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. 2023 Oct 18;14(2):173–181. doi: 10.1016/j.jtcme.2023.10.002

Qingguang'an-induced autophagy in TFs inhibits scar formation: A follow-up in vivo mechanistic investigation☆

Rong Hu a,b,c, Xian-jing Wang a,b,c, Shu Chen a,b,c, Yun Huang a,b,c, Juan Yu a,b,c,∗
PMCID: PMC10927378  PMID: 38481544

Abstract

Purpose

To investigate the mechanism by which Qingguang'an inhibits scar formation in rabbits administered glaucoma filtering surgery (GFS).

Methods

Combined trabeculectomy was performed in 100 rabbits diagnosed with glaucoma, which were assigned to five groups, including the no surgery, surgery only, mitomycin C (MMC; positive control), Qingguang'an (experimental) and PBS (negative control) groups. The animals were followed up at postoperative days 1–28. Ultrastructure was observed under a transmission electron microscope (TEM). Real-Time Polymerase Chain Reaction (RT-PCR), Western blot, Hematoxylin and Eosin (H&E) staining, Masson's trichrome staining and Immuno-histochemistry (IHC) were performed to assess the harvested blocks.

Results

In the Qingguang'an group, intraocular pressure (IOP) on postoperative D28 was significantly lower than values in the no surgery, surgery only and PBS groups (P < 0.05). Its blebs kept better filtering function and less complications in follow-up, which be detected to have less fibroblasts and collagen deposition histologically. Compared with the PBS group, ATG5, Beclin1 and LC3-II mRNA levels were significantly increased while P62 was downregulated in the Qingguang'an group (P < 0.05). Correspondingly, ATG5 and Beclin1 protein amounts in the Qingguang'an group were increased while P62 was downregulated. The LC3-II/Ⅰ ratio tended to rise to the process of autophagy. Abundant autophagosomes were captured under TEM in this condition.

Conclusions

Qingguang'an granules can inhibit scar formation in rabbits after GFS and restrain IOP increase by inducing autophagy in TFs.

Keywords: Qingguang'an, Autophagy, Fibrosis, Scar formation, Glaucoma filtering surgery

Graphical abstract

Image 1

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1. Introduction

Glaucoma is a progessive eye disease can eventually lead to blindness for its irreversible visual impairment.1 According to the reports,2 disease burden of glaucoma in China has been multiply increasing in the nearest three decades, and it is predicted that the number of glaucoma patients worldwide will rise to 112 million in 2040.3 However, GFS (glaucoma filtering surgery), the most predominant intervention of glaucoma, has a high failure rate of more than 50 % due to its complications.4 The curative efficacy can be greatly reduced by postoperative fibroblast proliferation in the Tenon capsule and subsequent bleb scarring, which gives rise to high IOP (intraocular pressure) and narrow filtering channel.5,6 Numerous methods such as the use of antimetabolites, application of beta radiation, amniotic membrane implants and so on are conducted to inhibit bleb scarring, yet the overall effect is unsatisfactory.7, 8, 9 Indeed, in the clinical application, complications including bleb leaking, corneal perforation, hypotony keratopathy, endophthalmitis, and maculopathy happen frequently.10, 11, 12 Individuals suffering from glaucoma hope for new safe and effective options.

Qingguang'an granules is a Chinese patent medicine comprised of Sheng-di 10g (Rehmannia glutinosa (Gaertn.)), Huang-qi 30g (Astragali Radix; Astragalus membranaceus (Fisch.)), Bai-zhu 10g (Atractylodes macrocephala Koidz), Che-qian-zi 20g (Plantago asiatica L), Fu-ling 20g (Poria cocos (Schw.)), Chi-shao-yao 10g (Paeoniae Radix Rubra; Paeonia lactiflora Pall), Hong-hua 5g (Carthami Flos; Carthamus tinctorius L) and Di-long 10g (Lumbricus; Lumbricus terrestris) to boost Qi, nourish Yin, activate blood and dredge meridians.13 It was developed by the First Affiliated Hospital of Hunan University of Chinese Medicine, and has been widely used in clinical care for over 20 years. Long-term clinical observation showed it inhibited scarring with less complications than intraoperative application of MMC (mitomycin C), a common antimetabolite used in GFS,14 and researches supported it can obviously lower IOP, reduce sub-conjunctival fibroblast proliferation as well as protect RGCs (retinal ganglion cells).15, 16, 17 The effect and safety can be well demonstrated, but the specific pharmacological mechanism remains elusive. By constructing biological networks in 2020, it was found that Qingguang'an was associated with 20 targets, 50 active compounds and many mechanisms, including cell autophagy.13

