|Year : 2022 | Volume
| Issue : 2 | Page : 241-246
Andrographolide protects retinal ganglion cells in rats with glaucoma by regulating the Bcl-2/Bax/caspase-3 signaling pathway
Jun Li, Jing Lu, Guang Chen, Dan Li
Department of Ophthalmology, Affiliated Hospital of Hebei University, Baoding, China
|Date of Submission||12-Dec-2020|
|Date of Acceptance||06-Apr-2021|
|Date of Web Publication||01-Mar-2022|
Affiliated Hospital of Hebei University, Baoding
Source of Support: None, Conflict of Interest: None
Objective: The aim is to investigate the protective effect of andrographolide (AP) on retinal ganglion cells (RGC) in rats with glaucoma and its mechanism. Methods: ninety-six adult male Wistar rats were randomly divided into normal control (NC) group, chronic ocular hypertension (CHOT) group, vehicle group, and AP group. The density of RGCs and the number of apoptotic cells in retinal slices were evaluated, and the function of RGC was evaluated by photopic negative wave response (PhNR). The expression of Bcl-2, Bax, and caspase-3 in the retina was detected. Results: (1) The density of RGC in the CHOT and vehicle groups were significantly lower than that in the NC and AP (P < 0.01). After AP intervention, the RGC density of the rats significantly increased (P < 0.01). The ganglion cell layer (GCL) of the CHOT and vehicle groups was obviously thinner, and the thickness of the GCL was partially restored in the AP group. (2) A large number of terminal deoxytransferase mediated dUTP nick end labeling (TUNEL) positive cells were found in the GCL of the CHOT and vehicle groups, but only a few TUNEL positive cells were found in the AP group. The percentage of TUNEL positive cells was 8.80 ± 4.97%, 37.00 ± 5.27%, 46.16 ± 6.50%, and 22.29 ± 3.52% for groups NC, CHOT, vehicle, and AP, respectively. (3) At 2 weeks, the amplitude of PhNR in the CHOT and vehicle groups decreased by 63.22 ± 13.89% and 57.88 ± 6.95%, respectively. The amplitude of PhNR in the AP group was only 22.56 ± 6.44% lower than that in the NC group. The AP intervention significantly reduced the decrease of the PhNR amplitude in CHOT eyes. (4) Compared with the NC group, the expression of anti-apoptotic protein Bcl-2 was decreased in the CHOT and vehicle groups, and the expression of the pro-apoptotic protein Bax and caspase-3 significantly increased in the CHOT and vehicle groups, which could be reversed by AP treatment. The trend of the reverse transcription-polymerase chain reaction was consistent with that of the western blot analysis. Conclusion: The protective effect of caspase-3/Bcl-2 may be achieved through the regulation of the Caspase-3/Bcl-2 pathway in the rat retina.
Keywords: Andrographolide, Bax, Bcl-2, caspase-3, glaucoma
|How to cite this article:|
Li J, Lu J, Chen G, Li D. Andrographolide protects retinal ganglion cells in rats with glaucoma by regulating the Bcl-2/Bax/caspase-3 signaling pathway. World J Tradit Chin Med 2022;8:241-6
|How to cite this URL:|
Li J, Lu J, Chen G, Li D. Andrographolide protects retinal ganglion cells in rats with glaucoma by regulating the Bcl-2/Bax/caspase-3 signaling pathway. World J Tradit Chin Med [serial online] 2022 [cited 2022 Aug 8];8:241-6. Available from: https://www.wjtcm.net/text.asp?2022/8/2/241/338794
| Introduction|| |
Glaucoma is the main cause of irreversible visual impairment, which is characterized by the progressive loss of retinal ganglion cells (RGC) and their axons. It was estimated that the number of glaucoma patients in the world will increase to 76 million in 2020 and will further increase to 112 million in 2040. The main goal of glaucoma research and clinical treatment is to prevent progressive RGC degeneration and protect the existing visual function. A variety of complex molecular signals, including mitochondrial dysfunction, glutamate-induced excitotoxic injury, and oxidative stress, are involved in the death of RGC. Blocking the harmful factors involved in RGC progressive death is an important neuroprotective strategy for glaucoma management. Andrographolide (AP) is the whole herb or leaf of Andrographis paniculata, which has the functions of clearing heat and detoxification, anti-inflammation, detumescence, and pain relief. It has been reported that AP can protect colon epithelial cells from oxidase-induced apoptosis by reducing the production of reactive oxygen species (ROS) and maintaining mitochondrial membrane potential in vitro. It can also reduce intracellular free radical concentrations to prevent cell death induced by hydrogen peroxide, and rescues primary rat cortical cells from glutamate-induced toxicity. However, it is not clear whether AP has a neuroprotective effect on RGC injury in glaucoma. Therefore, this study explored the neuroprotective effect of AP on RGC cell apoptosis and function and further explored its mechanism.
