD3-activated renal VDR signaling and LPS-activated renal NF-κ B signaling. The interaction between renal VDR and NF-κ B p65 provides a mechanistic explanation for VitD3-mediated anti-inflammatory activity during LPS-induced acute kidney injury.

Results
VitD3 pretreatment alleviates LPS-induced acute kidney injury in mice. The effects of VitD3 pretreatment on LPS-induced renal function were analyzed. As shown in Fig. 1A, the level of BUN was markedly increased 18 h and 24 h after LPS injection. Correspondingly, the level of serum creatinine was elevated 18 h and 24 h after LPS injection (Fig. 1B). Moreover, the levels of BUN and serum creatinine were higher at 18 h after LPS injection than at 24 h after LPS injection (Fig. 1A,B). Interestingly, VitD3 pretreatment protected against LPS-induced impairment of renal function (Fig. 1A,B). The effects of VitD3 pretreatment on LPS-induced renal pathological damage were then analyzed. As expected, an obvious renal pathological damage, including edema of renal tubular epithelial cells, dilation of renal capsule cavity, destruction of tubular structures, the epithelial cells of the local focal necrosis collapse and loss of tubular brush borders, was observed 18 h after LPS injection (Fig. 1C). VitD3 pretreatment obviously attenuated LPS-induced pathological damage in the kidneys (Fig. 1C,D).

Effects of VitD3 pretreatment on LPS-induced renal inflammatory cytokines, chemokines, icam-1, vcam-1 and cox-2.
The effects of VitD3 pretreatment on LPS-induced renal inflammatory cytokines were analyzed. As expected, renal tnf-α and il-6, two inflammatory cytokine genes, were markedly up-regulated 1 h after LPS injection and remained increased 6 h after LPS injection. Interestingly, VitD3 pretreatment significantly attenuated LPS-induced up-regulation of renal inflammatory cytokines ( Fig. 2A,B). The effects of VitD3 pretreatment on LPS-induced renal chemokines were then analyzed. As expected, renal mip-2, kc and mcp-1, three chemokine genes, were markedly up-regulated 1 h after LPS injection and remained increased 6 h after LPS injection ( Fig. 2C-E). Although VitD3 pretreatment had little effect on renal chemokines at 1 h after LPS, the expression of renal chemokines at 6 h after LPS injection was significantly repressed in VitD3-pretreated mice ( Fig. 2C-E). Next, the effects of VitD3 pretreatment on LPS-induced renal icam-1 and vcam-1 expression were analyzed. As expected, renal icam-1 and vcam-1 mRNAs were markedly up-regulated 1 h after LPS injection and remained increased 6 h after LPS injection (Fig. 2F,G). Although VitD3 pretreatment had little effect on the expression of renal icam-1 and vcam-1 mRNAs at 1 h after LPS injection, the expression of renal icam-1 and vcam-1 mRNAs at 6 h after LPS injection was significantly repressed in VitD3-pretreated mice (Fig. 2F,G). Finally, the effects of VitD3 pretreatment on LPS-induced renal cox-2 were analyzed. As shown in Fig. 2H, renal cox-2 mRNA was markedly up-regulated at 1 h after LPS injection and remained increased at 6 h after LPS injection. VitD3 alone had no effect on the expression of renal cox-2. Moreover, VitD3 pretreatment had little effect on LPS-induced upregulation of renal cox-2 (Fig. 2H).
Effects of VitD3 pretreatment on LPS-activated renal MAPK p38 and PI3K/Akt signaling. As shown in Fig. 4A, no significant difference on renal tlr4 mRNA was observed among different groups. Moreover, VitD3 alone did not affect renal myd88 expression (Fig. 4B). As expected, renal myd88 mRNA was up-regulated 6 h after LPS injection. Interestingly, VitD3 pretreatment had little effect on LPS-induced upregulation of renal myd88 (Fig. 4B). The effects of VitD3 pretreatment on LPS-induced renal Akt and MAPK p38 phosphorylation were analyzed. As shown in Fig. 4C, the level of renal phosphorylated Akt was obviously elevated 1 h after LPS injection. By contrast, the level of renal phosphorylated p38 was not elevated 1 h after LPS injection (Fig. 4D), indicating that renal MAPK p38 signaling is not activated 1 h after LPS injection. Unexpectedly, VitD3 pretreatment had little effect on LPS-induced renal Akt phosphorylation (Fig. 4C).

Effects of VitD3 pretreatment on LPS-activated renal NF-kB signaling.
