2 Results

2.1 Comparison of Refractive Error and Axial Length Among Groups

Compared with the normal group, the refractive error of rats in the FDM group, Cistanche formula group, and PIR group was significantly decreased, while the axial length was significantly increased (all P < 0.05). Compared with the FDM group, the refractive error of rats in the Cistanche formula group and PIR group was significantly increased, and the axial length was significantly decreased (all P < 0.05). Compared with the Cistanche formula group, the refractive error of rats in the PIR group was decreased, and the axial length was increased (all P < 0.05) (Table 2).

Table 2. Comparison of Diopter and Axial Length Among Groups of Rats

Note: Compared with normal group, *P < 0.05; compared with FDM group, P < 0.05; compared with blood supplement group, aP < 0.05.

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2.2 HE Staining

In the normal group, the structure of each retinal layer was clear, the cells were arranged uniformly and densely, and there were no abnormal morphological changes. In the FDM group, the retina was thinned, the ganglion cells were arranged loosely and irregularly, and vacuolar changes appeared. The retinal thickness of rats in the Cistanche formula group and PIR group was increased, the cell arrangement was basically regular, the number of ganglion cells was increased, and the cells were densely arranged, with the most significant performance in the Cistanche formula group (Figure 1).

A: Normal group; B: FDM group; C: Blood-nourishing and kidney-tonifying formula group; D: PIR group. ① indicates the nerve fiber layer; ② indicates the inner plexiform layer; ③ indicates the choroid layer; ④ indicates the sclera layer.
Figure 1 HE staining of rat eyes in each group (&times;400)

2.3 Comparison of TGF-&beta;2 Expression Levels in Scleral Tissue Among Groups

The expression levels of TGF-&beta;2 in the scleral tissue of rats in the normal group, FDM group, Cistanche formula group, and PIR group were 133.59 &plusmn; 8.46, 96.28 &plusmn; 5.23, 124.56 &plusmn; 7.59, and 115.79 &plusmn; 6.42, respectively. Compared with the normal group, the expression levels of TGF-&beta;2 in the scleral tissue of rats in the FDM group, Cistanche formula group, and PIR group were significantly decreased (all P < 0.05). Compared with the FDM group, the expression levels of TGF-&beta;2 in the scleral tissue of rats in the Cistanche formula group and PIR group were significantly increased (all P < 0.05). Compared with the Cistanche formula group, the expression level of TGF-&beta;2 in the scleral tissue of rats in the PIR group was decreased (P < 0.05) (Figure 2).

A: Normal group; B: FDM group; C: Blood-nourishing and kidney-tonifying formula group; D: PIR group. ① indicates nerve fiber layer; ② indicates inner plexiform layer; ③ indicates choroid layer; ④ indicates sclera layer.
Figure 2 Immunohistochemical staining to detect the expression level of TGF-&beta;2 in the scleral tissue of rats in each group (&times;400)

2.4 Comparison of AQP1 and CAV1 Protein Expression Levels in Scleral Tissue Among Groups

Compared with the normal group, the expression levels of AQP1 and CAV1 proteins in the scleral tissue of rats in the FDM group, Cistanche formula group, and PIR group were significantly decreased (all P < 0.05). Compared with the FDM group, the expression levels of AQP1 and CAV1 proteins in the scleral tissue of rats in the Cistanche formula group and PIR group were significantly increased (all P < 0.05). Compared with the Cistanche formula group, the expression levels of AQP1 and CAV1 proteins in the scleral tissue of rats in the PIR group were significantly decreased (all P < 0.05) (Table 3, Figure 3).

Table 3. Comparison of AQP1 and CAV1 Protein Expression in Retinal Tissue

Note: Compared with normal group, *P < 0.05; compared with FDM group, P < 0.05; compared with blood supplement group, aP < 0.05.

A: Normal group; B: FDM group; C: Blood-nourishing and kidney-tonifying formula group; D: PIR group.
Figure 3. Western blot analysis of AQP1 and CAV1 protein expression in scleral tissue of rats in each group.

2.5 Comparison of GLAST and GLT-1 mRNA Expression Levels in Scleral Tissue Among Groups

Compared with the normal group, the relative expression levels of GLAST and GLT-1 mRNA in the scleral tissue of rats in the FDM group, Cistanche formula group, and PIR group were significantly increased (all P < 0.05). Compared with the FDM group, the relative expression levels of GLAST and GLT-1 mRNA in the scleral tissue of rats in the Cistanche formula group and PIR group were significantly decreased (all P < 0.05). Compared with the Cistanche formula group, the relative expression levels of GLAST and GLT-1 mRNA in the scleral tissue of rats in the PIR group were significantly increased (all P < 0.05) (Table 4).