| Methods|| |
Preparation of Andrographis paniculata water extract
Two hundred grams of the traditional Chinese medicine A. paniculata was weighed, washed, and soaked in distilled water for 12 h, and then decocted. Then, 2000 ml of distilled water was added for the first time, heated until boiling, and then reduced to a lower heat. The boiling state was maintained, and the decoction was conducted for 60 min. After filtration, the first filtrate was obtained. The second and third filtrates were obtained by adding 1600 ml water the second time and 800 ml water the third time. The filtrates were combined for three times and concentrated to 200 ml by heating and evaporation. By this time, the concentration of traditional Chinese medicine was 1 g/ml.
Experimental animals and intervention methods
Ninety-six adult male Wistar rats of SPF grade, with body weights of 200–250 g, were purchased from the experimental animal Business Department of Shanghai Institute of Family Planning Science with the production license No.: Scxk (Shanghai) 2018-0006. All animals had unrestricted access to tap water and pellet feed. The rats were kept in standard cages, and the light/dark cycle was 12 h: 12 h. The experimental glaucoma model was induced by anterior chamber injection of magnetic microspheres. The specific procedures were as follows: 4.5 um magnetic beads (4 × 108/ml) were resuspended into a 4 × 109 magnetic beads/ml magnetic bead suspension. After satisfactory anesthesia, the right eye was selected as the glaucoma model eye and the left eye as the self-control. After withdrawing the needle, the syringe was inserted into the anterior chamber again after the aqueous humor flowed out. Then, the syringe was inserted into the anterior chamber again, the magnetic beads were injected slowly, and then the needle was slowly withdrawn. Normal saline (5 UL) was injected into the contralateral eye, and ofloxacin eye ointment was applied after the operation. Tonolab tonometer was used to measure the intraocular pressure (IOP) of the anesthetized rats, and the mean value was measured five times. All measurements were taken between 9 am and 11 am and were conducted by the same operator. A 5 mm Hg higher IOP was used as the success criterion. Methods: 96 Wistar rats were randomly divided into four groups: (1) normal control group (NC group, n = 24), normal feeding without any treatment; (2) chronic ocular hypertension group (CHOT group, n = 24), magnetic microspheres were injected into the anterior chamber to induce ocular hypertension; (3) CHOT + excipient group (vehicle group, n = 24), the construction method of ocular hypertension model was the same as before. During the establishment of the ocular hypertension model, 2 μl PBS phosphate balanced solution (manufactured by Shanghai Yuanye Bio-Technology Co. Ltd) was injected into the corresponding rat vitreous, and then the injection was repeated every week. (4) CHOT + A. paniculata extract group (AP group, n = 24), the construction method of the high IOP model was the same as before. During the establishment of the high IOP model, 2 μl of the Andrographis extract was injected into the corresponding rat vitreous, and then the injection was repeated every week. All animal procedures were carried out in compliance with the guidelines for scientific animal procedures approved by the ethics committee of the Department of Medicine, Hebei University (No. WYZYY201905026).
Retrograde labeling and quantification of retinal ganglion cells
After anesthesia was satisfied, the rats were placed in the stereotactic apparatus. Then, 2 μl fibrinogen was injected into the bilateral superior colliculus and lateral geniculate body according to the stereotactic map of the rat brain. Fibrinogen was then absorbed by the RGC axons and transported retrogradely to the retinal cell bodies. One week after injection, the animals were killed, and the retina was dissected and flattened. The images were taken at 1.2–2.0 mm of the optic disc, which was divided into four quadrants: upper quadrant, lower quadrant, nasal quadrant, and temporal quadrant. Four nonoverlapping images were captured from the midline of each quadrant and the number of cells in each retina in 16 microscope fields was counted. The number of RGCs was counted by ImageJ software.
