Objective:

2

To investigate the effect of Buyang Huanwu Tang in resisting lipopolysaccharide (LPS)-induced macrophage activation and autophagy through phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein (PI3K/Akt/mTOR) signaling pathway.

Method:

2

The cell counting kit-8 (CCK-8) method was used to screen out the optimal LPS concentration for inducing the activity of RAW264.7 macrophages. RAW264.7 macrophages were treated separately with PI3K blocker 3-methyladenine(3-MA) (5 mmol·L

-1

)

Akt blocker MK2206 (5 μmol·L

-1

)

mTOR blocker Rapamycin (10 μmol·L

-1

)

Beclin1 blocker Spautin-1 (5 μmol·L

-1

)

different doses of Buyang Huanwu Tang serum (5%

10%

20%) and the optimum concentration of LPS for 24 h. The concentrations of inflammatory factors interleukin-1

β

(IL-1

β

)

interleukin-6 (IL-6) and tumor necrosis factor-

α

(TNF-

α

) in RAW264.7 macrophages were detected by enzyme-lined immunosorbent assay(ELISA). Western blot was used to detect the expression levels of phosphorylated PI3K

phosphorylated Akt

phosphorylated mTOR protein

microtubule light chain protein 3 (LC3)

ubiquitin-binding protein 1 (p62) and Beclin-1. The autophagy flow of RAW264.7 cells was detected by transfection with autophagy double-labeled adenovirus.

Result:

2

Results of CCK-8 showed the highest cell viability when 10 mg·L

-1

LPS was applied. The concentrations of IL-1

β

IL-6 and TNF-

α

in the model group were significantly higher than those in the blank group (

P

<

0.01). Compared with the model group

Rapamycin significantly increased IL-6 concentration (

P

<

0.05)

and other administration groups could decrease the levels of IL-1

β

IL-6 and TNF-

α

(

P

<

0.05

P

<

0.01). The expression levels of p-Akt

p-PI3K and p-mTOR in the model group were significantly lower (

P

<

0.05)

and LC3 and p62 protein expressions were significantly higher than those in the blank group (

P

<

0.01). Compared with the model group

Rapamycin significantly decreased the expression of p-Akt protein

with no impact on the expressions of p-mTOR. However

Buyang Huanwu Tang and other blockers significantly increased p-Akt

p-PI3K and p-mTOR (

P

<

0.05

P

<

0.01)

while decreased the protein expressions of LC3 and p62 (

P

<

0.05

P

<

0.01). There was no significant difference in the expression level of Beclin-1 protein in each group. Compared with the blank group

there were obvious autophagosome spots in the model group

and the autophagic flow was smooth. Compared with the model group

the formation of autophagic spots in 3-MA

Spautin-1 group and Buyang Huanwu Tang groups were significantly decreased or disappeared

and the size of autophagosome spots in MK2206 and Rapamycin groups was significantly reduced

but the autophagy activity was still strong.

Conclusion:

2

Buyang Huanwu Tang can resist LPS-induced macrophages activation and autophagy

inhibit macrophage inflammatory response

regulate PI3K/Akt/mTOR signaling pathway

and inhibit the excessive occurrence of autophagy.