3.1. Effective composition and drug stability detection of XQLD
XQLD is composed of Ma Huang (Ephedra), Bai Shao (Radix Paeoniae Alba), Xi Xin (Asarum), Gan Jiang (Rhizoma Zingiberis), Zhi Gan Cao (Radix Glycyrrhizae Preparata), Gui Zhi (Cassia Twig), Wu Wei Zi (Schisandra Chinensis) and Fa Ban Xia (Rhizoma Pinellinae Praeparata). After all drugs are combined, the stability of the active ingredients needs to be determined. To identify the stability of the effective ingredients contained in XQLD, we detected the decoction by LC–MS. The XQLD mass spectrometry analysis data of different batches in this study is displayed in Fig. 1 (A, B).
A total of 1516 compounds were detected from LC–MS. Among them, 38 alkaloids, 3 catechins, 77 terpenes, 182 flavonoids, 38 cinnamic acids, 8 chalketes, 29 phenylpropanoid compounds, 25 benzenes and its substituted derivatives were included. Correlation analysis of those components detected in five batches of XQLD showed that the correlation coefficients were greater than 0.965, indicating that different batches of XQLD components had high correlation (Fig. 1C). We also screened 29 bioactive components from the detected compounds (Table 1). Comparing the signal intensity of 29 components in different batches, we found that the content of effective bioactive components in different batches of XQLD was stable (Fig. 1D).
3.2. Safety evaluation of XQLD in treating AR mice
Xiaoqinglong Decoction is administered orally and metabolizes mainly through the liver and kidney. Therefore, the effect of XQLD on liver and kidney function is the main index of its safety. CRE, BUN, ALT and AST levels in blood were detected to evaluate the effects of all drugs (different doses of XQLD, loratadine and the HDAC inhibitor JNJ-26481585) on mouse liver and kidney functions. The results showed that there were no significant differences in the expression of CRE, BUN, ALT and AST between all drug groups and the blank control group (P > 0.05), indicating that XQLD, loratadine and HDAC inhibitor intervention did not obviously affect the liver and kidney function of mice (Fig. 2A-D).
HE sections for mice in each group also showed no obvious change in the structure of the liver and kidney. Liver slices of each group showed a clear lobular structure, orderly arrangement of cell cords, monolayer arrangement of hepatocytes in clusters or cords, and polygonal shape of hepatocytes (Fig. 2E). The structure of the nephron was clear and complete, the glomerular mesangial cells and matrix did not proliferate, and the basement membrane did not thicken (Fig. 2E).
3.3 XQLD and HDAC inhibitor alleviates allergic symptoms in AR mice
The symptoms of AR mice were observed and recorded within 10 minutes after the last nasal drip. Times of nasal sensitivity symptoms (sneezing and scratching nose) in each group were statistically analyzed to evaluate the alleviation of symptoms after drug intervention. We observed that nasal sensitivity symptoms of the AR group were obvious. After drug intervention, the symptoms of sneezing and scratching nose in the medication groups were significantly reduced (P < 0.05) compared with the AR group, indicating that XQLD, loratadine and HDAC inhibitor could alleviate the nasal sensitivity symptoms of AR mice (Fig. 3).
3.4 XQLD inhibits the expression of HDACs in the nasal mucosa of AR mice
Our results found that XQLD and HDAC inhibitors could both alleviate nasal allergy symptoms in mice. The connection between XQLD and HDAC intrigued us, so we decided to examine regulatory molecules downstream of XQLD and HDAC inhibitors. We detected the mRNA expression of HDAC1-11 (Fig. 4A-K) in nasal mucosa to observe the changes in HDACs after XQLD intervention. The results showed that the mRNA expression levels of HDAC1, HDAC3 and HDAC4 (Fig. 4A, C, D) in the AR group were significantly higher than those in the control group (P < 0.05). However, after intervention with XQLD and HDAC inhibitors, the expression levels of HDAC1, HDAC3 and HDAC4 decreased significantly (Fig. 4A, C, D). Then, we verified the protein expression of HDAC1, HDAC3 and HDAC4 in mouse nasal mucosa by WB, and the result was consistent with the mRNA results (Fig. 4L). These results suggest that HDAC1, HDAC3 and HDAC4 are directly regulated by XQLD. XQLD may inhibit the progression of AR inflammation by regulating the expression of HDAC1, HDAC3 and HDAC4.