At room temperature, the eyeballs were removed and fixed with Davidson's fixative solution (37.5% ethanol, 9.3% paraformaldehyde, 12.5% acetic acid) for 24 h. After the lens was removed, the retina was embedded in paraffin and cut into 5 μm thick sections for h and e staining. The images were taken with a light microscope and measured at about 2–3 disc diameters from the optic nerve. According to the method of Bai et al. the number of neurons in the RGC layer (GCL) was counted at a distance of 1.0–1.5 mm from the optic disc.
Terminal deoxytransferase mediated dUTP nick end labeling detection
Frozen sections were fixed with 4% paraformaldehyde at room temperature for 20 min, and then 0.1% Triton X-100 was infiltrated on ice for 2 min. The samples were then incubated with the dUTP nick end labeling (TUNEL) reaction mixture in a humidifying chamber at 37°C for 60 min. After reverse staining with 4',6'-diamino-2-phenylindole (DAPI, 1:2000 dilution), the samples were observed under a confocal microscope with a magnification of ×400. The TUNEL and DAPI staining showed apoptotic cells, and the percentage of TUNEL positive cells was quantified by ImageJ software.
Measurement of the 1.5 photopic negative wave response
The photopic negative wave response (PhNR) was measured 2 weeks after the IOP induction to evaluate RGC function. The PhNR was recorded automatically by the Espion diagnostic system (Beijing henglu Science and trade Co. Ltd, origin: USA). After dark adaptation at night (12 h), the pupils were dilated with 0.5% norepinephrine and tropicamide, and the PhNR signals were recorded by placing two 3 mm platinum wire ring electrodes on the corneal surface. The subcutaneous needle electrode inserted into the bottom of the right leg was used as the grounding electrode, and another subcutaneous needle electrode placed on the nasal bone was used as the common reference. The retinal responses of both eyes were recorded within 30 min. The waveforms of PhNR were described by identifying the maximum peak and trough of the waveform and measuring the baseline trough and peak amplitude.
The tissue was frozen in radioimmunoprecipitation analysis (RIPA) lysate buffer. The lysed homogenate was centrifuged at 4°C and 13,000 g for 10 min. The retinal protein was extracted and detected by the BCA protein analysis kit (manufactured by Shanghai Yuanye Bio-Technology Co. Ltd). The protein samples were separated by 10% sodium dodecyl sulfate (SDS) gel electrophoresis, then transferred to the polyvinylidene fluoride (PDEF) membrane, sealed with 5% skim milk, and incubated with mouse anti-β-actin (1:1000 dilution), rabbit anti-Bcl2 (1:1000), rabbit anti-lysis cysteine aspartate proteinase-3 (1:1000 dilution), and rabbit anti-Bax (1:1000 dilution) overnight. On the 2nd day, the goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase was incubated with TBST solution three times. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference and the bands were detected by an enhanced chemiluminescence detection system.
Reverse transcription polymer polymerase chain reaction
The total RNA was extracted according to Trizol solution instructions (manufactured by Shanghai Yuanye Bio-Technology Co. Ltd), DNA contamination was removed by RNase, and the concentration and quantity of the total RNA were detected. The cell RNA was reverse transcribed into cDNA and then amplified into DNA. The operation was carried out in strict accordance with the instructions of the kit. The polymerase chain reaction (PCR) primers used are shown in [Table 1]. β-actin was used as an internal reference. The RT-PCR reaction conditions were as follows: predenaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 50°C for 30 s, 40 cycles, and extension at 70°C for 10 min.
Frozen 10 μm sections of the retina were fixed with 4% paraformaldehyde for 20 min, then incubated with 0.1% Triton X-100 and 3% bovine serum albumin for 40 min. The sections were incubated with rabbit anti-caspase-3 antibody (1:300 dilution) overnight and then incubated with the goat anti-rabbit IgG secondary antibody (1:500 dilution) coupled with Alexa fluor 488 for 1 h at room temperature. The sections were stained with Hoechst 33,258 (1:1000) and were observed and photographed using a confocal microscope.
All data were analyzed using SPSS 20.0 software (SPSS, Chicago, IL, USA). The count data were expressed as mean ± standard deviation. Univariate analysis of variance was used, and a Bonferroni multiple test was used for analysis among the different groups. A Kolmogorov–Smirnov test was used to compare the distribution of the amplitude and interval between events. Differences where P < 0.05 were considered statistically significant.
| Results|| |
Induction of experimental glaucoma
On the 3rd day after the injection of the magnetic beads, the IOP of each intervention group was significantly increased. The IOP of the NC group was 10.80 ± 1.47 mm Hg, while the IOP of the CHOT group, vehicle group, and AP group was 27.27 ± 5.86 mm Hg, 26.93 ± 7.44 mm Hg, and 26.86 ± 7.57 mm Hg respectively. At each time point during the experiment, the IOP of each intervention group was significantly higher than that of the NC group [Figure 1].
