Study On Neuroendocrine-Immune Function Of Cistanche Deserticola And Its Rice Wine Steaming Products in Glucocorticoid-Induced Rat Model-Ⅱ

3.2.4. Immunohistochemistry

Neuronal apoptosis is controlled by many apoptotic and antiapoptotic genes, mainly the apoptosis-promoting gene Bax and the antiapoptotic genes of the Bcl-2 gene family. Bcl-2 is localized in reactive oxygen-producing organelles such as the mitochondria, endoplasmic reticulum, and nuclear membranes. Bcl-2 has little impact on oxygen free radicals but can prevent oxidative damage in cells. Bax transports from the cytoplasm to the mitochondrial membrane, changes the mitochondrial membrane structure, and promotes the release of cytochrome c and the induction of apoptosis.

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There was an expression in the adrenal cytoplasm of Bax and Bcl-2 protein. The staining showed all yellow particles. Compared to that of the BC group, the average optical density of the Bax protein in the MC group increased significantly (P < 0.01). Compared to that of the MC group, the average optical density in the CD-LD and WCD-LD groups decreased significantly (P < 0.01) (Figure 5(a)).

Figure 5(b) shows that compared to that of the BC group, the average optical density of Bcl-2 protein in the MC group decreased significantly (P < 0.05). Compared to that of the MC group, the average optical density in the WCD-HD and CD-HD groups increased significantly (P < 0.01), while those of the WCD-MD and CD-MD groups increased (P < 0.05).

The caspase-3 protein is localized mainly in the cytoplasm in its original form in normal tissues and has low expression. Positive signals are pale yellow or brownish yellow. Compared to that of the BC group, the expression of caspase-3 in the MC group increased significantly (P < 0.05). Compared to that of the MC group, the expression of caspase-3 in the WCD-HD and WCD-MD groups increased (P < 0.05) (Figure 5(c)).

In normal tissues, the expression of the Fas gene was reduced, and the positive signal for in situ hybridization was brownish yellow. As shown in Figure 5(d), compared to that of the BC group, the average optical density of Fas protein in the MC group increased (P < 0.05). Compared to that of the MC group, the average optical density in the WCD-HD and WCD-LD groups improved (P < 0.05) (Figure 5(d)).

The FasL protein is located mainly in the cell membrane or cytoplasm, and its positive signals are brown. Compared to that of the BC group, the average optical density of the FasL gene in the MC group increased significantly (P < 0.05). Compared to that of the MC group, the average optical density of the FasL gene in the WCD-HD, WCD-MD, CDHD, and CD-MD groups increased significantly (P < 0.01) (Figure 5(e)).

The results demonstrated that CD and WCD could induce apoptosis in inflammatory cells and reduce adrenal damage by promoting the expression of the FasL protein and binding to the Fas protein, activating caspase-3 protein and triggering a caspase cascade. The effect of the WCD-HD group was the best.

3.3. Determination Of Immune Function 

3.3.1. Immune Organ Index

Compared to those of the BC group, the thymus coefficient and the spleen index of the MC group decreased significantly (P < 0.01). Compared to the MC group, the WCD-MD group had the greatest difference (P < 0.05). The thymus coefficient was almost back to the normal level. For the spleen index, the WCDMD, WCD-LD, and CD-MD groups showed significant differences compared with that of the MC group (P < 0.01) (see Figure 6). Therefore, WCD and CD treatment could protect the immune organs, and the effects of WCD were better. 

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3.3.2. Detection Of T Lymphocyte Subsets In Peripheral Blood

The percentage of CD4+ cells in the upper left quadrant and CD8+ cells in the right lower quadrant in the BC and each drug group was higher than that in the MC group. Compared to that of the BC group, the ratio of CD4+ThCD8+ decreased significantly in the MC group (P < 0.01). Compared to that of the MC group, the ratio of CD4+ThCD8+ increased significantly in the PC group, the WCD group, and the CD-HD group (P < 0.01) (Figure 7).

3.3.3. Determination Of The Level Of IL-10, IL-6, IL-2, TNF-Α, And IFN-C In Serum

Figure 8 shows that compared to that of the BC group, the level of IL-6, TNF-α, and IFN-c increased significantly (P < 0.01). The level of IL-10 and IL-2 decreased in the MC group. Compared to that of the MC group, the level of IL-6, TNF-α, and IFN-c decreased and IL-10 and IL-2 increased in serum in each of the drug groups. The effect of WCD was better than the effect of CD, especially in the WCD-MD group, in which the effect was close to that of the PC group.

3.3.4. Results Of Lymus Tissue Microscope Observation Experiment

Figure 9 shows that in the BC group, a large number of lymphocytes were present. Lymphocytes and reticulocytes were loosely arranged in the center with scattered round or oval thymocytes. There was no necrosis, no inflammatory cell infiltration, and no abnormal changes.

The MC group showed a large amount of lymphoid necrosis tissue, with a disordered arrangement of lymphocytes and reticulocytes in the center. Round or oval thymocytes disappeared, massive cell degeneration or necrosis was evident, and there was inflammatory cell infiltration with abnormal pathological changes.

In the CD groups, the morphological characteristics of lymphocytes had returned to normal, and the arrangement of central lymphocytes and reticulocytes was almost normal. Round or oval thymocytes had recovered, but a small number of degenerative or necrotic lymphocytes and infiltrating inflammatory cells remained.

In the WCD groups and the PC group, the morphological characteristics of lymphocytes were better repaired. The lymphocytes and reticulocytes in the center were well aligned, the round or oval thymocytes were almost normal, but there remained a small number of infiltrating and degenerative lymphocytes.