Autophagy is an intracellular degradation process not only maintaining IOP homeostasis and preventing trabecular meshwork damage, but also protecting RGCs, and inhibiting scarring in blebs after GFS, which is closely related to the physiopathology of glaucoma.18 Many drugs have been proven to prevent fibrosis of filtering passage by inducing autophagy in recent years, such as metformin eye drops,19 rosiglitazone,20 and hydroxycamptothecin, a chinese herbal medicinal ingredient.21 Similarly, our previous in vitro study has made known that high-dose Qingguang'an containing serum had the strongest effect on inhibiting HTFs (Human Tenon Capsule fibroblasts) proliferation, simultaneously the autophagy positive cells and average fluorescence intensity in Qingguang'an group were significantly increased with time-dependent manner versus the control group, substantiating the link between Qingguang'an and autophagy at the molecular level.22,23

This is our second report examining the mechanism of Qingguang'an, addressing its anti-scarring effect in vivo and investigating whether these effects involve autophagy in TFs (Tenon Capsule fibroblasts). Based on previous experiments, we established animal models after GFS and conducted follow-up studies to further verify the hypothesis that Qingguang'an granules can reduce scar formation by autophagy induction of TFs. Qingguang'an is expected to be a prospective candidate for glaucoma treatment in the future.

2. Materials and methods

2.1. Animal models

To establish the chronic intraocular hypertension model,24 we administrated intravenous anesthesia with pentobarbital sodium (30 mg/kg) in healthy adult New Zealand rabbits (Provided by The Animal Experiment Center of Hunan University of Chinese Medicine; Male, 2 years old, weighing 2.5–3 kg), who were further injected 0.2 ml of 2 % methylcellulose into the anterior chamber of the right eye, once a week for four weeks. IOP was measured with a Tonopen (Medtronic, Minneapolis, MN, USA) every 4 days. Only operated rabbits with stable IOP in the range of 25–35 mmHg were further examined. Eventually, 100 rabbits with high IOP were included as animal models of scar formation after GFS. We performed trabeculectomy in the superior quadrant of eyes after injecting 2 % pentobarbital sodium (30 mg/kg) via the ear vein for general anesthesia. A rectangular scleral flap was made in the base of limbus corneae, with dimensions of 4 × 3 mm2 approximately. In 1.5-mm thickness of the scleral flap, we performed resection of tissues, including the internal sclera, the trabeculum and the peripheral iris, measuring about 2 × 2 mm2. The superficial scleral flap was sutured by 10-0 nylon sutures. All the operations were performed under a microscope by the investigator, who has rich experience in ophthalmic surgeries in animals. All animal studies (including euthanasia) were performed in compliance with Hunan University institutional animal care regulations and guidelines, and conducted according to the AAALAC and IACUC guidelines. All the rabbits were applied Tobradex eye ointment (Alcon, Fort Worth, TX, SA) after GFS and given Tobradex eye drops (Alcon) four times per day postoperatively for a week.

