The effect of modified Qiyuan paste on mice with low immunity and sleep deprivation by regulating GABA nerve and immune system

Abstract

Background: Low immunity and sleep disorders are prevalent suboptimal health conditions in contemporary populations, which render them susceptible to the infiltration of pathogenic factors. LJC, which has a long history in traditional Chinese medicine for nourishing the Yin and blood and calming the mind, is obtained by modifying Qiyuan paste. Dendrobium officinale Kimura et Migo has been shown to improve the immune function in sleep-deprived mice. In this study, based on the traditional Chinese medicine theory, LJC was prepared by adding D. officinale Kimura et Migo to Qiyuan paste decoction.

Methods: Indicators of Yin deficiency syndrome, such as back temperature and grip strength, were measured in each group of mice; furthermore, behavioral tests and pentobarbital sodium-induced sleep tests were performed. An automatic biochemical analyzer, enzyme-linked immunosorbent assay kit, and other methods were used to determine routine blood parameters, serum immunoglobulin (IgG, IgA, and IgM), cont (C3, C4), acid phosphatase (ACP) and lactate dehydrogenase (LDH) levels in the spleen, serum hemolysin, and delayed-type hypersensitivity (DTH) levels. In addition, serum levels of γ-aminobutyric acid (GABA) and glutamate (Glu) were detected using high-performance liquid chromatography (HPLC). Hematoxylin-eosin staining and Nissl staining were used to assess the histological alterations in the hypothalamus tissue. Western blot and immunohistochemistry were used to detect the expressions of the GABA pathway proteins GABRA1, GAD, GAT1, and GABAT1 and those of CD⁴⁺ and CD⁸⁺ proteins in the thymus and spleen tissues.

Results: The findings indicated that LJC prolonged the sleep duration, improved the pathological changes in the hippocampus, effectively upregulated the GABA content in the serum of mice, downregulated the Glu content and Glu/GABA ratio, enhanced the expressions of GABRA1, GAT1, and GAD, and decreased the expression of GABAT1 to assuage sleep disorders. Importantly, LJC alleviated the damage to the thymus and spleen tissues in the model mice and enhanced the activities of ACP and LDH in the spleen of the immunocompromised mice. Moreover, serum hemolysin levels and serum IgG, IgA, and IgM levels increased after LJC administration, which manifested as increased CD⁴⁺ content, decreased CD⁸⁺ content, and enhanced DTH response. In addition, LJC significantly increased the levels of complement C3 and C4, increased the number of white blood cells and lymphocytes, and decreased the percentage of neutrophils in the blood.

Conclusions: LJC can lead to improvements in immunocompromised mice models with insufficient sleep. The underlying mechanism may involve regulation of the GABA/Glu content and the expression levels of GABA metabolism pathway-related proteins in the brain of mice, enhancing their specific and nonspecific immune functions.

Keywords: GABA; Low immunity; Modified Qiyuan paste (LJC); Nervous immune system; Sleep deprivation.

Fig. 1

The HPLC profile of the active constituents in LJC. A HPLC plot of polysaccharide, 1: mannose, 2: rhamnose, 3: glucosamine, 4: glucose, 5: arabinose; B Non-polysaccharide HPLC plot, 1: chlorogenic acid, 2: vetsenin-2, 3: rutin, 4: naringin, 5: hesperidin, 6: naringenin

Fig. 2

Effects of LJC treatment on body temperature, grip strength, and salivary flow rate. A Thermal imaging; B Holding power; C Salivary flow speed. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with the control group; ^*P < 0.05 and ^**P < 0.01 compare with the model group

Fig. 3

Sedative and hypnotizing effects of LJC in model mice. A Sleep duration; B Total autonomous activity; C Autonomous activity ratio; D Horizontal scores; E Vertical scores; F Travel distance; G Enter open arm time; H Times of enterring the open arm; I Open field track diagram. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with the control group; ^*P < 0.05 and ^**P < 0.01 compare with the model group

Fig. 4

Effects of LJC on immune cells and immune molecules in the blood. A Serum hemolysin levels; B Toe thickness; C Peripheral white blood cell count; D The percentage of peripheral blood lymphocytes; E The percentage of neutrophils in peripheral blood; F Serum complement C3 level; G Serum complement C4 level; H Serum IgA level; I Serum IgG levels; (J) Serum IgM levels. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with the control group; ^*P < 0.05 and ^**P < 0.01 compare with the model group

Fig. 5

Histopathological evaluation of brain tissue, thymus and spleen. A H&E staining of hippocampus; B Nissant staining; C Thymus H&E staining (200 ×), R: thymus medulla, S: thymus cortex; D H&E staining of spleen (400 ×), m: white pulp area of spleen, n: red pulp area of spleen

Fig. 6

Quantitative histopathological evaluation of brain tissue, thymus and spleen. A H&E score of hippocampus; B Thymus index; C Spleen index; D Nissl bodies; E Spleen ACP viability; F Activity of LDH in spleen. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with the control group; ^*P < 0.05 and ^**P < 0.01 compare with the model group

Fig. 7

The effects of LJC on GABA and Glu contents. A HPLC diagram of Glu standard; B HPLC diagram of GABA standard; C HPLC representative map of serum samples; D Glu content; E GABA content; F Glu/GABA. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with the control group; ^*P < 0.05 and ^**P < 0.01 compare with the model group

Fig. 8

The effects of LJC on GABA pathway proteins in brain tissuecretion. A Immunohistochemical representation of GABA pathway proteins (400 ×); B Statistical diagram of GAD protein expression; C Statistical map of GABAT1 protein expression; D Statistical diagram of GAT1 protein expression; E Statistical diagram of GABRA1 protein expression; F Representative diagrams of GABA pathway protein expression. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with the control group; ^*P < 0.05 and ^**P < 0.01 compare with the model group

Fig. 9

The expression of CD⁴⁺ and CD⁸⁺ proteins. A Representative diagram of splenic CD⁴⁺ immunohistochemistry (400 ×); B thymic CD⁴⁺ immunohistochemical representative plot (400 ×); C CD⁸⁺ immunohistochemical representative diagram of spleen (400 ×); D thymic CD⁸⁺ immunohistochemical representation (40 ×); E Statistical graph of splenic CD⁴⁺ expression; F Statistical diagram of thymic CD⁴⁺ expression; G Statistical diagram of splenic CD⁸⁺ expression; H Statistical plot of thymic CD⁸⁺ expression. Data are expressed as the mean ± SD. ^#P < 0.05, ^##P < 0.01 compare with control group; ^*P < 0.05 and ^**P < 0.01 compare with model group

Fig. 10

Schematic representation of the mechanism underlying the ameliorative effects of LJC on immunocompromised and sleep-deprived mice through modulation of the GABAergic neuro-immune system