Intervention and mechanism of Xiaoyin Anshen Yin in treatment of psoriasis combined with sleep disorders

Abstract

Objective: To explore the therapeutic mechanisms of Xiaoyin Anshen Yin (, XYAS) in treating psoriasis associated with sleep focusing on melatonin and the regulation of the nuclear factor kappa-B (NF-κB) pathway.

Methods: Forty Sprague-Dawley rats were randomly divided into four groups, and administered distilled water, XYAS and its two different disassembly prescriptions by gavage respectively. Four types of drug-containing serums corresponding to the four groups were then prepared. Tumor necrosis factor (TNF)-α stimulated HaCaT was used to establish a psoriasis cell model, and the serums and the retinoid related orphan receptor alpha (RORα) inverse agonist were used respectively to intervene in the model. Enzyme-linked immunosorbent assay was used to detect the levels of interleukin (IL)-6 and melatonin in each group; flow cytometry was used to detect the levels of reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis; Western blot was used to evaluate the levels of superoxide dismutase 2 (SOD2), cytochrome-c (Cyt-c), inhibitor of kappa-B alpha (IκBα), p65 and phosphorylated p65.

Results: XYAS and its disassembly prescriptions inhibited the secretion of inflammatory factors such as IL-6, reduced the ROS content and Cyt-c expression, increased the mitochondrial membrane potential and SOD2 content, promoted the apoptosis in HaCaT cells and inhibited the activation of the NF-κB pathway. XYAS was also found increase the melatonin content. The above effects are beneficial in the treatment of psoriasis combined with sleep disorders. Meanwhile, XYAS no longer had a significant ameliorative effect after applying the RORα inverse agonist, suggesting that the therapeutic effect of XYAS is related to RORα.

Conclusions: The results of this study confirm that XYAS can be utilized for the treatment of psoriasis combined with sleep disorders via inhibiting the NF-κB pathway, anti-inflammatory, antioxidant and pro-apoptotic, which is in part related to the regulatory role of melatonin and its receptor RORα.

Keywords: NF-kappa B; melatonin; nuclear receptor subfamily 1, group F, member 1; psoriasis; sleep disorder.

Figure 1. Effect of drug-containing serum on oxidative stress indexes

A: cells of each group were stained with 2', 7'-dichlorodihydrofluorescein diacetate probe, the average fluorescence intensity was used to represent the level of intracellular ROS for statistical analysis. A1: flow cytometry analysis results; A2: relative levels of ROS. B: cells of each group were stained using JC-1 probe, Q2 is JC-1 multimer and its percentage, Q3 is JC-1 monomer and its percentage, and mitochondrial membrane potential was statistically analyzed by multimer/monomer to represent mitochondrial membrane potential. B1: NC; B2: Model; B3: XYAS; B4: QXAS; B5: LXJD; B6: mitochondrial membrane potential levels. C: The expression levels of SOD2 and Cyt-c in each group was detected via western blot method. C1: immunoblot bands for SOD2 and Cyt-c; C2: relative protein levels of SOD2; C3: relative protein levels of Cyt-c. NC: 10% NC serum; Model: 25 ng/mL TNF-α + 10% NC serum; XYAS: 25 ng/mL TNF-α + 10% XYAS serum; QXAS: 25 ng/mL TNF-α + 10% QXAS serum; LXJD: 25 ng/mL TNF-α + 10% LXJD serum. SOD2: superoxide dismutase 2; Cyt-c: Cytochrome-c; NC: Negative control; XYAS: Xiaoyin Anshen; QXAS: Qinxin Anshen; LXJD: Liangxue Jiedu; TNF-α: tumor necrosis factor‐α. Statistical analyses were measured using one-way analysis of variance for multiple comparisons. Data were presented as mean ± standard deviation (n ≥ 3). Compared with the Model group, ^aP < 0.001, ^cP < 0.01, ^dP < 0.05; compared with the NC group, ^bP < 0.001, ^eP < 0.05.

Figure 2. Effect of drug-containing serum on apoptosis

A: the cells of each group were stained using AnnexinV-FITC/PI double staining. Q1-Q4 represented cell debris and necrotic cells, late apoptotic cells, early apoptotic cells, non-apoptotic cells and the percentage of apoptotic cells respectively. A1: NC; A2: Model; A3: XYAS; A4: QXAS; A5: LXJD. B: The percentage of Q2 + Q3 (early apoptotic cells + late apoptotic cells) was used for statistical analysis. NC: 10% NC serum; Model: 25 ng/mL TNF-α + 10% NC serum; XYAS: 25 ng/mL TNF-α + 10% XYAS serum; QXAS: 25 ng/mL TNF-α + 10% QXAS serum; LXJD: 25 ng/mL TNF-α + 10% LXJD serum. NC: Negative control; XYAS: Xiaoyin Anshen; QXAS: Qinxin Anshen; LXJD: Liangxue Jiedu; TNF-α: tumor necrosis factor‐α; FITC: fluorescein isothiocyanate; PI: propidium iodide. Statistical analyses were measured using one-way analysis of variance for multiple comparisons. Data were presented as mean ± standard deviation (n = 3). Compared with the Model group, ^aP < 0.05, ^cP < 0.001; compared with the NC group, ^bP < 0.05, ^dP < 0.001, ^eP < 0.01.

Figure 3. Effect of drug-containing serum on the activation of NF-κB pathway

A: immunoblot bands for p-p65, p65 and IκBα; B: ratios of p-p65 to p65; C: relative protein levels of IκBα. NC: 10% NC serum; Model: 25 ng/mL TNF-α + 10% NC serum; XYAS: 25 ng/mL TNF-α + 10% XYAS serum; QXAS: 25 ng/mL TNF-α + 10% QXAS serum; LXJD: 25 ng/mL TNF-α + 10% LXJD serum. NF-κB: nuclear factor kappa-B; p-p65: phosphorylated p65; IκBα: inhibitor of kappa-B alpha; NC: Negative control; XYAS: Xiaoyin Anshen; QXAS: Qinxin Anshen; LXJD: Liangxue Jiedu; TNF-α: tumor necrosis factor‐α. Statistical analyses were measured using one-way analysis of variance for multiple comparisons. Data were presented as mean ± standard deviation (n ≥ 3). Compared with the Model group, ^aP < 0.01, ^cP < 0.001, ^eP < 0.05; compared with the NC group, ^bP < 0.01, ^dP < 0.05.

Figure 4. Effects of drug-containing serum on melatonin and RORα

A: The melatonin content in the drug-containing serum of each group tested by ELISA method; B: immunoblot bands for RORα; C: relative protein levels of RORα. NC: 10% NC serum; Model: 25 ng/mL TNF-α + 10% NC serum; XYAS: 25 ng/mL TNF-α + 10% XYAS serum; QXAS: 25 ng/mL TNF-α + 10% QXAS serum; LXJD: 25 ng/mL TNF-α + 10% LXJD serum. MLT: melatonin; RORα: retinoid related orphan receptor alpha; NC: Negative control; XYAS: Xiaoyin Anshen; QXAS: Qinxin Anshen; LXJD: Liangxue Jiedu; TNF-α: tumor necrosis factor‐α; ELISA: enzyme linked immunosorbent assay. Statistical analyses were measured using one-way analysis of variance for multiple comparisons. Data were presented as mean ± standard deviation (n ≥ 3). Compared with the NC serum, ^aP < 0.001; compared with the Model group, ^bP < 0.05; compared with the NC group, ^cP < 0.05.