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Small interference RNA targeting TLR4 gene effectively attenuates pulmonary inflammation in a rat model.

Wu F, Liu Y, Lv X, Miao X, Sun Y, Yu W - J. Biomed. Biotechnol. (2012)

Bottom Line: Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo.TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury.These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Eastern Hepatobiliary Hospital, Shanghai, China.

ABSTRACT

Objective: The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA) targeting Toll-like receptor 4 (TLR4) gene in ameliorating lipopolysaccharide- (LPS-) induced acute lung injury (ALI).

Methods: In vitro, alveolar macrophages (AMs) were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL) for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group), 300 μL of Ad-EGFP (Ad-EGFP group), or 300 μL of Ad-siTLR4 (Ad-siTLR4 group) and then were intravenously treated with LPS (50 mg/kg) to induce ALI.

Results: Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals.

Conclusion: TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

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Effects of Ad-siTLR4 on LPS-induced ALI. Lungs were collected at 4 h after injection of normal saline or LPS for histological examination (a, b, c). Sections were stained with hematoxylin-eosin. Magnification: ×100 Wet/Dry ratio (d), total protein content in BALF (e) as well as MPO activity (f) were measured to determine the lung edema, microvascular permeability, and neutrophil sequestration, respectively. *P < 0.05 versus Ad-EGFP group, #P < 0.05 versus control group.
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fig5: Effects of Ad-siTLR4 on LPS-induced ALI. Lungs were collected at 4 h after injection of normal saline or LPS for histological examination (a, b, c). Sections were stained with hematoxylin-eosin. Magnification: ×100 Wet/Dry ratio (d), total protein content in BALF (e) as well as MPO activity (f) were measured to determine the lung edema, microvascular permeability, and neutrophil sequestration, respectively. *P < 0.05 versus Ad-EGFP group, #P < 0.05 versus control group.

Mentions: In the present study, pathological features of the lungs of rats with LPS-induced lung injury were observed following Ad-siTLR4 treatment. Lung histological examination showed that, in animals treated with saline, the lung was almost intact (Figure 5(a)). By contrast, in the Ad-EGFP group, LPS treatment significantly increased the exudation and thickened the alveolar walls accompanied by alveolus congestion, hemorrhage as well as massive neutrophil infiltration (Figure 5(b)). The pathological changes in the lungs were not obvious in the Ad-siTLR4 group (Figure 5(c)). The wet/dry ratio of the lungs was measured to represent the extent of lung edema. Results revealed LPS dramatically increased the wet/dry ratio, while Ad-siTLR4 treatment markedly reduced the lung edema when compared with the Ad-EGFP group (Figure 5(d)). Moreover, in the Ad-EGFP group, the content of total proteins was significantly increased as compared to the control group. However, treatment with Ad-siTLR4 markedly decreased the total protein content in the BALF, indicating intratracheal injection of Ad-siTLR4 can improve the LPS-induced microvascular leakage (Figure 5(e)). In order to examine the leukocyte infiltration in response to LPS, the lung MPO activity, an index of neutrophil sequestration, was determined. Findings displayed there was no significant difference in the lung MPO activity between the saline group and the Ad-siTLR4 group indicating no obvious neutrophil infiltration into both the alveolar space and the whole lung, while MPO activity in the Ad-EGFP group was significantly increased when compared with the former two groups (Figure 5(f)). These observations indicate that Ad-siTLR4 treatment can improve the lung injury in response to LPS through reducing microvascular damage, decreasing neutrophil influx, and improving lung histology.


Small interference RNA targeting TLR4 gene effectively attenuates pulmonary inflammation in a rat model.

Wu F, Liu Y, Lv X, Miao X, Sun Y, Yu W - J. Biomed. Biotechnol. (2012)

Effects of Ad-siTLR4 on LPS-induced ALI. Lungs were collected at 4 h after injection of normal saline or LPS for histological examination (a, b, c). Sections were stained with hematoxylin-eosin. Magnification: ×100 Wet/Dry ratio (d), total protein content in BALF (e) as well as MPO activity (f) were measured to determine the lung edema, microvascular permeability, and neutrophil sequestration, respectively. *P < 0.05 versus Ad-EGFP group, #P < 0.05 versus control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303866&req=5

fig5: Effects of Ad-siTLR4 on LPS-induced ALI. Lungs were collected at 4 h after injection of normal saline or LPS for histological examination (a, b, c). Sections were stained with hematoxylin-eosin. Magnification: ×100 Wet/Dry ratio (d), total protein content in BALF (e) as well as MPO activity (f) were measured to determine the lung edema, microvascular permeability, and neutrophil sequestration, respectively. *P < 0.05 versus Ad-EGFP group, #P < 0.05 versus control group.
Mentions: In the present study, pathological features of the lungs of rats with LPS-induced lung injury were observed following Ad-siTLR4 treatment. Lung histological examination showed that, in animals treated with saline, the lung was almost intact (Figure 5(a)). By contrast, in the Ad-EGFP group, LPS treatment significantly increased the exudation and thickened the alveolar walls accompanied by alveolus congestion, hemorrhage as well as massive neutrophil infiltration (Figure 5(b)). The pathological changes in the lungs were not obvious in the Ad-siTLR4 group (Figure 5(c)). The wet/dry ratio of the lungs was measured to represent the extent of lung edema. Results revealed LPS dramatically increased the wet/dry ratio, while Ad-siTLR4 treatment markedly reduced the lung edema when compared with the Ad-EGFP group (Figure 5(d)). Moreover, in the Ad-EGFP group, the content of total proteins was significantly increased as compared to the control group. However, treatment with Ad-siTLR4 markedly decreased the total protein content in the BALF, indicating intratracheal injection of Ad-siTLR4 can improve the LPS-induced microvascular leakage (Figure 5(e)). In order to examine the leukocyte infiltration in response to LPS, the lung MPO activity, an index of neutrophil sequestration, was determined. Findings displayed there was no significant difference in the lung MPO activity between the saline group and the Ad-siTLR4 group indicating no obvious neutrophil infiltration into both the alveolar space and the whole lung, while MPO activity in the Ad-EGFP group was significantly increased when compared with the former two groups (Figure 5(f)). These observations indicate that Ad-siTLR4 treatment can improve the lung injury in response to LPS through reducing microvascular damage, decreasing neutrophil influx, and improving lung histology.

Bottom Line: Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo.TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury.These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Eastern Hepatobiliary Hospital, Shanghai, China.

ABSTRACT

Objective: The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA) targeting Toll-like receptor 4 (TLR4) gene in ameliorating lipopolysaccharide- (LPS-) induced acute lung injury (ALI).

Methods: In vitro, alveolar macrophages (AMs) were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL) for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group), 300 μL of Ad-EGFP (Ad-EGFP group), or 300 μL of Ad-siTLR4 (Ad-siTLR4 group) and then were intravenously treated with LPS (50 mg/kg) to induce ALI.

Results: Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals.

Conclusion: TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Show MeSH
Related in: MedlinePlus