Andrographolide improves the survival rate of retinal ganglion cell in experimental glaucoma rats
In the AP group, FG-labeled RGC s showed bright and diffuse golden fluorescence, while in the CHOT group and vehicle group, the golden fluorescence intensity was significantly weaker [Figure 2]. In the 2nd and 4th weeks of the experiment, the RGC density of the four groups was significantly different (P < 0.01). After a pairwise comparison, the RGC density of the CHOT and vehicle groups was significantly lower than the NC and the AP (P < 0.01). After the AP intervention, the RGC density of the rats significantly increased (P < 0.01). This suggests that AP can increase the survival rate of RGC induced by ocular hypertension [Table 2].
In the NC group, the structure of the GCL was normal and the thickness was standard, containing a large number of cells; in the CHOT and vehicle groups, the GCL was obviously thinner and the number of cells was less; in the AP group, the thickness of the GCL was partially restored and the number of cells was relatively increased. At 2 weeks, the density of HE stained cells in the ganglion cells of the NC, CHOT, vehicle, and AP groups were 10.23 ± 2.54 cells/200 μm, 6.28 ± 2.54 cells/200 μm, 6.13 ± 2.00 cells/200 μm, and 8.92 ± 2.20 cells/200 μm, respectively, which was significantly different (P < 0.05). This suggests that AP can prevent harmful changes to the retinal thickness and increase the number of RGCs [Figure 3].
Protective effect of andrographolide on apoptosis of the retinal ganglion cells in chronic ocular hypertension rats
After 2 weeks of CHOT induction, the apoptosis of RGC was evaluated by TUNEL staining. The TUNEL staining of RGC cells in the NC group was basically negative; a large number of TUNEL positive cells were found in GCL of the CHOT and vehicle groups, while only a few TUNEL positive cells were found in the AP group. The percentages of TUNEL positive cells in the NC, CHOT, vehicle, and AP groups were 8.80 ± 4.97%, 37.00 ± 5.27%, 46.16 ± 6.50%, and 22.29 ± 3.52%, respectively, which were significantly different (P < 0.01). This suggests that the intravitreal injection of AP can significantly reduce RGC apoptosis induced by ocular hypertension [Figure 4].
|Figure 4: dUTP nick end labeling staining of retinal ganglion cells apoptosis in rats|
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Effects of andrographolide on retinal dysfunction in chronic ocular hypertension rats
Two weeks after the operation, the amplitude of PhNR in the CHOT and vehicle groups decreased by 63.22 ± 13.89% and 57.88 ± 6.95%, respectively. The amplitude of PhNR in the AP group was only 22.56 ± 6.44% lower than that in the NC group. The AP intervention significantly reduced the decrease of the PhNR amplitude in the CHOT eyes. This suggests that AP can reduce retinal dysfunction in experimental glaucoma rats [Figure 5].
|Figure 5: Andrographolide improvement of retinal dysfunction in chronic ocular hypertension rats|
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Detection of apoptosis-related proteins by western blot and reverse transcription polymer polymerase chain reaction
The western blot analysis showed that compared with the NC group, the expression of the anti-apoptotic protein BCL2 was decreased, and the expression of the pro-apoptotic protein Bax and caspase-3 was significantly increased in the CHOT and vehicle groups, which could be reversed by AP treatment. The RT-PCR showed that Bcl-2 mRNA expression in the CHOT and vehicle groups was significantly lower than in the NC group by 0.53 ± 0.2 and 0.49 ± 0.05 times, respectively. The expression of Bcl-2 mRNA was upregulated in the AP group, which was 1.05 ± 0.19 times higher than in the NC group. The Bax mRNA expression levels in the CHOT and vehicle groups were 1.23 ± 0.24 and 1.30 ± 0.33 times that of the NC group, respectively. The expression of Bax mRNA in the AP group was 0.77 ± 0.2 times higher than that in the NC group [Figure 6].
|Figure 6: Western blot and reverse transcription polymer polymerase chain reaction detection of apoptosis-related proteins|
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| Discussion|| |
Glaucoma is the most common cause of irreversible blindness, characterized by the progressive loss of RGC. At present, the main purpose of glaucoma treatment is to reduce IOP; however, in recent years, more studies have emphasized the role of neuroprotection. Some studies have found that α-lipoic acid treatment can prevent RGCs from dying in the retina of DBA/2J mice with glaucoma, while the application of another antioxidant, Tempol, can protect the RGCs by inhibiting neuritis. In the glaucoma rat model, Ginkgo biloba extract intervention can protect RGC function, while the lack of antioxidants can promote the loss of RGC. Treatment by reducing the oxidative stress of the RGCs may be a new strategy for treating glaucoma; however, there is no effective neuroprotective agent available for the treatment of glaucoma.