2.2. Groups and medicine

Divide the 100 rabbits (100 eyes) which stable IOP in the range of 25–35 mmHg into five groups by the random number table method, including the (1) no surgery (blank group with no treatment), (2) surgery only (combined trabeculectomy with no other treatment provided intraoperatively or postoperatively), (3) MMC (positive control; 0.4 mg/ml MMC cotton pad placed under the conjunctiva and scleral flap for approximately 3 min after scleral flap production in GFS, with the operation area flushed with 50 ml saline subsequently), (4) Qingguang'an (experimental group, with rabbits administered Qingguang'an granules (batch No. 940425) provided by the Department of Pharmacy, the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine (Changsha, China), by gavage since the second postoperative days, twice daily for 15 days) and PBS (phosphate buffer saline; negative control group, administered an equal amounts of PBS in the same way as the experimental group) groups. Each group included 20 rabbits. The operated eyes were followed up for 4 weeks after operation (D1 - D28). We evaluated the morphology and function of filtering blebs and monitored IOP at different times to observe bleb scarring progress. The ultrastructure of TFs was observed under a transmission electron microscope (TEM), with 4 in each group. Then, hematoxylin and eosin (H&E) staining, Masson's trichrome staining, immunohistochemistry (IHC), Real-Time Polymerase Chain Reaction (RT-PCR) and Western blot were performed on D28 postoperatively.

2.3. Histopathological observation

4 eyeballs per group were enucleated and paraffin-embedded for histopathological observation. H&E as well as Masson's trichrome staining were conducted to observe histological changes and the content of collagen fibers. In H&E staining, the tissue blocks were incubated in xylene (10 min, 2 times), followed by gradient ethanol hydration (100 %, 100 %, 95 %, 85 %, and 75 %), hematoxylin-eosin stain, gradient alcohol(95–100 %), xylene dehydration(10 min, 2 times) and then neutral gum sealing, while in Masson staining, the sections were dropped proper amount of nuclear dye solution after conventional dewaxing. Besides, IHC (immunohistochemistry) was performed to detect the degree of fibrosis by antigen-antibody reaction. In Masson's trichrome staining, muscle fibers were stained by red while collagen fibers are blue. In IHC, positive staining was brown. All images were acquired by ordinary computers with IPP (Image-pro-Plus).

2.4. Quantitative RT-PCR

6 tissue blocks per group containing the bleb, conjunctiva, Tenon capsule and sclera were separated out for RT-PCR. Total RNA was extracted from harvested cells with TRIzol (Invitrogen, California, USA) and subsequently reverse transcribed into cDNA. Quantitative RT-PCR was carried out with the obtained cDNA. The whole procedure was conducted with SYBR green expression master mix (Beijing Puboxin Biology, Beijing, China). All experiments were performed in triplicate. The ΔΔCt method was applied to calculate the relative differences between the control and treatment groups, therefore reflecting the target gene expression levels of the four treatment groups. The results were expressed as fold change. The primers were designed using Primer5 and synthesized by Sangon Biotech (Shanghai). The forward and reverse primers were as follows: GAPDH, Forward 5′-TGGAATCCACTGGCGTCTTCAC-3′ and Reverse 5′-AGGATGCGTTGCTGACAATCTTGA-3’; ATG5, Forward 5′-CAGATGACAAAGATGTGCTTC-3′ and Reverse 5′-CAAATAACTTACTCTTGGCAAT-3’; Beclin1, Forward 5′-TGCCCTATGGAAATCACTCGT-3′ and Reverse 5′-GTCTCGCCCTTTTCAACCTC-3’; LC3-II, Forward 5′-ACGTCCACCCAATCAGTCC-3′ and Reverse 5′-TTACTGGCGACTCAGAATGCAA-3’; P62, Forward 5′-CACAGCAAGCTCGCCTTCC-3′ and Reverse 5′-AAGTTCACGTTGGGATCCTCT-3’.

2.5. Western blot

The prepared samples containing the bleb, conjunctiva, Tenon capsule and sclera, 6 per group, were washed thrice with ice-cold PBS and then lysed with cold RIPA lysis buffer containing 1.74 mg/ml (10 mmol/L) PMSF (phenyl methyl sulfonyl fluoride). The blocks were repeatedly homogenized on ice for 10 min. After centrifugation for 15 min at 12,000 rpm, the supernatants were collected and transferred to 0.5-mL centrifuge tubes. Immunoreactive proteins were visualized by autoradiography after treatment with the super ECL plus kit (Thermo pierce, USA). Monoclonal antibodies to Beclin1, ATG5, LC3-II, P62 and β-actin were obtained commercially (Proteintech, USA; CST, USA). Anti-β-actin antibodies (Proteintech, USA) were used as a loading control.

