Objective

2

To observe the effects of Wendantang on the expression of inflammatory cytokines， autophagy markers， and key molecules of phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway in the adipocytes of the rat model of obesity （syndrome of phlegm-dampness） and to explore the material basis of inflammation in obesity （syndrome of phlegm-dampness） and the underlying mechanism of Wendantang intervention.

Method

2

A total of 126 SD rats were randomized into 2 groups： 16 rats in the blank group and 110 rats in the modeling group. The blank group was fed with a basic diet while the modeling group with a high-fat diet to establish the animal model of obesity （syndrome of phlegm-dampness） for 8 weeks. After successful modeling， 48 obese rats were selected according to their body mass and randomized into a model control group， an orlistat （ORLI， 32.40 mg·kg

-1

） group， a rapamycin （RAPA， 2 mg·kg

-1

） group， and low-， medium-， and high-dose （4.45， 8.90， 17.80 g·kg

-1

， respectively） Wendantang groups， with 8 rats in each group. In addition， 8 rats were randomly selected from the blank group to be set as the normal control group. The corresponding agents in each group were administrated by gavage and the model and control groups were administrated with equal amounts of distilled water once daily for 6 weeks. The body mass， Lee's index， body fat ratio， and obesity rate were measured or calculated. The expression of UNC51-like kinase-1 （ULK1）， Beclin1， human autophagy-related protein 5 （Atg5）， p62， and microtubule-associated protein 1 light chain 3 （LC3） Ⅰ/Ⅱ （markers of autophagy in adipocytes） was detected by the immunohistochemical two-step method. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the expression of tumor necrosis factor （TNF）-

α

， interleukin-6 （IL-6）， IL-1

β

， monocyte chemotactic protein-1 （MCP-1）， IL-4， IL-10， IL-13， and transforming growth factor （TGF）-

β

in adipocytes. Western blot was employed to measure the protein levels of classⅠ-PI3K， phosphatidylinositol triphosphate （PIP3）， Akt， mTORC1， ULK1， TSC1， and TSC2 in adipocytes.

Result

2

Compared with the blank group， the modeling group showed increased body mass and Lee's index （

P

<

0.01）， the obesity rate

>

20%， and phlegm-dampness syndrome manifestations such as physical obesity， decreased mobility， decreased appetite， lusterless and tight fur， loose stools， decreased responsiveness to the outside world， and decreased water intake. Compared with the normal control group， the model control group showed increased body mass， Lee's index， body fat ratio， adipocyte autophagy marker expression， pro- and anti-inflammatory cytokine levels （

P

<

0.05，

P

<

0.01）， down-regulated protein levels of classⅠ-PI3K， PIP3， Akt， mTORC1， TSC1， and TSC2 （

P

<

0.01）， and up-regulated protein level of ULK1 （

P

<

0.01）. The intervention groups showed lower body mass， body fat ratio， adipocyte autophagy marker protein expression， and protein levels of TNF-

α

， IL-6， IL-1

β

， MCP-1， IL-4， and IL-13 than the model control group （

P

<

0.05，

P

<

0.01）. Moreover， the RAPA and Wendantang （medium and high dose） groups showed lowered levels of IL-10 and TGF-

β

（

P

<

0.01）， and the ORLI group showed down-regulated expression of TGF-

β

（

P

<

0.01）. The expression of key molecules of the signaling pathway was up-regulated （

P

<

0.05，

P

<

0.01） while that of ULK1 was down-regulated （

P

<

0.01） in all the intervention groups. Compared with the RAPA group， the Wendantang groups showed up-regulated expression of all autophagy marker proteins in adipocytes （

P

<

0.01）. In addition， the low-dose Wendantang group showed elevated levels of inflammatory cytokines （except TNF-

α

） （

P

<

0.05，

P

<

0.01） and down-regulated expression of all key molecules of the signaling pathway （

P

<

0.05，

P

<

0.01）. The levels of inflammatory cytokines （except IL-16， MCP-1， and IL-10） were elevated in the medium-dose Wendantang group （

P

<

0.05，

P

<

0.01）. The expression of key molecules except PI3K of the signaling pathway was down-regulated in the medium- and high-dose Wendantang groups （

P

<

0.05，

P

<

0.01）. Compared with the ORLI group， low- and medium-dose Wendantang groups showed up-regulated expression of autophagy markers in adipocytes （

P

<

0.01）， and the low-dose group showed elevated levels of inflammatory cytokines （IL-6， IL-4， and TGF-

β

） （

P

<

0.01） and down-regulated expression of all key molecules of the signaling pathway （

P

<

0.01）. The medium-dose Wendantang group showed up-regulated expression of IL-4 （

P

<

0.01） and down-regulated expression of key molecules except PI3K of the signaling pathway （

P

<

0.05，

P

<

0.01）. The high-dose Wendantang group showed increased body mass， up-regulated expression levels of autophagy markers （ULK1， LC3 Ⅰ/Ⅱ） （

P

<

0.05，

P

<

0.01）， down-regulated expression of PIP3， mTORC1， and TSC1 （

P

<

0.05，

P

<

0.01）， and lowered levels of Beclin1， Atg5， TNF-

α

， and IL-13 （

P

<

0.05，

P

<

0.01）.

Conclusion

2

The inflammation in obesity （syndrome of phlegm-dampness） is closely associated with the PI3K/Akt/mTOR pathway-mediated adipocyte autophagy. Wendantang can treat the chronic inflammation in obese rats with the syndrome of phlegm-dampness by regulating this signaling pathway and thus improve adipocyte autophagy.