AP is the main effective component of the plant A. paniculata. It has the effect of dispelling heat, detoxification, anti-inflammatory action, and analgesic effects. It has a special curative effect on bacterial and viral upper respiratory tract infections and dysentery, and is known as a natural antibiotic drug. According to reports, AP also has antioxidant, anti-inflammatory, and anti-neuroexcitotoxicity properties, and can improve mitochondrial function. Therefore, we speculate that AP may have a certain protective effect against glaucoma. High IOP is the main risk factor of glaucoma. In this study, we established a glaucoma model by injecting magnetic microspheres into the anterior chamber of rats. We confirmed that AP has neuroprotective effects against experimentally induced glaucoma in rats, and can play a neuroprotective role by reducing the apoptosis of RGC cells and saving retinal dysfunction.
Retrograde labeling is a reliable method for the identification of RGC groups. The label FG or its analog hydroxystilbene mesylate are small molecules with similar fluorescence, which is the preferred tracer in related research. Consistent with previous studies, this study found that the number of RGCs decreased and the thickness of the GCL decreased in the CHOT group. After AP intervention, the number of surviving RGCs was increased and the normal thickness of the GCL was maintained. The recovery of the RGC function plays an important role in neuroprotection. RGC dysfunction occurs earlier and precedes the loss of optic nerve tissue and the decrease of RGC density. The TUNEL staining showed that AP can significantly reduce the apoptosis rate of rats with glaucoma, which shows the unique protective effect of AP. The PhNR originated from the inner layer of the retina. It refers to the negative wave that appears slowly after b wave when stimulated by bright light under the condition of light adaptation. It is widely used in glaucoma diagnosis and the evaluation of RGCs and their axon function. The PhNR amplitude is significantly correlated with the thickness of the ganglion cell complex in the central macula, which provides a direct and objective evaluation tool for RGC function changes. In this study, the PhNR amplitude in the CHOT and vehicle groups was significantly lower than that in the NC group, but AP treatment improved RGC dysfunction. This suggests that AP can not only reduce the apoptosis of RGC but also improve the dysfunction of RGC.
The genes Bax and Bcl-2 belong to Bcl-2 gene family. The Bcl-2 protein is an inhibitor of the apoptosis gene. The Bax protein not only antagonizes the inhibitory effect of Bcl-2 on apoptosis, but also promotes apoptosis. It plays a key role in apoptosis induced by mitochondrial stress. In this study, AP can upregulate the expression of the anti-apoptotic protein Bcl-2, downregulate the expression of the pro-apoptotic protein Bax and caspase-3, and effectively prevent the apoptosis of RGC. The mitochondrial pathway leading to caspase activation is the general pathway of the cell response to apoptosis. The activation and oligomerization of Bax lead to the formation of voltage-dependent anion channel pores or the increase of mitochondrial membrane permeability, thus initiating the release of cytochrome c and activating the downstream effector, cysteine protease. Progressive caspase-independent mitochondrial dysfunction can lead to cell death. AP can reverse the activation of caspase-3, downregulate Bcl-2, and upregulate Bax in the rat glaucoma model, suggesting that AP inhibits RGC apoptosis by regulating cell death related to the mitochondrial pathway.
| Conclusion|| |
This study found that AP has a neuroprotective effect on damaged RGCs in the glaucoma rat model, which may be achieved by regulating the activity of Bcl-2/Bax/caspase-3 pathway. However, the limitation of this study is that the mechanism by which AP protects RGCs was not fully elucidated. In some neurodegenerative diseases, AP can maintain the mitochondrial membrane in the mitochondria, inhibit the formation of voltage-dependent anion channel pores, reduce the production of intracellular ROS, and reduce the release of cytochrome c and apoptosis-inducing factor. However, the mechanism of AP on glaucoma needs further study.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 1], [Table 2]