Vitro UPLC analysis and mass method identification, and in vivo or cellular immune anti-inflammatory function of Sanhuang Xiexin Decoction (SHXD)

Sanhuang Xiexin Decoction (SHXD), consisting of Coptis chinensis Franch., Scutellaria baicalensis Georgi and Rheum palmatum L., is traditionally used for relieving fever, purging fire for removing toxins, eliminating phlegm and haemostasis, eliminating the wetness-evil from the upper warmer, clearing away the heat-evil and expelling superficial evils. Each of the three herbs contained in SHXD has been indicated to have anti-inflammatory effects in vivo, but its effects on rat NK-cell phenotypes remain unexplored, and the comprehensive mechanism of this compound SHXD in curing the inflammation induced by lipopolysaccharides (LPS) remains to be revealed. The study aim was to assess the effect of SHXD on LPS-induced fever and inflammation in a rat model, reduce NLRP3 activation in NK cells expressing specific cell phenotype antibodies and determine the therapeutic value of this approach in vivo. SHXD extract was prepared and analysed by the developed ultra-performance liquid chromatography (UPLC) method for the simultaneous detection of 14 compounds. The main peaks were firstly identified on an Orbitrap via high resolution tandem mass spectrometry (MS). Then, the extract was used in the rat model of LPS-induced inflammation and fever for pharmacologically study the effects of drug treatment. Peripheral blood lymphocyte cells were isolated from the animals, including those subjected to the SHXD extract treatment, and the cell phenotype was determined prior to cell culture and after treating the cell cultures with the extract. The phenotypes of cells harvested using CD3, CD4, CD8a, CD81, CD161 and CD86 antibodies were used to verify the enhanced memory of the peripheral blood lymphocytes cells (PBMC) that were induced into nature killer (NK) cells. The SHXD extract was prepared, analysed and identified via quality control equipment and was observed to have pharmacological effects that reduced NLRP3 activation and fever in rats. The production of NK cells and peripheral blood lymphocytes was induced by the SHXD extract, which manifested as increased levels of CD4+, CD8a+, CD81+, CD161+ and CD86+ cells. The levels of CD3+ cells were significantly different between the model group and the drug-treated or control groups (p < 0.01) with dose independence, while the levels of CD4+ cells were not significantly different between the drug-treated and control groups, with a trend towards lower levels in the model group with dose independence. The levels of CD4+ cells was significantly different between the drug-treated group and the model groups with dose independence (p < 0.05). The levels of CD86+ cells were not significantly different between the drug-treated group and the model and control groups. The levels of CD8a + cells was significantly different between the model group and the drug and control groups (p < 0.05, dose 2.0 μg/ml), with higher levels in the drug-treated group. The levels of CD3+, CD4+, CD8a + cells in the drug treated group have dose dependence with SHXD. This experiment revealed that SHXD reduced NLRP3 activation in the blood of LPS-treated rats, which occurred through the activation of NK cells that expressed CD3, CD8a and CD161. SHXD may possess anti-inflammatory effect via activacting the one of major pharmacology effcet of NK cells that expressed CD3, CD8a and CD161 phenotypes expression. This result demonstrates that SHXD may possess ability to enhance the memory of peripheral blood lymphocytes and natural killer cells.