Abstract

Objective: To investigate the effects of a Cistanche-containing formula (replacing the original blood-nourishing and kidney-tonifying herbs) on transforming growth factor-β2 (TGF-β2) expression, scleral remodeling, and water transport in form-deprivation myopia (FDM) rats by regulating the caveolin-1 (CAV1)/aquaporin 1 (AQP1) pathway.

Methods: Forty SPF-grade male SD rats were selected, among which 30 were used to establish the FDM model. After successful modeling, the rats were divided into the FDM group, pirenzepine (PIR) group, and Cistanche formula group, with 10 rats in each group. The remaining 10 non-modeled rats served as the normal group. The normal group and FDM group received no intervention. The PIR group was treated with PIR eye drops twice a day (morning and evening), while the Cistanche formula group was given intragastric administration of the Cistanche-containing formula. Refractive error and axial length of rats were detected using a retinoscope and diagnostic instrument. HE staining was used to observe the morphological changes of scleral tissue. Immunohistochemical staining was employed to detect the expression of TGF-β2 in scleral tissue. Western blot was used to determine the relative expression levels of AQP1 and CAV1 proteins in scleral tissue. RT-PCR was performed to measure the relative expression levels of glutamate-aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) mRNA in scleral tissue. Results: Compared with the normal group, the refractive error, TGF-β2 expression level, and relative expression levels of AQP1 and CAV1 proteins in the FDM group, Cistanche formula group, and PIR group were significantly decreased, while the axial length and relative expression levels of GLAST mRNA and GLT-1 mRNA were significantly increased (all P < 0.05). Compared with the FDM group, the refractive error, TGF-β2 expression level, and relative expression levels of AQP1 and CAV1 proteins in the Cistanche formula group and PIR group were significantly increased, while the axial length and relative expression levels of GLAST mRNA and GLT-1 mRNA were significantly decreased (all P < 0.05). Compared with the Cistanche formula group, the refractive error, TGF-β2 expression level, and relative expression levels of AQP1 and CAV1 proteins in the PIR group were significantly decreased, while the axial length and relative expression levels of GLAST mRNA and GLT-1 mRNA were significantly increased (all P < 0.05). HE staining results showed that there were no abnormal changes in the structure of each retinal layer in the normal group; the retina of the FDM group was thinned and severe vacuolar changes appeared in the ganglion cell layer; the retinal thickness of 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 in each layer were densely arranged, with the most significant performance in the Cistanche formula group. Conclusion: The Cistanche-containing formula has a certain positive effect on the treatment of FDM, and its mechanism may be related to regulating the expression of CAV1, AQP1, and TGF-β2, as well as balancing and improving scleral remodeling and water transport functions.

 

 

CISTANCHE HERB SUPPLEMENT FOR IMPROVING KIDNEY FUNCTION

 

 

Keywords

Form-deprivation myopia; Cistanche-containing formula; Transforming growth factor-β2; Aquaporin 1; Caveolin-1; Scleral remodeling; Water transport

 

In recent years, with the changes in people's lifestyles and the continuous development of electronic devices, the incidence of myopia has been on the rise. As a common disease, it has become a global health problem. Form-deprivation myopia (FDM) is a special type of myopia, which is usually caused by the deprivation or reduction of visual stimulation to the eyes. FDM not only interferes with the scleral remodeling process but also leads to abnormal changes in eyeball shape and disorders of water transport function, which may further aggravate scleral tissue damage and deformation. Aquaporin 1 (AQP1) is a widely existing protein whose main function is to promote the rapid transport of water molecules across cell membranes. In the eye, the distribution and function of AQP1 are related to the dynamic balance of intraocular fluid. Caveolin-1 (CAV1) is a protein involved in the formation of cell membrane microstructures and signal transduction. In the eye, CAV1 is mainly present in retinal pigment epithelial cells and photoreceptor cells, and is related to the transmission of visual signals and the stability of the intraocular environment. Transforming growth factor-β2 (TGF-β2) is a pleiotropic cytokine with multiple biological effects such as promoting cell proliferation, differentiation, and migration. In the intraocular environment, TGF-β2 plays an important role in the development and maintenance of the cornea and sclera. At present, the main treatment for FDM is drug therapy.

In traditional Chinese medicine (TCM), the formation of myopia is believed to be related to factors such as insufficient qi and blood, and kidney essence deficiency. The kidney is regarded as the "congenital foundation" and is closely related to the function of the eyes. TCM holds that "the kidney governs essence, and essence nourishes the eyes; the liver opens into the eyes, and sufficient liver blood ensures clear vision". Therefore, tonifying the kidney and nourishing blood has always been an important therapeutic principle for eye diseases caused by deficiency syndromes in TCM.