2.6. Statistical analysis

All experiments were performed in triplicate. Data are mean ± SD. Statistical analysis was performed with SPSS 23.0 or GraphPad Prism 8. Differences were assessed by ANOVA and P < 0.05 was considered statistically significant. Futhermore, P < 0.01 and P < 0.001 were considered extremely significant statistical difference.

3. RESULTS

3.1. IOP changes

Pre-to post-operative IOP changes in the five groups were fully recorded and statistically analyzed. Results showed that, IOP in all rabbits was significantly increased (P < 0.05) after modeling and was stable in the range of 25–35 mmHg after D8 (Fig. 1). The modeling was successful.

Fig. 1.

Fig. 1

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IOP before and after modeling in each group (mean ± SD, n = 20)

Notes: The range framed by the yellow rectangular represents the target high IOP values; ∗∗∗P < 0.001 versus the IOP on D0 per group.

Before GFS, IOP in five groups was over 30 mmHg on average and showed no significant difference between each group (P > 0.05). Compared with the no surgery group, IOP in rabbits performed with trabeculectomy in the four treatment groups were overtly decreased on postoperative days (P < 0.05). Nevertheless, IOP values in the surgery only and PBS groups gradually increased with time, yet values in the MMC and Qingguang'an groups was relatively well-controlled throughout (Fig. 2, A). On postoperative D28, IOP in rabbits treated with Qingguang'an granules was obviously lower than IOP in the surgery only or PBS groups (P < 0.001), and showed a equivalent level with the MMC group (P > 0.05) (Fig. 2, B).

Fig. 2.

Fig. 2

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IOP changes after GFS in each group (mean ± SD, n = 20)

Notes: A: IOP on postoperative D1 - D28; B: IOP difference in five groups on D28. nsP>0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the Qingguang'an group.

3.2. Comparison of filtering bleb condition

Filtering bleb condition was assessed from four dimensions: vascularization, corkscrew vessels, encapsulation, and microcysts.25 On postoperative D28, blebs in the surgery only as well as PBS groups had vascularized to different degrees, with massive corkscrew vessels between sclera and conjunctiva. Non-functional filtering blebs were observed because of the entire encapsulation and large-area scarring in the surgery only group. In contrast, blebs in the MMC and Qingguang'an groups served function of filtration all along. In the MMC group, filtering blebs remained diffuse but flat, and some of them had mild vascularization, poor shape of scleral flap and became thin-wall, which suggested intraocular inflammation. By comparison, blebs in the Qingguang'an group were slightly bulging and retained better function owing to the smooth drainage in the filtering passage. Although there were a small amount of neovascularization between the conjunctiva and corneal limbus, the peripheral operation site kept clear (Fig. 3). Blebs treated with Qingguang'an granules had the best morphology and function among all groups.

Fig. 3.

Fig. 3

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Filtering bleb conditions on postoperative D28.

3.3. Relative gene expression of ATG5, Beclin1, LC3-II and P62

RT-qPCR was performed to detect the relative expression of autophagy genes (ATG5, Beclin1, LC3-II and P62) in ocular tissue blocks including conjunctiva, Tenon capsule and sclera harvested on postoperative D28. All amplification curves had flat baselines, obvious exponential areas and large slopes which remained stable. The melting curves emerged as single peaks respectively, which suggested the accuracy of quantification.

In the Qingguang'an group, ATG5, Beclin1 and LC3-II mRNA amounts were obviously upregulated compared with the no surgery, surgery only or PBS group, while P62 degradation was increased (Fig. 4). Most of the differences in groups were statistically significant (P < 0.05). A small number of insignificant difference might be caused by insufficient sample size or cell loss during sampling. Autophagy in filtering blebs treated with Qingguang'an granules had a tendency to increase.

Fig. 4.

Fig. 4

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Relative mRNA expression in each group (mean ± SD, n = 3)

Notes: In the Qingguang'an group, ATG5 (A), Beclin1 (B) and LC3-II (C) expression levels were increased, while P62 (D) expression was decreased versus other groups. nsP>0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

3.4. Protein amounts of ATG5, Beclin1, LC3-II and P62

Western blot was performed to assess the expression levels of autophagy-related proteins. As shown in Fig. 5, ATG5, Beclin1 and LC3-II proteins showed higher expression levels in the Qingguang'an group versus the no surgery, surgery only or PBS group, while expression of the soluble proteins P62 showed the opposite trend. LC3-II/LC3-I ratio tended to increase, indicating autophagy was induced. The WB results corroborated qPCR data.