A recent study demonstrated that renal NF-κ B signaling was involved in the pathogenesis of LPS-induced acute kidney injury 27 . The effects of VitD3 pretreatment on LPS-activated renal NF-κ B signaling were analyzed. As expected, renal phosphorylated Iκ B level was significantly increased in LPS-treated mice (Fig. 5A,B). Correspondingly, renal I-κ B level was significantly decreased in LPS-treated mice (Fig. 5A,B). By contrast, the levels of nuclear NF-κ B p65 and p50 subunits in the kidneys were markedly elevated 1 h after LPS injection (Fig. 5C,D), indicating that LPS could rapidly evoke renal NF-κ B p65 and p50 translocation from cytoplasm to nuclei. Immunohistochemistry observed a strong immunoreactivity in the nuclei of the distal convoluted tubules (DCT) and a relatively weak immunoreactivity in the nuclei of the proximal convoluted tubules (PCT) (Fig. 5E). These results suggest that LPS-evoked nuclear translocation of renal NF-kB p65 subunit was mainly distributed in the DCT, and to a lesser extent, in the PCT. Of interest, VitD3 pretreatment had little effect on LPS-induced I-κ B phosphorylation. Moreover, VitD3 pretreatment did not inhibit LPS-induced reduction of renal Iκ Bα level (Fig. 5A,B). In addition, VitD3 Effects of LPS on VitD3-activated renal VDR signaling. The effects of LPS on VitD3-activated renal VDR signaling were analyzed. As shown in Fig. 6A, VitD3 alone had no effect on renal vdr expression. Interestingly, renal vdr mRNA was slightly upregulated at 1 h after LPS injection and then downregulated at 6 h after LPS injection. The effects of LPS on renal cyp27b1, an enzyme gene that transformed 25(OH)D3 into 1,25(OH)2D3, were analyzed. Although no significant difference on renal cyp27b1 mRNA was observed among different groups (Fig. 6B), the level of CYP27B1 protein in proximal tubule of renal cortex was markedly elevated at 1 h after LPS injection and remained increased at 6 after LPS injection (Fig. 6C). Interestingly, LPS had no effect on the level of CYP27B1 protein in renal medulla (Fig. 6C). The effects of LPS on renal cyp24a1, a target gene of VDR signaling, are presented in Fig. 6D. As expected, renal cyp24a1 mRNA was upregulated by more than 12 folds in VitD3-pretreated mice. Interestingly, VitD3-induced upregulation of renal cyp24a1 was obviously inhibited at 1 h after LPS injection and remained repressed at 6 h after LPS injection (Fig. 6D). Finally, the effects of LPS on renal nuclear VDR were analyzed. As expected, nuclear VDR level in the kidneys was significantly elevated in VitD3-pretreated mice (Fig. 6E), indicating that VitD3 pretreatment promotes renal VDR translocation from the cytoplasm to the nucleus. LPS significantly inhibited VitD3-evoked nuclear VDR translocation in the kidneys (Fig. 6E).
The interaction between renal VDR and NF-κB p65. The interaction between renal VDR and NF-κ B p65 was determined by CoIP. As expected, VitD3 plus LPS treatments increased the level of NF-κ B p65 in the immunocomplexes precipitated by anti-VDR antibody (Fig. 7). Correspondingly, VitD3 plus LPS treatments increased the level of VDR in the immunocomplexes precipitated by anti-p65 antibody (Fig. 7). These results suggest that VitD3 pretreatment reinforces the interaction between VDR and NF-κ B p65 in the kidneys.

Discussion
An earlier study indicated that paricalcitol, a vitamin D analog, prevented rats from cisplatin-induced acute renal injury by suppressing apoptosis and proliferation 28 . According to a recent report, paricalcitol protected against ischemia/reperfusion-induced acute kidney injury by suppressing TLR4-NF-κ B mediated inflammation 29 . The present study investigated the effect of VitD3 pretreatment on LPS-induced acute kidney injury. We showed that VitD3 prevented LPS-induced reduction of renal function. In addition, VitD3 pretreatment obviously alleviated
It is increasingly recognized that inflammatory cytokines, such as TNF-α , are the major mediators of sepsis-induced acute kidney injury 10,11 . MCP-1 and IL-8, two chemokines, play key roles in the recruitment of inflammatory cells into renal interstitium in sepsis-induced acute kidney injury 30 . The present study showed that renal tnf-α and il-6 were rapidly up-regulated after LPS injection. Moreover, renal mcp-1 mRNA was markedly elevated after LPS injection. In addition, renal mip-2 and kc, two functional analogues of human IL-8, were obviously up-regulated at 1 and 6 h after LPS injection. Interestingly, LPS-induced up-regulation of renal tnf-α and il-6 was significantly attenuated by VitD3. Moreover, LPS-induced up-regulation of renal mcp-1, mip-2 and kc was obviously repressed in VitD3-pretreated mice. The role of adhesion molecules, such as ICAM-1 and VCAM-1, in LPS-induced acute kidney injury is well established 7,31 . Indeed, the present study showed that the expression of renal icam-1 and vcam-1 was up-regulated after LPS injection. Interestingly, LPS-induced up-regulation of renal icam-1 and vcam-1 was markedly attenuated by VitD3 pretreatment. These results suggest that VitD3 pretreatment inhibits early inflammatory response during LPS-induced acute kidney injury.
