*4.10. Statistical Analysis*

Data are presented as the mean ± SD derived from at least three independent experiments. ANOVA analysis was used to examine the statistical significance of the differences between the groups, and the criterion for significance for all the experiments was *p* < 0.05.

#### **5. Conclusions**

Our study indicated that coelonin is one of the active components of *Bletilla striata*. Furthermore, we showed that using a PEX100 antibody microarray, a total of 32 different phosphorylation proteins were downregulated by coelonin pre-treatment on LPS-induced RAW264.7 cells. The maximum number of proteins belonged to the PI3K/AKT signal pathway, and three of them, PTEN, p65 and p27 Kip1 were confirmed by western blot, and more proteins and signaling pathways need to be verified. Western blot and confocal microscopy analysis revealed that coelonin inhibits the expression of IL-1β, IL-6 and TNF-α in a dose-dependent manner by eliminating lipopolysaccharide-induced NF-κB activity. However, besides inhibiting IκBα degradation, which pathways coelonin mainly inhibits the activation

of NF-κB still need to be further studied. While, we did confirm that coelonin inhibit LPS-induced p27 Kip1 degradation and block RAW264.7 cells in the G1 phase in a PTEN dependent manner (Figure 9). Overall, our results suggest that traditional Chinese medicine *Bletilla striata* has anti-inflammatory activity, and coelonin is one of the main active components. It may play a potential role in treating silicosis by inhibiting the proliferation of macrophages and the secretion of inflammatory factors. Additionally, PTEN may play an important role during this process, our following work will use modified RAW264.7 cells to address this issue.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/18/ 4422/s1.

**Author Contributions:** Conceptualization: G.Y.L., methodology: X.B.Y. and Z.S.D.; software: F.S.J. and H.Y.W.; formal analysis: M.Y.L. and B.D.; writing—original draft preparation: F.S.J. and M.Y.L.; writing—review and editing: F.S.J., M.Y.L. and C.C.Z.; supervision: G.Y.L. and X.B.Y.; funding acquisition: F.S.J., M.Y.L. and Z.S.D. All authors have read and approved the final version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (81603360, 81503329, 81673672) and the Natural Science Foundation of Zhejiang province (LY16H310005).

**Acknowledgments:** We thank the public platform of the Medical Research Center, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University for its instrumentation and equipment, and Jiangjiang Qin for his guidance in revising the manuscript.

**Conflicts of Interest:** The authors declare no conflicts of interest.