Cistanche, known as the "desert ginseng" and one of the "nine immortal medicines" in TCM, has a history of more than 2,000 years as a medicinal ingredient. Recorded as a top-grade nourishing medicinal material in ancient TCM classics, Cistanche is highly valued 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. The unique desert environment endows it with rich active ingredients, including echinacoside, verbascoside, and flavonoids, which have been scientifically proven to have strong antioxidant properties, reduce cell apoptosis, and promote cell proliferation.

Modern research has shown that Cistanche has significant kidney-nourishing effects. It can reduce kidney cell apoptosis, promote kidney cell proliferation, repair damaged kidney cells, and dilate renal blood vessels, thereby improving kidney function. In addition, Cistanche can enhance immunity by promoting lymphocyte proliferation, resist fatigue by increasing liver glycogen production, and exert anti-aging effects through scavenging free radicals. These biological activities are highly consistent with the TCM theory of "tonifying the kidney and nourishing essence", and also provide a scientific basis for its application in eye disease treatment. Based on the TCM therapeutic principle of tonifying the kidney and nourishing blood, and combining the modern pharmacological characteristics of Cistanche, this study replaced the original blood-nourishing and kidney-tonifying herbs with Cistanche to form a new Cistanche-containing formula, and explored its efficacy and mechanism in the treatment of FDM, aiming to provide a new idea for the development of natural herbal-based therapies for myopia.

1 Materials and Methods

1.1 Experimental Animals

Forty SPF-grade male SD rats, weighing 180-200 g and aged 6-8 weeks, were purchased from Beijing Keyu Animal Breeding Center [License No.: SCXK (Jing) 2022-0004] and routinely reared in the Animal Laboratory of the Eye Hospital of China Academy of Chinese Medical Sciences. This experiment complied with the relevant provisions of the "Regulations on the Administration of Experimental Animals" and was approved by the Medical Ethics Committee of the Eye Hospital of China Academy of Chinese Medical Sciences (Batch No.: YKEC-KT-2024-017-P001).

1.2 Main Reagents and Instruments

Cistanche tubulosa (purchased from Chengdu WeCistanche Bio-Tech. Co., Ltd., with active ingredients including echinacoside ≥30.0%, verbascoside ≥12.0%, and total phenethyl alcohol glycosides ≥75.0%) was used as the core medicinal material. Pirenzepine (PIR, Suzhou Hongsen Pharmaceutical Co., Ltd.); Compound Tropicamide Eye Drops (Hebei Jinyao Yongguang Pharmaceutical Co., Ltd., specification 5 mL); 4 g·L⁻¹ Oxybuprocaine Hydrochloride Eye Drops (Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd.); AQP1 antibody (Wuhan Fine Biotech Co., Ltd.); CAV1 antibody (Wuhan Botu Biotechnology Co., Ltd.); β-actin antibody (Shanghai Jingke Chemical Technology Co., Ltd.); Goat anti-rabbit IgG (Wenzhou Kemiao Biotechnology Co., Ltd.); HE Staining Kit (Shanghai Zeye Biotechnology Co., Ltd.).

1.3 Modeling Method and Grouping

Thirty rats were used to establish the FDM model. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg·kg⁻¹). After cleaning and disinfecting the right eye with povidone-iodine, an opaque eye mask was glued to the right eye. The eye mask was checked daily for detachment or light leakage, and re-glued if loose. The left eye was left untreated. After 4 weeks of monocular form deprivation, binocular retinoscopy and axial length measurement were performed on the rats. The success criteria for modeling: after cycloplegia, refractive error was measured with a retinoscope or automatic refractometer, and the deprived eye showed a significant myopic shift in refractive status [i.e., the difference in binocular refractive error ≥2 D was considered successful modeling, and the average myopic shift of the deprived eye compared with the control eye was about (-4.5 ± 1.2) D]. All 30 rats were successfully modeled and divided into the FDM group, PIR group, and Cistanche formula group, with 10 rats in each group. The remaining 10 non-modeled rats served as the normal group.

1.4 Intervention Methods

The normal group and FDM group received no intervention. The PIR group was given 5 drops of PIR eye drops in the right eye twice a day (morning and evening) with an interval of 2 minutes for 15 days. The Cistanche formula group was intervened with the Cistanche-containing formula. Cistanche tubulosa (50 g, the core ingredient) was decocted with water and concentrated to a medicinal solution with a concentration of 1000 g·L⁻¹. The rats were given intragastric administration at a dose of 12.70 g·kg⁻¹ once a day for 15 days. The dosage of Cistanche was determined based on its clinical application experience and modern pharmacological research, ensuring effective concentrations of active ingredients such as echinacoside and verbascoside.