It is well known that both adaptive and innate immune cells express VDR 17 . Indeed, immune cells play a predominant role in the pathogenesis of sepsis. To demonstrate whether renal VDR also plays an important role in VitD3-mediated anti-inflammatory effect, the effects of 1,25(OH)2D3, an active form of VitD3, on LPS-evoked inflammatory cytokines and chemokines in human HK-2 cells, a renal tubular epithelial cell line, were analyzed. As expected, TNF-α and IL-1β, two inflammatory cytokines, and IL-8 and MCP-1, two chemokines, were markedly upregulated in LPS-treated human HK-2 cells. Moreover, pretreatment with 1,25(OH)2D3 significantly attenuated LPS-evoked inflammatory cytokines and chemokines in human HK-2 cells. To further determine the functional role of renal VDR in modulating LPS-induced inflammatory cytokines and chemokines, siRNA was used to inhibit VDR expression in human HK-2 cells. As expected, TNF-α, IL-1β, IL-8 and MCP-1 were obviously up-regulated   in LPS-stimulated VDR-siRNA-transfected human HK-2 cells. Interestingly, 1,25-(OH)2D3 had little effect on LPS-induced inflammatory cytokines and chemokines in VDR-siRNA-transfected human HK-2 cells. These results suggest that renal VDR is involved in the regulation of early renal inflammatory responses during LPS-induced acute kidney injury. The present study does not exclude the role of VDR in both adaptive and innate immune cells on VitD3-mediated anti-inflammatory effect in LPS-induced acute kidney injury. Thus, additional work is required to determine the role of immune cells in VitD3-mediated protection against LPS-induced acute kidney injury.
The mechanism through which VDR plays its anti-inflammatory activity remains with debate. According to an earlier report, 1,25(OH)2D3, an active VitD3 that activates VDR signaling, stimulates MAPK p38 phosphorylation in aortic smooth muscle cells 32 . A recent study showed that 1,25(OH)2D3 activated MAPK p38 and PI3K/ Akt signaling in cultured endothelial cells 33 . However, two recent studies found that 1,25(OH)2D3 obviously down-regulated LPS-evoked inflammatory cytokines through inhibiting MAPK p38 and Akt phosphorylation in macrophages 34,35 . The present study investigated the effects of VitD3 pretreatment on renal inflammatory signaling in LPS-induced acute kidney injury. Although renal MAPK p38 was not activated 1 h after LPS injection, renal inflammatory cytokines were significantly up-regulated, indicating that LPS-induced upregulation of inflammatory cytokines is independent of renal MAPK p38 signaling. Interestingly, the level of renal phosphorylated Akt was significantly elevated in LPS-treated mice. Moreover, renal NF-κ B was activated 1 h after LPS injection, as determined by the reduced I-κ B level and the elevated nuclear NF-κ B p65 and p50 levels. Unexpectedly, VitD3 pretreatment did not repress LPS-induced renal Akt phosphorylation. By contrary, VitD3 inhibited LPS-induced renal NF-κ B activation. These results suggest that VitD3 down-regulates renal inflammatory cytokines by inhibiting renal NF-κ B signaling.