3.5 XQLD and HDAC inhibitors reduce the mucosal immune response in AR mice
3.5.1 Effects of XQLD and HDAC inhibitors on the expression of OVAsIgE and Th1/Th2 cytokines in the serum of AR mice
To further confirm XQLD can inhibit AR inflammation by regulating expression of HDACs, we observed the changes of mucosal immune response after intervention with XQLD and HDAC inhibitors simultaneously in AR mice. Serum OVA sIgE was an important criterion for determining the effect of drug therapy in OVA sensitized mice. We first detected the serum OVAsIgE expression of each group by ELISA after drug intervention. Our results showed that the expression of Th1/Th2-related cytokines IL2, IgG, IL4, Il5 and IL13 was also detected. We found that the content of serum OVAsIgE in each treatment group (XQLD, loratadine and HDAC inhibitor group) significantly decreased compared with that in the AR group (P < 0.05). This result indicated that different doses of XQLD, loratadine and HDAC inhibitor could inhibit the expression of serum-specific OVAsIgE in AR mice (Fig. 5A). The levels of serum IL2 and IgG in the AR group were obviously lower than those in the blank control group (P < 0.05), while IL2 and IgG levels were significantly upregulated in the XQLD, loratadine and HDAC inhibitor groups compared with the AR group (Fig. 5B, C) (P < 0.05). The high levels of IL4, IL5 and IL13 in the AR groups were significantly inhibited after XQLD, loratadine and HDAC inhibitor intervention (P < 0.05) (Fig. 5D-F).
3.5.2. XQLD and HDAC inhibitors downregulate the expression of Th2 inflammatory factors and restore the epithelial integrity of nasal mucosa in AR mice.
AR is an allergic reaction dominated by type 2 inflammatory response. We confirmed that XQLD at different concentrations could inhibit the high levels of type 2 crucial inflammatory factors (IL4, IL5, IL13) in serum, but the effect on local nasal mucosa still remains unknown. We found that after interference with the XQLD and HDAC inhibitor groups, the expression levels of IL4, IL5 and IL13 in the nasal mucosa were obviously downregulated in mice (Fig. 6A-D). Pathological sections of nasal mucosa also confirmed that XQLD could restore the integrity of nasal mucosa in AR mice, which was also observed in the HDAC inhibitor group (Fig. 6E).
3.6. XQLD repairs nasal mucosal epithelial function in AR mice
XQLD has been demonstrated it directly regulate the expression of HDACs in our previous results. HDACs have also been reported to cause certain damage to the nasal epithelium [4]. We then explored the curable effects of XQLD and HDAC inhibitor on epithelial function. The results showed that the expression of Muc5ac and Muc5b (predominant gel-forming mucins of nasal mucosa) in AR mice was significantly higher than that in control mice. After XQLD and HDAC inhibitor intervention, Muc5ac and Muc5b expression decreased significantly (Fig. 7A, B). Moreover, the mRNA expression of ZO-1 and Claudin-1 (main tight junction markers) in the nasal mucosa of AR mice was significantly lower than that of the control group (P < 0.05) but significantly increased with XQLD and HDAC inhibitor intervention (Fig. 7C, D). Western blotting results also confirmed this conclusion (Fig. 7E).
IHC staining was performed on the nasal mucosa of mice in each group. The results showed that the expression of Muc5ac and Muc5b in the nasal mucosa of AR mice was higher than that of the control group, and the expression of Muc5ac and Muc5b in AR mice was inhibited after medication. The expression of Claudin-1 and ZO-1 in the nasal mucosa of mice in the AR group was lower than that in the control group, while the expression of Claudin-1 and ZO-1 was upregulated after treatment (Fig. 8, 9).