Fig. 5.

Fig. 5

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Electrophoresis diagrams of Beclin1, LC3-II, ATG5 and P62 protein expression by western blot in each group. β-actin was used as a loading control.

3.5. Histological Effects

H&E staining showed few fibroblasts in the no surgery group under a light microscope, while a higher number of fibroblasts were found in the surgery only and PBS group, surrounded by several inflammatory cells and red blood cells. Collagen fibers were abundant and arranged disorderly. The MMC and Qingguang'an groups had less fibroblasts with relatively obvious cleft of the filtering passage. The latter arranged in better order (Fig. 6, A1 - A5).

Fig. 6.

Fig. 6

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Histological Effects in each group

Notes: A1 - A5: H&E staining ( × 100); B1 – B5: Masson's trichrome staining ( × 100); C1 – C5: IHC staining ( × 100).

Masson's trichrome staining indicated tissues in the no surgery group had low content of collagen fibers, while those of the surgery only group had massive collagen fibers with disordered arrangement which indicated severe fibrosis, and so did the PBS group. The MMC group had less collagen fibers with regular arrangement. Additionally, collagen fibers were least with minimum blue staining area in the Qingguang'an group, and fiber arrangement was neat and relatively loose (Fig. 6, B1 – B5).

IHC was performed to further detect fibrosis progress. In the no surgery group, α-SMA (α-smooth muscle actin) expression under the blebs was weak but wide, while collagen deposition was scattered. In the surgery only and PBS groups, α-SMA was highly positive and collagen deposition was extensive. In the MMC and Qingguang'an groups, α-SMA expression was lower and collagen deposition was relatively less (Fig. 6, C1 – C5). The histological outcome demonstrated that Qingguang'an inhibited de novo collagen formation in vivo to a certain extent.

3.6. Ultrastructural findings under TEM

Tissues in the surgery only group had severe fibrosis and a large area of scar formation (Fig. 7, A). In the PBS group, massive and thick fibrils were observed (Fig. 8, B). On the contrary, there were many structured vacuoles with a bilayer membrane in the cytoplasm in the Qingguang'an groups, which was considered the typical characteristic of APs (autophagosomes) (Fig. 7, D). These findings verified directly in terms of morphology that autophagy induction could be one of the mechanisms of Qingguang'an.

Fig. 7.

Fig. 7

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Representative images of ultrastructure observation under TEM.

Notes: A: The surgery only group, framed indicate large areas of scar formation; B: The negative control group, arrows indicate many fibrils; C: The MMC group, blue arrows indicate the presence of several APs, and red ones point at nuclei; D: The Qingguang'an group, blue arrows indicate multiple APs, and red ones point at nuclei.

Fig. 8.

Fig. 8

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The whole autophagy process and related proteins.

4. Discussion

GFS's therapeutic effect is not ideal enough resulting from bleb scarring, and intraoperative application of MMC is one of the most common and effective anti-scar formation interventions at the current stage. However, long-term observation suggests a bleak prognosis for its high risk of secondary hypotony keratopathy, bleb leaking, scleritis and late-onset endophthalmitis caused by MMC.5,6 Prevent fibrosis and inhibit scarring after GFS, especially by means of integrated traditional Chinese and western medicine treatment, has become a hot topic recently. Over the past 20 years of clinical practice and extensive researches, Qingguang'an granules has shown good therapeutic effect on glaucoma.13, 14, 15, 16 Moreover, enhanced autophagy was found in HTFs treated with Qingguang'an medicated serum in our in vitro study, providing clues for its anti-scarring mechanism.22,23 In this in vivo study, accordingly, we divided 100 chronic intraocular hypertension models into five groups and administered different treatments, using MMC as the positive control group, to further research how Qingguang'an works in filtering passage. For better comparison, all experimental rabbits were operated under a microscope by one surgeon and fed in the same environment during the whole period, reducing the impact of subjective factors as well as enhancing the reliability of experimental indices.