According to a recent report using GST pull-down assay, the activated VDR physically interacts with Iκ B kinase β (IKKβ ), abrogates IKKβ phosphorylation and abolishes IKK to phosphorylate I-κ B, thus prevents nuclear translocation of NF-κ B p65/p50 subunits 26 . The present study analyzed the effects of VitD3 pretreatment on LPS-evoked Several studies have demonstrated that the activated nuclear receptors, such as pregnane X receptor and liver X receptor, repress NF-κ B signaling in macrophages 36,37 . Conversely, LPS-activated NF-κ B inhibits nuclear receptor signaling in hepatocytes and intestinal epithelial cells 38,39 . Indeed, VDR, also a nuclear receptor, is highly expressed in tubular epithelial cells of human and rodent kidneys 23,40 . The present study hypothesizes that the activated VDR inhibits LPS-activated renal NF-κ B signaling through its interaction with NF-κ B p65 subunit. Following evidence demonstrates the hypothesis that VitD3 inhibits renal NF-κ B signaling through the interation between VDR and NF-κ B p65 subuni. First, VitD3, which activated renal VDR, simultaneously blocked LPS-evoked nuclear NF-κ B p65 translocation and repressed its downstream target genes in the kidneys. Second, LPS, which activated renal NF-κ B, simultaneously abolished VitD3-evoked nuclear VDR translocation and inhibited its downstream target genes in the kidneys. Third, LPS-evoked nuclear NF-kB p65 translocation was mainly observed in the nuclei of the distal convoluted tubules, and to a lesser extent, in the nuclei of the proximal convoluted tubules. Correspondingly, VDR was highly expressed in the distal convoluted tubules, and to a relatively lower level, in the proximal convoluted tubules 23 . To elucidate the mechanism through which VitD3-activated VDR inhibits LPS-evoked nuclear NF-κ B p65 translocation in the kidneys, CoIP was used to test physical association between renal VDR and NF-κ B subnuits in LPS-induced acute kidney injury. As expected, VitD3 pretreatment reinforced the interaction between renal VDR and NF-κ B p65 during LPS-induced acute kidney injury. Taken together, these results suggest that VitD3-activated VDR inhibits LPS-evoked renal NF-κ B activation through its interaction with NF-κ B p65 subunit.
Renal VDR as a regulator of inflammatory response may have preventive and therapeutic implications. Several studies showed that vitamin D status was negatively associated with acute kidney injury in critically ill patients 41,42 . According to a recent report, vitamin D deficiency aggravated the progression of chronic kidney disease after ischemia/reperfusion -induced acute kidney injury 43 . The present study showed that VitD3 pretreatment protected against LPS-induced acute kidney injury by regulating early renal inflammatory responses. Therefore, VitD3 may be used as a potential protective agent for clinical prevention and therapy especially in high-risk situations in which the patients are infected with bacteria.
In summary, the present study investigated the effects of VitD3 pretreatment on early renal inflammatory response during LPS-induced acute kidney injury. Our results showed that VitD3 pretreatment down-regulated renal inflammatory response during LPS-induced acute kidney injury. We demonstrated for the first time that there was a mutual repression between VitD3-activated renal VDR and LPS-activated renal NF-κ B. The interaction between renal VDR and NF-κ B p65 subunit provides a mechanistic explanation for VitD3-mediated anti-inflammatory activity during LPS-induced acute kidney injury. Overall, the present study provides evidence Figure 7. The interaction between renal VDR and NF-κB p65. In VitD3 + LPS group, mice were pretreated with three doses of VitD3 (25 μ g/kg) at 48, 24 and 1 h before LPS (1.0 mg/kg) injection. Kidney samples were collected 1 h after LPS injection. The nuclear fractions were prepared from kidneys and incubated with agaroseconjugated either VDR or NF-κ B p65 antibody. NF-κ B p65 and VDR were measured using immunoblots. All experiments were duplicated for four times. All data were expressed as means ± S.E.M. (n = 4). **P < 0.01. for roles of renal VDR partially as an important regulator of renal inflammatory response in sepsis-induced acute kidney injury.

Animals and treatments.
Adult male CD-1 mice (8 week-old, 28-32 g) were purchased from Beijing Vital River whose foundation colonies were all introduced from Charles River Laboratories, Inc. The animals were allowed free access to food and water at all times and maintained on a 12-h light/dark cycle in a controlled temperature (20-25 °C) and humidity (50 ± 5%) environment. Mice were divided into four groups randomly. In LPS group, mice were intraperitoneally (i.p.) injected with a single dose of LPS (1.0 mg/kg). In control group, mice were i.p. injected with normal saline (NS). In VitD3 + LPS group, all mice were pretreated with three doses of VitD3 (each 25 μ g/kg) by gavage, first dose at 48 h before LPS, second at 24 h before LPS, and third at 1 h before LPS. In VitD3 group, all mice were pretreated with three doses of VitD3 (each 25 μ g/kg) by gavage at 48, 24 and 1 h before NS injection. The doses of VitD3 used in the present study referred to others 44,45 . Preliminary experiment showed that inflammatory cytokines were significantly up-regulated at 1 h after LPS injection and remained elevated at 6 h after LPS injection. Thus, mouse kidneys were collected 1 h and 6 h after LPS injection for the measurement of inflammatory cytokines and inflammatory signaling. Some mice were euthanized with carbon dioxide and cervical dislocation 18 h and 24 h after LPS injection. Blood samples were collected for measurement of renal function. The left kidneys were collected for histopathology. The right kidneys were collected and kept at -80 °C for subsequent experiments.