Table 4. Relative mRNA Expression of GLAST and GLT-1 in Retinal Tissue

Note: Compared with normal group, *P < 0.05; compared with FDM group, P < 0.05; compared with blood supplement group, aP < 0.05.

3 Discussion

FDM seriously affects patients' visual function and quality of life. In traditional Chinese medicine (TCM) theory, the occurrence of myopia is closely related to liver and kidney yin deficiency. Insufficient liver blood leads to malnutrition of the eyes, and deficiency of kidney essence results in inadequate marrow supply to the brain, ultimately leading to decreased vision [5]. Based on the TCM theory that "the liver opens into the eyes, and the kidney stores essence to produce marrow", the method of tonifying the kidney and nourishing blood has provided an important idea for the treatment of myopia.

More Details About Cistanche For Improving Kidney Function

Cistanche, known as "desert ginseng" and one of the "nine immortal medicines" in TCM, has a history of more than 2,000 years of medicinal use. It is recorded as a top-grade nourishing medicinal material in ancient TCM classics and is highly respected for its effects of tonifying the kidney, replenishing essence, nourishing blood, and moistening dryness. Unlike many other TCM herbs, Cistanche grows exclusively in deserts, mainly parasitic on the roots of Tamarix ramosissima in regions such as Xinjiang, Inner Mongolia, and Gansu in China. This unique growth environment endows it with rich active ingredients, including echinacoside, verbascoside, and flavonoids. Modern scientific research has confirmed that these active ingredients have strong antioxidant properties, can reduce cell apoptosis, promote cell proliferation, repair damaged cells, and dilate blood vessels-biological activities that are highly consistent with the TCM effect of "tonifying the kidney and nourishing essence" [16]. In addition, Cistanche has been proven to enhance immunity by promoting lymphocyte proliferation, resist fatigue by increasing liver glycogen production, and exert anti-aging effects by scavenging free radicals, making it a promising natural ingredient for the development of health products and pharmaceuticals [16].

Refractive error is an indicator of the eye's focusing ability. In FDM, the continuous adjustment of the lens focal length and changes in eyeball shape to see objects clearly can lead to axial length growth, which further exacerbates myopia and forms a vicious cycle [6-7]. The Cistanche-containing formula, with Cistanche tubulosa as the core ingredient, inherits the TCM therapeutic principle of tonifying the kidney and nourishing blood. Cistanche's active ingredients, such as echinacoside and verbascoside, can improve ocular microcirculation, enhance nutrient supply to the retina, and relieve ciliary muscle tension, thereby reducing visual fatigue and regulating refractive error [16]. The results of this study show that the Cistanche-containing formula can significantly regulate axial length, which may be related to its ability to improve periorbital blood circulation, reduce intraocular pressure, and promote the repair and regeneration of retinal cells and ciliary muscles. Moreover, Cistanche's effect of promoting cell proliferation and repairing damaged cells may help regulate scleral collagen fiber remodeling, thereby inhibiting abnormal eyeball expansion [16].

The pathogenesis of FDM involves the interaction of various growth factors and cytokines. As a key factor, TGF-&beta;2 activates downstream signaling pathways by binding to cell surface receptors, participating in the regulation of corneal and scleral cell proliferation, promoting axial length extension, and thus exacerbating myopia progression [11]. The Cistanche-containing formula exerts its therapeutic effect by regulating the expression level of TGF-&beta;2 in FDM. Its mechanism may include inhibiting the abnormal activation of the TGF-&beta;2/Smad signaling pathway and reducing the expression of extracellular matrix remodeling-related proteins, thereby delaying scleral thinning and axial length extension. This effect is likely related to the antioxidant and anti-fibrotic active ingredients in Cistanche, such as flavonoids and echinacoside [16]. Previous TCM studies have shown that tonifying the kidney and nourishing blood can improve the state of blood deficiency and kidney deficiency, and the mutual promotion of sufficient blood and a healthy kidney helps maintain normal physiological functions of the human body [12]. In addition, the Cistanche-containing formula also has the effects of improving visual function, regulating immune function, and delaying axial length extension, which together provide a guarantee for the treatment of FDM.

