A comparative proteomic study of secretomes in kaempferitrin-treated CTX TNA2 astrocytic cells

Kaempferitrin is extracted in significantly high quantities from the leaves of Cinnamomum osmophloeum (C.O) and Bauhinia forficata, and are used as an antidiabetic herbal remedy in China and Brazil. Commercial product using dry Cinnamomum osmophloeum leaves has been sold locally in Taiwan. Oral administration of kaempferitrin reduced blood sugar in diabetic rats.

Though previously demonstrated to activate the classical insulin signaling pathways, a mechanism for kaempferitrin is still not fully understood. Also, studies on kaempferitrin on immune related cells have been inconclusive, and people consuming extract containing kaempferitrin often happen to be at high risk of diabetes and neurodegenerative diseases. Therefore, for kaempferitrin to be used every day, a comprehensive study is needed.

Astrocytic cell line was used as a model to test the differentially regulated secretomes, to test kaempferitrin effect on CNS glia, on pro-inflammatory cytokines, and to test how different the mechanism of kaempferitrin is from that of insulin. CTX TNA2 astrocytic cells were differentially treated with and without 10 µM kaempferitrin for 24 h, and the conditioned medium was collected. For the proteomic study, protein in conditioned medium was trypsin digested, and resulting peptides in kaempferitrin/non-treated sample pair were differentially dimethyl labeled. The labeled peptides were further fractionated by StageTip-based strong-exchange method before LC-MS/MS analyses. Levels of interesting proteins were verified using Western or Eliza. C.O. leaf crude extract treated samples were included for a comparison of effects of purified kaempferitrin vs. kaempferitrin containing crude extract.

Data were obtained via ProteomeXchange with identifier PXD002814. It was found that no pro-inflammatory cytokines or inhibitory ECM were elevated upon treatment of kaempferitrin or a crude extract of C.O. leaves. This suggests that prolonged use of kaempferitrin containing herbs may not increase pro-inflammatory reaction. LDL-R trafficking between the cell membrane and the extracellular niche was regulated by kaempferitrin toward reduced secretion. Our proteomic study also demonstrated that molecules related to plasma membrane recycling were regulated by kaempferitrin. Our discoveries provide evidence that link kaempferitrin regulation for LDL-R and membrane recycling to the blood lipid regulation by the C.O. leaves extract. However, these proteins were differently regulated when cells were treated with crude extract. This demonstrates that the molecular interactions within crude extract of herbs are complex and may not act similar to the compound purified from the crude extract.