Renal histology. Renal tissues were fixed in 4% formaldehyde and embedded in paraffin according to the standard procedure. Paraffin-embedded renal tissues were serially sectioned. At least five consecutive longitudinal sections were stained with periodic acid-schiff (PAS). Renal histopathologic alterations were evaluated using the criteria established by Solez and used previously by Conger 46 . Changes were graded on a 0 to 2 scale in a double blind fashion. There was a uniform correlation on sections from the same animal. Cell culture and treatments. Human HK-2 cell is a renal tubular epithelial cell line, which was purchased from the American Type Cell Collection (ATCC). HK-2 cell was grown in Nunc flasks in Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12, GIBCO) supplemented with 100 U/mL penicillin, 100 μ g/mL streptomycin, 1% (v/v) insulin-transferrin-selenium, and 10% (v/v) heat-inactivated FBS in a humidified chamber with 5% CO 2 /95% air at 37 °C. HK-2 cells were seeded into 6-well culture plates at a density of 5 × 10 5 cells/well and incubated for at least 12 hr to allow them to adhere to the plates. After washing three times with medium, human HK-2 cells were pre-incubated with 1,25-(OH)2D3 for 24 h. Human HK-2 cells were incubated with LPS (5.0 μ g/ ml) for 2 h in the presence or absence of 1,25-(OH)2D3 (100 nM). The concentration of 1,25-(OH)2D3 used in the present study referred to others 48 . The cells were washed with chilled PBS for three times and then harvested for real-time RT-PCR and immunoblots.
Real-time RT-PCR. Total RNA in renal tissues was extracted using TRI reagent. RNase-free DNase-treated total RNA (1.0 μ g) was reverse-transcribed with AMV (Pregmega). Real-time RT-PCR was performed with a LightCycler 480 SYBR Green I kit (Roche Diagnostics GmbH) using gene-specific primers as listed in Table 1. The amplification reactions were carried out on a LightCycler 480 Instrument (Roche Diagnostics GmbH) with an initial hold step (95 °C for 5 minutes) and 50 cycles of a three-step PCR (95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds).
Immunoblots. Renal lysate was prepared by homogenizing 50 mg renal tissue in 300 μ l lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecylsylphate, 1 mM phenylmethylsulfonyl fluoride) supplemented with a cocktail of protease inhibitors (Roche). For nuclear protein extraction, renal lysate was suspended in hypotonic buffer and then kept on ice for 15 min. The suspension was then mixed with detergent and centrifuged for 30 s at 14,000 × g. The nuclear pellet obtained was resuspended in complete lysis buffer in the presence of the protease inhibitor cocktail, incubated for 30 min on ice, and centrifuged for 10 min at 14,000 × g. Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay reagents (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. For immunoblots, same amount of protein (40 ~ 80 μ g) was separated electrophoretically by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membranes were incubated for 2 h with the following antibodies: pAkt, Akt, pp38, p38, p-Iκ B, I-κ B, NF-κ B p65, NF-κ B p50 and VDR. For total proteins, β -actin, Akt or p38 was used as a loading control. For nuclear protein, lamin A/C was used as a loading control. After washes in DPBS containing 0.05% Tween-20 four times for 10 min each, the membranes were incubated with goat anti-rabbit IgG or goat anti-mouse antibody for 2 h. The membranes were then washed for four times in DPBS containing 0.05% Tween-20 for 10 min each, followed by signal development using an ECL detection kit.

Immunohistochemistry (IHC).
For IHC, paraffin-embedded renal sections were deparaffinized and rehydrated in a graded ethanol series. Antigen retrieval was achieved by microwave method using sodium citrate solution with pH 6.0. After antigen retrieval and quenching of endogenous peroxidase, sections were incubated with NF-κ B p65 monoclonal antibody (1:200 dilution) or CYP27B1 monoclonal antibody (1:200 dilution) at 4 °C overnight. The color reaction was developed with HRP-linked polymer detection system (Golden Bridge International, WA, USA) and counterstaining with hematoxylin 50 .
Statistical analysis. All data were expressed as means ± SEM. SPSS 13.0 statistical software was used for statistical analysis. All statistical tests were two-sided using an alpha level of 0.05. ANOVA and the Student-Newmann-Keuls post hoc test were used to determine differences among different groups. Student t test was used to determine differences between two groups.
Ethics statement. This study was approved by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University (Permit Number: 14-0017). All procedures on animals followed the guidelines for humane treatment set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University.