Ligustrazine protects against chronic hypertensive glaucoma in rats by inhibiting autophagy via the PI3K-Akt/mTOR pathway

Chemicals and reagents

Ligustrazine (TMP; sigma, 183,938-25G) was purchased from Sigma-Aldrich (St. Louis, MO). Ly294002 (MCE, #HY-10108, 2-morpholin-4-yl-8-phenylchromen-4-one) and rapamycin (MCE, #HY-10219) were obtained from MedChem Express (MCE, Monmouth Junction, NJ). All other reagents were obtained from commercial vendors and were of the highest purity available.

Animals

The experimental protocol was approved by the Animal Ethics Committee at the Affiliated Traditional Chinese Medicine Hospital of Guangzhou Medical University. All experimental procedures were performed in compliance with the National Institute of Health’s guide for the care and use of laboratory animals. Specific-pathogen-free adult Sprague-Dawley (SD) rats (weight: 200–300 g, both male and female) were purchased from Southern Medical University (Guangdong, China; Batch No. 44002100023747), and housed in an environmentally controlled animal breeding facility (temperature: 22 ± 2 °C, humidity: 60 ± 5%, 12h dark/light cycle). All rats were given water and food ad libitum.

Episcleral vein cauterization

The glaucoma model with high IOP was induced by EVC on the rats’ right eyes, as previously described [16]. In brief, rats were anesthetized with xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (10 mg/kg), and three drops of a topical anesthetic (alcaine) were instilled into the right eyes. A radial surgical incision was cut along the limbus. The fascia, muscles, and upper bulbar conjunctiva were separated to expose the underlying episcleral vein. A heated cautery pen was used to cauterize the three episcleral veins. The unoperated eye served as a control. After the operation, the conjunctival sac was flushed with normal saline and treated with antibiotic ointment.

Animal treatments

A total of 25 rats were randomly assigned to five groups (n = 5/per group). 1) Control group: The right eyes in the control group were sham-operated by similarly isolating the veins without cauterization. Once a day, an equal amount of normal saline was injected intraperitoneally from two days before the sham operation to one month after the sham operation. 2) Operated group: Once a day, an equal amount of normal saline was injected intraperitoneally from two days before EVC to one month after EVC. 3) TMP group: Once a day, TMP at a dose of 80 mg/kg was injected intraperitoneally from two days before EVC to one month after EVC. 4) TMP + Ly294002 group: Once a day, TMP at a dose of 80 mg/kg was injected intraperitoneally from two days before EVC to one month after EVC. Ly294002 at a dose of 10 mg/kg was injected intraperitoneally at the same time, from two days before EVC (once a day) to one month after EVC (twice a week). 5) TMP + rapamycin group: TMP at a dose of 80 mg/kg was injected intraperitoneally from two days before EVC to one month after EVC (once a day). Rapamycin at a dose of 5 mg/kg was injected intraperitoneally at the same time, from two days before EVC (once a day) to one month after EVC (once every other day). In previous studies, 80 mg/kg ligustrazine was used to improve atherosclerosis or suppress pulmonary artery smooth muscle cell proliferation in rats [17,18]. Thus, we used 80 mg/kg ligustrazine in this study. Additionally, the dosages of rapamycin and Ly294002 were also chosen in accordance with previous literature [19,20]. No animals were excluded during the study.

Measurement of intraocular pressure

The IOP of the experimental and control eyes was measured using a Tono-Pen AVIA Handheld Tonometer once every six days. The rats were anesthetized with xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (10 mg/kg), and three drops of alcaine were instilled into the experimental and control eyes, respectively. IOP was measured five times per eye at the same time by the same person. No animals were excluded from the study.

Tissue preparation

All rats were deeply anesthetized using a single intraperitoneal injection of xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (75 mg/kg), and euthanized one month after EVC. The eyes were enucleated carefully, leaving 2 mm of the optic nerve attached to the globe. After the eyes were washed with saline, the extra-orbital muscles and fat were removed. the resulting eyes were fixed in a freshly prepared fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for 2 h at room temperature. The cornea was punctured to expedite the penetration of fixative. Following additional 24 h fixation, the lens were removed, and the remaining eyes were dehydrated using graded ethanol (70% ethanol for 1 h, 80% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol twice for 1 h, 100% ethanol twice for 30 min) and embedded in paraffin at 60 ºC.