3.4. CaM Protein Gene Expression

RQ value (CaM mRNA β-actin mRNA) was considered to examine the expression of CaM mRNA. The RQ of the BC group was 1. The expression of CaM mRNA in the hypothalamus in the MC group increased significantly (P < 0.01), while the value decreased in the WCD-HD, WCD-MD, WCDLD, and CD-HD groups (P < 0.05) (Figure 10). The expression of CaM mRNA in the hypophysis in the MC group was lower than that in the BC group (P < 0.01). The expression in the WCD-HD, WCD-MD, WCD-LD, CD-HD, and CD-LD groups increased (P < 0.05), and the effect in the WCD groups was better than the effect in the CD groups.

4. Discussion 

The Chinese Pharmacopoeia [2] records that CD and WCD can be used in the clinic as the main types of decocting pieces. After being steamed with rice wine, the antiaging and tonifying kidney-yang effects were enhanced. Through UPLC-QqQ-MS, we detected the quantitative changes in the chemical material base during the process of rice wine steaming. Iridoid glycosides were unstable and the content of these glycosides decreased, even becoming undetectable. The content of phenylethanol glycosides also changed, which could induce changes in the pharmacodynamics and the clinical effect.

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The classic kidney-yang deficiency model was replicated by subcutaneous injection of corticosterone [19]. Overdose corticosterone could inhibit the secret of CRH in the hypothalamus. Meanwhile, the secret of ACTH was also inhibited. The level of adrenal cortex hormone decreased, and thus, the function of the HPA axis was reduced. Hypofunction of the HPA axis is a common characteristic of kidney-yang deficiency [20]. Then the rats show "depletion" phenomena and the symptoms are in accord with those of kidney-yang deficiency in the clinic.

In the MC group, the level of CRH, ACTH, T, and CORT in rat serum decreased significantly (P < 0.01). After being treated by CD and WCD, the content of CRH, ACTH, T, CORT, and cortisol all increased to some degree, and the effect of WCD-MD was the best, almost equal to that in the PC group. These results indicated that the relative hormone balance in the HPA axis had been destroyed in the MC group, CD and WCD could improve the functional recovery of abnormal adrenal axis hormone levels, but the effect of WCD was better.

Modulating the proportion of Bcl-2 family members is one of the core mechanisms by which FasThFasL mediates the death receptor pathway, and the Bcl-2 family regulates the mitochondrial pathway [21]. Apoptosis-inhibiting factor Bcl-2 and apoptosis-inducing factors, such as Bax, all belong to the Bcl-2 family [22]. Cell apoptosis may be determined by the relative ratio of apoptosis-inhibiting factors and apoptosis-inducing factors [23]. Mitochondria have the function of central checkpoints for many forms of apoptosis [24]. The present study showed that CD-MD and WCDs could deduce the expression of Bax and increase Bcl-2 to improve the kidney-yang deficiency. Fas is the major death receptor that induces apoptosis by ligation with the Fas ligand (FasL) [25]. The expression of Fas and FasL in the MC group increased, while this expression increased significantly in the WCD-MD group (P < 0.01) compared with the MC group. Both FasThFasL and BaxThBcl-2 systems transduce apoptotic signals to the caspase family [26]. Caspase-3 is one of the pivotal proteinases that initiate cell apoptosis. The activation of caspase-3 indicates that apoptosis enters an irreversible stage [27]. The expression of caspase-3 in the MC group increased and increased significantly in the WCD-HD and WCD-MD groups (P < 0.05).

Deficiency of immune function is one of the important manifestations of kidney-yang deficiency [28]. In this study, we found that the index of the spleen and thymus of the WCD-MD group recovered significantly (P < 0.01, P < 0.05). The immune response of the immune system is regulated mainly by the Th subgroups [29]. IL-2 is the growth factor for T cells, secreted by T cells and macrophages, participates in inflammatory regulation, regulates immunity, and increases infection resistance [30]. The level of IL-2 in the MC group decreased (P < 0.01), and after treatment with CD and WCD, the level of IL-2 recovered. IL-6 and TNF-α are proinflammatory cytokines. Increased levels of IL-6 and TNF-α cytokines cause cytokine imbalances and exacerbate inflammation. The levels of IL-6 and TNF-α increased in the MC group, while after treatment, the levels of IL-6 and TNF-α decreased. T cells are divided into CD4 cells and CD8 cells, and the CD4+ThCD8+ ratio can be used as an important index of immune regulation [31]. CD4+ThCD8+ in the MC group decreased (P < 0.01), while in the WCD and CDHD groups increased (P < 0.01).

The HPA axis is the most important regulatory system because it regulates glucocorticoid secretion and the endocrine system to maintain the secretory function of the adrenal cortex [32]. Overexpression of CaM mRNA in hypothalamus tissue was related to deficiency of kidney yang. CD and WCD could block the calcium channels, inhibit CaM mRNA expression in the hypothalamus, and decrease calcium influx, thus decreasing Ca2+·CaM complex activity.

The formation of kidney-yang deficiency is a chronic disease development process. Kidney is no longer a purely anatomical concept, and it is a general term for neuroendocrine-immunity and genitourinary systems. Neuroendocrine-immune (NEI) network is an important integrated regulatory system [33]. CD and WCD can regulate the function of the HPAT axis and improve the recovery of kidney-yang deficiency. This result provides the experimental basis for the clinical prevention and treatment of kidney-yang deficiency.

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