As shown above, IOP was kept well on postoperative D28 in the Qingguang'an group, effect of lowering IOP being equivalent to MMC (P > 0.05), and being significantly better than the surgery only or PBS groups (P < 0.001). Blebs treated with Qingguang'an granules had smooth drainage in the filtering passage and retained the best morphology and function among all groups. No obvious inflammatory reaction of anterior segment was observed over time, and even if there were a small amount of neovascularization near corneal limbus, the peripheral operation site kept clear, correspondingly with less fibroblasts and collagen deposition between conjunctiva and sclera from histological anlysis. This indicated Qingguang'an can be a prospective candidate to inhibit scar formation after GFS and was likely to reach a better prognosis than MMC with less complications. Meanwhile, abundant APs were captured under TEM, and a higher autophagy level was observed both in RT-qPCR as well as Western blot. Therefore, different from anti-scarring mechanism of MMC, which was invloved in inhibiting DNA synthesis and inducing apoptosis of HTFs,26 we speculated that the specific mechanism of Qingguang'an was involved in autophagy induction.

The whole autophagy process can be divided into four phases (Fig. 8), including induction, AP nucleation, autolysosome formation and degradation.27,28 At the initial stage, a flat membrane structure that is similar to a “liposome” takes shape in the cytoplasm under various stresses, namely the phagophore. This autophagic vacuole subsequently extends, forming a AP under regulation from a battery of upstream signaling and then engulfing redundant damaged organelles, followed by the fusion with lysosomes and final degradation. The four biomarkers we chosen in this study: ATG5 (autophagy-related gene5), Beclin1, LC3 (microtubule-associated protein 1 light chain 3) and P62 are all indispensable participants in the lifetime of APs. ATG5, a crucial protein of upstream signaling, is related to the elongation of phagocyte membrane and regulates it from a semi-closed structure to a closed structure, therefore forming a AP29; Beclin1 is the earliest member involved in the assembly process of phagophore and formation of APs as well, besides, it can also determine the initiation of the whole autolysosome degradation progress by combination with different proteins30,31; Beclin1 phosphorylation is the key link of autophagy, and LC3 is a indicator of autophagy activation. When changes occur, LC3-I will be processed into LC3-II, and then widely distributed throughout APs, regarded as autophagic flux to evaluate the proportion and quantity of APs.32,33 The expression levels of ATG5, Beclin1 and LC3-II are positively associated with autophagy. Instead, P62 shows a different change. As a transporter protein, it interacts with LC3-II and forms a complex served as a bridge for assisting APs to swallow the transported substances. P62 in cytoplasm continuously degrades when autophagy is enhanced, and if the activity weakens, p62 protein will accumulate, which brings it an important marker of the cargo. P62 decrease indicates ongoing autophagy flux and degradation, consistent with the upregulation of LC3 expression, indicating that autophagy is induced.34 According to our experimental results this time, ATG5, Beclin1 and LC3-II mRNA and protein amounts were obviously upregulated compared with the no surgery, surgery only or PBS group in qPCR and WB, yet P62 accumulation was rescued. Most of the differences in groups were statistically significant (P < 0.05). A small number of insignificant difference might be caused by insufficient sample size or cell loss during sampling. Hence our initial hypothesis was verified: Qingguang'an granules inducing autophagy could be one of the mechanism to inhibit scar formation in TFs.