AQP1 is an aquaporin expressed in the eye and is involved in intraocular fluid metabolism and transport. In FDM, abnormal expression of AQP1 may cause disorders of intraocular fluid metabolism and affect eyeball shape. By regulating the hydration state and osmotic pressure of the extracellular matrix, AQP1 can indirectly activate TGF-&beta;2 signaling, thereby regulating the MMPs/TIMPs system and participating in scleral collagen degradation and scleral thinning. In addition, as a protein involved in intracellular signaling and vesicular transport, abnormal expression of CAV1 may be related to retinal cell damage and dysfunction in FDM [13]. The results of this study show that the Cistanche-containing formula can regulate the expression levels of AQP1 and CAV1 in the eyes of FDM rats. Specifically, the formula can up-regulate the expression of AQP1, promote the balance of intraocular fluid metabolism, and improve the hydration state of the extracellular matrix; at the same time, it regulates the expression of CAV1, improves intracellular signal transduction and vesicular transport, and reduces retinal cell dysfunction. The synergistic effect of AQP1 and CAV1 can inhibit the abnormal activation of the TGF-&beta;2 signaling pathway, thereby delaying scleral extracellular matrix remodeling and axial length extension. Previous clinical studies have also shown that myopia patients treated with kidney-tonifying and blood-nourishing formulas have improved vision and fundus conditions, which is consistent with the results of this study [14].

Abnormal water transport is one of the important pathological mechanisms of FDM, which can change the water content of scleral tissue and exacerbate scleral deformation [15]. The active ingredients in Cistanche, such as echinacoside and verbascoside, have strong biological activities. They can improve ocular blood circulation, promote normal metabolism and functional recovery of the sclera [16]. The results of this study show that after intervention with the Cistanche-containing formula, the expression levels of GLAST and GLT-1 in scleral tissue are significantly reduced. The possible reasons are as follows: first, the ingredients in Cistanche improve the oxygen and nutrient supply of the ocular microenvironment by improving ocular blood circulation, thereby providing a good environment for the normal work of GLAST and GLT-1. Second, the Cistanche-containing formula may directly act on GLAST and GLT-1, inhibit the release of abnormal neurotransmitters by regulating their activity, and promote the reuse of normal neurotransmitters. In the process, it may also reduce ocular inflammatory reactions by regulating ocular immune function, thereby protecting the structural integrity and functional stability of transporters.

4 Conclusion

The Cistanche-containing formula can effectively improve the refractive status of FDM rats and inhibit excessive axial length growth. Its mechanism of action may be multi-targeted and multi-pathway: on the one hand, by tonifying the kidney and nourishing blood (in line with TCM theory), it improves ocular microcirculation and nutrient supply, and relieves ciliary muscle tension; on the other hand, by regulating the expression of AQP1 and CAV1, it restores the balance of intraocular fluid metabolism and retinal function, inhibits the abnormal activation of the TGF-&beta;2/Smad signaling pathway, and reduces extracellular matrix degradation, thereby delaying scleral thinning. In addition, the formula may also maintain the homeostasis of retinal neurotransmitters by regulating the function of glutamate transporters such as GLAST and GLT-1, indicating its potential to intervene in FDM at multiple levels from molecules to the whole body.

For researchers and developers engaged in herbal product development, the results of this study highlight the potential of Cistanche as a natural ingredient for myopia intervention. Cistanche's clear active ingredients (echinacoside, verbascoside, etc.), definite pharmacological effects (promoting cell proliferation, antioxidant, regulating signal pathways), and long history of safe use in TCM make it an ideal candidate for the development of functional foods, health supplements, or even pharmaceutical preparations for myopia prevention and treatment. Moreover, Cistanche tubulosa, with the highest content of active ingredients among Cistanche species, has been industrially produced by enterprises such as Chengdu WeCistanche Bio-Tech. Co., Ltd., which provides stable raw material supply and quality control support for product development [16].

However, the therapeutic effect of the Cistanche-containing formula is affected by various factors, such as individual differences and medication duration. This study also has certain limitations, and the specific mechanisms of the formula's regulation need to be further verified. Future research can focus on optimizing the dosage form and dosage of Cistanche, exploring its combined use with other natural ingredients, and conducting clinical trials to evaluate its safety and efficacy in humans, so as to promote the transformation and application of Cistanche-based products in the field of myopia prevention and treatment.

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