HE staining and TUNEL staining of retina sections

Thick sections (5 μm) of paraffin-embedded eyes were cut with a microtome, placed on glass slides. Conventional hematoxylin and eosin (H&E) staining was performed. Briefly, the obtained eye sections were deparaffinized with xylene and rehydrated using an ethanol gradient. Sections were stained in hematoxylin for 10 min, washed under running tap water for 10 min before 3 fast dips in 1% acid alcohol to enhance differentiation. Sections were re-washed under running tap water for 10 min, counterstained with eosin for 2 min prior to examination under light microscopy. Apoptotic cells were evaluated by using terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling (TUNEL)-staining according to the manufacturer's protocol. Cells with brown or dark-brown nuclei were identified as TUNEL-positive cells. Cells with blue nuclei were identified as TUNEL-negative cells. TUNEL-positive cells were quantified at least five x400 power fields of randomly selected tissue sections, and were expressed as the percentage of positive cells per 100 total cells.

Transmission electron microscopy

Retinal sections (100 μm) were cut with a vibratome, fixed with 4% glutaraldehyde in a 0.1 mmol/l cacodylate buffer (pH 7.4) for 1 h, and then with 1% osmium tetroxide in a 0.1 mmol/l cacodylate buffer for 2 h. Sections were rinsed with distilled water and treated with 1% aqueous uranyl acetate overnight. Next, they were dehydrated in an ascending series of ethanol to 100%, followed by dry acetone, and then embedded in Durcupan ACM. Ultrathin sections (0.1 μm) were cut and mounted on Formvar-coated slot grids and stained with 3% lead citrate. Sections were examined using a Zeiss transmission electron microscope (Zeiss Inc., Thornwood, NY).

Western blot

Retinas were homogenized in RIPA lysis buffer (Cwbio Bio Inc., Beijing, China) to extract the total protein according to the manufacturer’s protocol. Equally loaded proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with a solution of nonfat powdered milk for 1.5 h at room temperature and incubated overnight with the appropriate primary antibodies (p-Akt [Abcam, ab38449], AKT [Abcam, ab182729], p-PI3K [Abcam, ab127617], PI3K [Abcam, ab189403], p-mTOR [Abcam, ab131538], mTOR [Abcam, ab2732], LC3 [Abcam, ab48394]), followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. Blot imaging was conducted using ECL western blotting detection reagents. Bands of western blot were quantified using the open-source ImageJ software, version 1.53 (NIH, Bethesda, MD). Each bank intensity was normalized relative to its respective loading control bands. Values were expressed as the ratio of the average of the control group.

Immunohistochemistry

ATG5 protein expression was assessed with immunohistochemistry (IHC). The paraffin retinal sections were deparaffinized, rehydrated, and stained with ATG5 antibody (Abcam, ab108327). All sections were performed and stained simultaneously. Images of immunohistochemical sections were magnified to x40 and examined using the open-source ImageJ software version 1.53 (NIH, Bethesda, MD). Five randomly selected areas were captured for analysis. The pixel count for each area was used, and a score was assigned to each area based on a pixel-by-pixel analysis. The positive area ratio was scored as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (76%–100%) [21].

Statistical analysis

All data are presented as mean± SEM (n=5). The data were statistically analyzed using GraphPad Prism software, version 7.00. The differences between groups were analyzed using a one-way analysis of variance (ANOVA) followed by two-by-two comparisons using Tukey’s test. p<0.05 is considered significant.

Ligustrazine has no effect on intraocular pressure in chronic hypertensive glaucoma rats

As shown in Figure 1, the average IOP was 7.94 ± 1.39 mm Hg in rats before surgery. It should be noted that the IOP in this study is lower than the previously reported IOP of awake rats (ranging from 18 to 52 mm Hg) [22]. Although the exact causes for such a difference are unknown, the IOP values should be comparable because all the experiments were performed with the same instrument and under the same conditions. The IOP in the control rats with sham surgery did not show elevation throughout the study. In contrast, EVC gradually increased the IOP, which reached a plateau around six days after surgery. Consequently, the IOP of the operated rats was about 2.6 times higher than that of the control rats throughout the experiment. Nonetheless, the IOP in operated rats was not significantly altered by TMP, TMP + rapamycin, or TMP + Ly294002. These findings suggest that TMP has no effect on the IOP of EVC-induced glaucoma rats.