Generally, TCM (Traditional Chinese medicine) thinks patients after GFS have Qi-deficiency and blood-stasis, consequently precipitating meridians dystrophy, Xuanfu stagnation as well as aqueous humor silting, which raises the risk of IOP rebound and aggravation of condition.35 Despite a few studies have chosen to explore the pathogenesis and treatment of glaucoma from other perspectives, such as, Yu Jing et al. considered the surgical bleb after GFS corresponds to the so-called “Bai Jing” according to the Five-Wheel theory in TCM, in which its dysfunction refers to “lung” problems, especially “lung heat” and chose Modified Cortex Mori Capsules (Sang Bai Pi Tang) to treat the condition, it still lacks of in vitro or in vivo experiments to explore the specific mechanism to date.36 To our knowledge, Qingguang'an is a modified prescription derived from Buyang Huanwu decoction which can successfully boost Qi, nourish Yin, promote blood circulation and dredge meridians.37 It has the characteristics of benefiting the healthy Qi without causing stagnation, leading to harmony of Qi and blood, thus the aqueous humor and blood circulating freely in eye orifices. Moreover, Miraculous Pivot once said, “The fluid and humor produced by nutrient Qi seeps into the meridians and then will be transformed into blood.” It also emphasizes that “Hemorrhgia shunning diaphoresis, and polyhidrosis shunning hemorrhagia.“38 The body fluid and blood share the same origin whether in physiological or pathological state, and they influence with each other. Therefore, in addition to regulating Qi, Yin and blood, the regulation of body fluid metabolism is equally considerable. Based on this theory, Qingguang'an granules formula contains Huang-qi to invigorate Qi and then activate blood stasis, Chi-shao-yao, Hong-hua and Di-long to enhance efficacy, Sheng-di to clear heat as well as cool the blood, Bai-zhu, Fu-ling along with Che-qian-zi to fortify the spleen, drain dampness and improve vision. All the Chinese medicinal herbs combined together, Qingguang'an produced a marked effect on treating glaucoma and reducing blebs' scar formation,39 and findings in this in vivo research can provide complementary to its anti-scarring effect and mechanism from a new perspective.

5. Problems and prospects

As the first report to investigate the in vivo anti-scarring effect of Qingguang'an on TFs, this study still had some limitations. On the one hand, larger sample size and longer follow-up period might be a consideration, and it is yet uncertain that which pathways are involved in the change induced by Qingguang'an. Since Beclin1 amounts were overtly increased in both PCR and WB analyses, can it be boldly hypothesized that Qingguang'an granules activates the PI3KC3/Beclin1 pathway to control subconjunctival scarring? These questions will be addressed in our next project. Additionally, autophagy and apoptosis could share common signaling pathways.40,41 Apoptosis-related indices were not examined in this study, and most of the previous findings concerning Qingguang'an focused on apoptosis in RGCs42 not TFs. Whether apoptosis can be further activated or inhibited by cell autophagy in TFs is unknown.

On the other hand, many details could be ameliorated. In this research, rabbits in the experimental group were administered Qingguang'an granules by gavage from the second postoperative day, while those of the positive control group were provided a MMC cotton during GFS, which maybe impact the comparability of the two groups. In future studies, the drug administration route could be changed for a better comparison, e.g., placement of Qingguang'an sustained-release membranes under the conjunctiva during GFS, which can also achieve a long-lasting pharmacological impact and less side effects. Besides, autophagic flux could be tested by dual-fluorescence mRFP-eGFP-LC3 in future to obtain a more complete autophagy change.43

Nowadays, researches on autophagy induction by TCM to reduce scarring are still in the infancy stage despite tremendous potential. More efforts are needed to alleviate the suffering of patients who develop scar formation after GFS. We hope this study could further promote TCM use for glaucoma treatment, and Qingguang'an granules can be a great potential candidate for controlling bleb scar formation.

6. Conclusion

Qingguang'an granules can restrain IOP increase after GFS and inhibit scar formation, with limited adverse reactions compared with MMC. It can also upregulate the ATG5, Beclin1 and LC3-II mRNA and proteins, while rescue P62 accumulatiopn in the filtering passage by results of RT-PCR and WB. Therefore, autophagy induction may be one of the mechanisms by which Qingguang'an granules inhibit the proliferation of TFs.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 81603665) and Training Program for Excellent Young Innovators of Changsha, China (kq2107014).

Footnotes

☆

Supported by the National Natural Science Foundation of China (Inducing effect of Qingguang'an on Autophagy of Tenon's Capsule Fibroblasts after Glaucoma Filtration Surgery, No. 81603665); Training Program for Excellent Young Innovators of Changsha (kq2107014).

Peer review under responsibility of The Center for Food and Biomolecules, National Taiwan University.

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