Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

Abstract

Xuebijing (XBJ) is a type of traditional Tibetan medicine, and previous pharmacological studies have shown that the ethanol extract is derived from Chuanxiong, Chishao, Danshen and Honghua. Chuanxiong, Chishao, Danshen and Honghua possesses potent anti-inflammatory activity, and has been used in the treatment of inflammatory infectious diseases. In the present study, we investigated the effects of XBJ on pulmonary permeability and lung injury in cecal ligation and puncture (CLP)-induced sepsis in rats. A CLP sepsis model was established for the control and treatment groups, respectively. Approximately 2 h prior to surgery, an amount of 100 mg/kg XBJ injection was administered to the treatment group. Reverse transcription polymerase chain reaction (PT-PCR) and western blot analysis were used to examine the expression of Toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase 1 (IRAK1), Toll-like receptor 4 (TLR4), nuclear factor-κB65 (NF-κB65) and TNF receptor-associated factor 6 (TRAF6) in lung tissue. ELISA was applied to detect changes of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interleukin-4 (IL-4) and interleukin-10 (IL-10) levels in bronchoalveolar lavage (BAL) fluid, and intercellular adhesion molecule 1 (ICAM-1) and von wille-brand factor (vWF) in serum. The number of neutrophils, albumin and total cells in the BAL fluid were measured. For histological analysis, hematoxylin and eosin (H&E) stains were evaluated. Lung permeability, the wet/dry weight ratio (W/D) and the lung pathology score were determined following the induction of ALI by CLP for 24 h. The results demonstrated that XBJ upregulated Tollip expression and blocked the activity of IRAK1, TLR4, NF-κβ65 and TRAF6. Additionally, the number of neutrophils and total cells were significantly decreased in the XBJ group compared to that in the control group. Lung permeability, the wet/dry weight ratio (W/D) and the lung pathology score were significantly decreased in the XBJ group. The histological results also demonstrated the attenuation effect of XBJ on CLP-induced lung inflammation. The results of the present study indicated that XBJ has a significantly reduced CLP-induced lung permeability by upregulating Tollip expression. The protective effects of XBJ suggest its therapeutic potential in CLP-induced acute lung injury treatment.

Figure 1

Effect of XBJ on the mRNA expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Representative RT-PCR shows the level of Tollip, IRAK1, TLR4, NF-κB65, and TRAF6 expression in the four rat groups. M, marker; A, normal control group; B, sham operation group; C, control group; D, treatment group.

Figure 2

Administration of XBJ led to increased expression levels of Tollip mRNA, and inhibition of TLR4, NF-κB65 and TRAF6 mRNA expression in lung tissue in CLP-ALI mice. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Statistical summary of the densitometric analysis of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ^*P<0.05 and ^**P<0.01 vs. normal control group and sham operation group. ^#P<0.05 and ^##P<0.01 vs. control group.

Figure 3

Administration of XBJ enhanced the expression of Tollip protein, and inhibition of TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Representative western blots show the level of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. A, normal control group; B, sham operation group; C, control group; D, treatment group.

Figure 4

Administration of XBJ enhanced the expression of Tollip protein protein, and inhibition TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Statistical summary of the densitometric analysis of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ^**P<0.01 vs. the normal control group and sham operation group. ^#P<0.05, ^##P<0.01 vs. the control group.

Figure 5

Administration of XBJ upregulated Tollip positive protein in lung tissue in CLP-ALI mice. Groups of mice were challenged with CLP and treated with XBJ 24 h later. Tollip positive protein levels of lung tissue were determined using immunohistochemistry and the average proportion of positive expression in each field was counted using the true color multi-functional cell image analysis management system. Values are expressed as mean ± SD; ^**P<0.01 vs. normal control group and sham operation group. ^#P<0.05 and ^##P<0.01 vs. control group.

Figure 6

Administration of XBJ attenuated LPS-induced pulmonary inflammatory cell infiltration in lung tissue in CLP-ALI mice. Groups of mice were challenged with CLP and treated with XBJ 24 h later. MPO activity, total cell and neutrophil count were measured. Values are expressed as mean ± SD, ^*P<0.05, ^**P<0.01 vs. normal control and sham operation groups; ^#P<0.05 vs. control group.

Figure 7

Administration of XBJ attenuated LPS-induced pulmonary inflammation. Groups of mice were challenged with CLP and treated with XBJ 24 h later. TNF-α, IL-6, IL-1β, IL-4 and IL-10 levels in BAL were determined using ELISA. Values are expressed as mean ± SD; ^**P<0.01 vs. normal control and sham operation groups. ^#P<0.05, ^##P<0.01 vs. control group.

Figure 8

Administration of XBJ attenuated LPS-induced pulmonary oxidative stress response. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The protein expression of (A) Nrf2, (B) MDA and (C) O₂⁻ weremeasured. All the values are expressed as mean ± SD; ^*P<0.05, ^**P<0.01 vs. the normal control and sham operation groups; ^#P<0.05 vs. control group.

Figure 9

Administration of XBJ reduced the CLP-induced VEGF-α production and expression. Groups of rats were treated as described in Materials and methods. VEGF-α levels in (B) BAL fluid and (A) serum, and (C) the protein expression in lung tissue were determined at 24 h after the CLP challenge. Data are presented as mean ± SD of one experiment consisting of three replicates. ^*P<0.05, ^**P<0.01 vs. normal control and sham operation groups; ^#P<0.05, ^##P<0.01 vs. control group.

Figure 10

XBJ decreased the CLP-induced ICAM and vWF production. Groups of rats were treated as described in Materials and methods and then ICAM and vWF levels in serum were determined at 24 h after the CLP challenge. Data are presented as mean ± SD of one experiment consisting of three replicates. ^*P<0.05, ^**P<0.01 vs. the normal control and sham operation groups; ^#P<0.05, ^##P<0.01 vs. control group.

Figure 11

XBJ reduced the CLP-induced lung permeability. Groups of rats were treated as described in Materials and methods. FITC-labeled albumin in the BAL fluid, wet/dry lung weight ratio and lung water content in lung tissue were determined at 24 h after the CLP challenge. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. ^*P<0.05, ^**P<0.01 vs. normal control and sham operation groups; ^#P<0.05, ^##P<0.01 vs. control group.

Figure 12

Administration of XBJ ameliorated the histopathologic changes of lung in CLP-ALI mice. Groups of mice were treated as described in Materials and methods. Histological evaluation of the therapeutic potential of XBJ on LPS-induced lung injury in mice was analyzed at 24 h after the LPS challenge. Representative images of hematoxylin and eosin-stained lung sections from four experimental groups. (A) Sham operation, (B) control and (C) treatment groups.

Figure 13

Administration of XBJ decreased the acute lung injury histopathologic score of lung in CLP-ALI mice. Groups of mice were treated as described in Materials and methods and a histological evaluation of the therapeutic potential of salidroside on LPS-induced lung injury in mice was analyzed at 24 h after LPS challenge. Lung injury score was determined. Data are presented as mean ± SD of one experiment consisting of three replicates. The ALI pathology score is expressed as mean ± SD; ^**P<0.01 vs. the normal control and sham operation groups; ^#P<0.05 vs. control group.

Figure 14

Effect of administration of XBJ on LPS-induced arterial blood gas in the four rat groups. Groups of mice were challenged with CLP and treated with XBJ 24 h later the arterial blood gas were measured. Values are expressed as mean ± SD; ^*P<0.05, ^**P<0.01 vs. the normal control and sham operation groups; ^#P<0.05 vs. control group.