1.5 Detection Methods

1.5.1 Evaluation of Refractive Error and Axial Length Changes

Morphological and refractive error detection: Twenty-four hours after the end of form deprivation, 3 rats were randomly selected from each group for detection. Before the experiment, rats were placed in a dark-adapted environment for 30 minutes, then anesthetized by isoflurane inhalation and fixed on a special animal bracket. A high-resolution OCT imaging system was used for panoramic scanning of the eyeball. During scanning, the probe was adjusted to be perpendicular to the cornea to obtain clear cross-sectional images of the retina and lens. Each eye was scanned 3 times consecutively, and the axial distance from the anterior corneal surface to the retinal pigment epithelium layer (i.e., axial length) was automatically measured by the system's built-in software to calculate the spherical equivalent refractive error. To ensure data accuracy, two professionally trained researchers independently analyzed the images in a blinded manner. If the difference between the two measurement results was greater than 0.50 D, the data was discarded and re-measured. Confirmatory detection of axial length: To verify the OCT measurement results, A-scan ultrasound diagnostic instrument was also used. First, oxybuprocaine hydrochloride eye drops were instilled into both eyes of the rats for topical anesthesia, and then the A-scan probe was gently placed in contact with the cornea. The instrument was adjusted to manual mode, and the accurate ultrasonic propagation speed was set according to the intraocular media: 1540 m·s⁻¹ for the anterior chamber and vitreous. During operation, each interface was located by manual marking, and each eye was measured 3 times repeatedly to take the average value. This experiment was performed in a blinded manner, and all operators received unified training to ensure consistent measurement techniques. The instrument was calibrated before measurement, and some samples were randomly selected for repeatability testing to evaluate the intraclass and interclass correlation coefficients (ICC). Strict standardized operating procedures were followed during the experiment to ensure the scientificity and reliability of the data.

1.5.2 HE Staining

After detecting refractive error and axial length, rats were sacrificed by excessive anesthesia. The eyelids and surrounding tissues of both eyes were disinfected, and the nasal and temporal limbus perpendicular to the midpoint of the cornea were marked. The conjunctiva was incised according to the marks, and the eyeball was enucleated. The limbus was incised to remove the anterior segment tissue and vitreous, and the scleral tissue was retained. The scleral tissue was fixed with 40 g·L⁻¹ paraformaldehyde for 24 hours, dehydrated with gradient ethanol, routinely embedded in paraffin, cleared with xylene, mounted with neutral balsam, sectioned at 5 μm, stained with HE, and observed for morphological changes of scleral tissue under an optical microscope.

1.5.3 Immunohistochemical Staining for TGF-β2 Expression in Scleral Tissue

Two rats were selected from each group. Scleral tissue sections were subjected to antigen retrieval and blocking, incubated with mouse anti-TGF-β2 (1:3000) for 24 hours, washed, incubated with secondary antibody (1:2000) for 1 hour, routinely developed and counterstained, mounted with neutral balsam, and 2-3 images were taken under an optical microscope to observe tissue changes. The gray value of the positive part was detected by an image analysis system.

1.5.4 Western Blot Detection of AQP1 and CAV1 Protein Expression Levels in Scleral Tissue

Two rats were selected from each group. Scleral tissue was excised, homogenized, and total protein was extracted by adding RIPA lysis buffer. Protein denaturation, quantification, electrophoresis, membrane transfer, and blocking were performed. Primary antibodies AQP1 (1:1000), CAV-1 (1:3000), and β-actin antibody (1:2000) were added and incubated overnight at 4 ℃. Secondary antibody (1:2000) was added and incubated at room temperature for 1 hour. ECL reagent was added, and the protein gray value was analyzed with Image Lab software. The ratio of the gray value of the target band to that of β-actin was its relative expression level.

1.5.5 RT-PCR Detection of GLAST and GLT-1 mRNA Expression in Scleral Tissue

The remaining scleral tissue of rats was taken, and total RNA was extracted according to the Trizol method. The total RNA was measured with a spectrophotometer, and cDNA was reverse-transcribed according to the kit instructions. RT-PCR reaction system was prepared with the following reaction conditions: 75 ℃ for 30 s, 85 ℃ for 60 s, 80 ℃ for annealing for 30 s, 65 ℃ for extension for 30 s, a total of 40 cycles. With β-actin as the internal reference, the relative expression levels of glutamate-aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) were calculated by the 2⁻ΔΔCt method. Primer sequences are shown in Table 1. One-way analysis of variance was used for comparison between groups, and LSD-t test was used for pairwise comparison between groups. The test level: α = 0.05.