Ligustrazine rescues apoptotic retinal cells in chronic hypertensive glaucoma rats

Figure 2A shows representative histological appearance of retinal cross-sections observed by H&E staining. Control rats displayed a well-organized outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). RGCs in the control rats were arranged in a single layer with regular shapes. In contrast, EVC-operated rats showed loose retinal layers, and their RGCs were disorganized with irregular shapes.

Ligustrazine significantly rescued the retinal injury induced by EVC in operated rats, as evidenced by the organized cell layers. Although TMP + rapamycin and TMP + Ly294002 also attenuated retinal injury in operated rats, they were less effective compared to TMP alone.

Apoptosis is thought to play an important role in the degeneration of the retina. To further elucidate the effect of TMP, TUNEL staining was performed on retina sections, and the percentages of TUNEL-positive cells were quantified (Figure 2B,C). Compared to control rats, numerous TUNEL-positive cells were observed in the INL and GCL of operated rats. Ligustrazine significantly reduced TUNEL-positive cells in operated rats, and the retinal staining in ligustrazine rats was almost as light as that of control rats. Compared with operated rats treated with ligustrazine alone, more TUNEL-positive cells were observed in operated rats treated with TMP + rapamycin and TMP + Ly294002. Quantitative analysis of TUNEL confirmed that ligustrazine completely rescued apoptotic cells in operated rats, whereas rapamycin and Ly294002 significantly attenuated the effect of ligustrazine. Taken together, these findings suggest that the anti-apoptotic effect of ligustrazine on retina cells in EVC-induced glaucoma rats is suppressed by mTOR or PI3K inhibitors.

Ligustrazine inhibits autophagy in the retinal cells of chronic hypertensive glaucoma rats

Autophagy and apoptosis are known to be interconnected through multiple crosstalk mechanisms. To determine whether autophagic activity was altered by ligustrazine, we examined autophagosomes (APs) in retinal sections using transmission electron microscopy (EM). As shown in Figure 3A, double- and multiple-membrane APs were occasionally observed in the cytoplasma of RGCs and were scattered in the dendrites of RGCs in the IPL of control rats. The number of RGCs displaying APs was markedly increased in the operated rats. Notably, ligustrazine almost reduced the number of APs to the normal control level in the retinas of operated rats. Although TMP + rapamycin and TMP + Ly294002 also reduced the number of APs in the retinas of operated rats, their inhibitory effects on autophagy were much less potent than ligustrazine alone. These findings suggest that the inhibitory effect of ligustrazine on autophagy of retinal cells in EVC-induced glaucoma rats is partially reversed by mTOR and PI3K inhibitors.

Ligustrazine activates the PI3K-Akt/mTOR signaling pathway in retinal cells of chronic hypertensive glaucoma rats

To further evaluate the autophagic mechanisms, we evaluated the expression of several autophagy-related proteins in rat retinas. ATP5 is indispensable for the formation of the autophagic vesicle, and its dysfunction results in autophagy inhibition [23]. IHC staining showed that the ATP5 protein level was significantly increased in the retinas of operated rats compared to control rats (Figure 4A,B). Ligustrazine decreased the protein expression of ATP5 back to the control level in operated rats, but the effect was significantly attenuated by rapamycin and Ly294002. Western blot analysis revealed that operated rats had lower protein levels of p-mTOR, p-Akt and p-PI3K, but higher protein levels of LC3B-I and -II than the control rats did (Figure 4C,D). Such alterations of autophagy-related proteins in operated rats were almost completely reversed by ligustrazine. Notably, both rapamycin and Ly294002 suppressed the effect of ligustrazine on the protein expression of autophagy-related proteins in operated rats. Taken together, the anti-autophagic effect of ligustrazine on the retina in EVC-induced glaucoma rats is due to the activation of the PI3K-Akt/mTOR signaling pathway.