Effect of Qingguangan Ⅱ on Rho/ROCK associated factors in the retina of DBA/2J mice

Jian Shi, Jun Peng, Xiao-Lei Yao, Jia-Hui Sun, Yin-Xin Li, Qing-Hua Peng ,

1.Hunan University of Chin Med, Changsha, Hunan 410007, China

2.The First Hospital of Hunan University of Chin Med, Changsha, Hunan 410007, China

3.The First Hospital of Guangxi University of Chin Med, Nanning, Guangxi 530022, China

4.Key Laboratory of Hunan Province for Prevention and Treatment of Eye, Ear,Nose and Throat Diseases with Traditional Chin Med, Changsha,Hunan 410208, China

Keywords:Qingguangan Ⅱ prescription DBA/2J mice Rho/ROCK signaling pathway Caspase-3 mRNA Bcl-2 mRNA

ABSTRACT Objective: The effect of Qingguangan Ⅱ on the transcription of RhoA mRNA, ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retina of DBA / 2J mice was observed. Methods:Forty-eight DBA/2J mice were randomly divided into six groups: model groups, Qingguangan II decoction group, low concentration, medium concentration and high concentration group of Qingguangan II effective ingredient and positive control group (Yimaikang tablet group), and eight C57BL / 6 mice were used as blank group, DBA/2J mice were fed until 38 weeks before forming a glaucoma model, The transcription of RhoA mRNA, ROCK mRNA, Caspase-3 mRNA and Bcl-2 mRNA in the retinal of DBA/2J mice was detected using real-time fluorescence quantitative PCR (Quantitative Real-time PCR) after 4 weeks of intervention.Results: Four weeks after the intervention, In the transcription of the RhoA mRNA, ROCK mRNA and the Caspase-3 mRNA, Compared to the blank groups, Relative expression was increased in the other 6 groups, There are statistical differences in the model group, Yimaikang tablet group and low concentration group (P＜0.05); In comparison to the model groups,The other 6 groups were lower than the model group, Among them, there are statistical differences between the effective groups of Qingguangan II decoction and high concentration group of Qingguangan II effective ingredient in RhoA mRNA transcription (P＜0.05); In the transcription of the ROCK mRNA and the Caspase-3 mRNA, Statistics have differences between the model group and the effective component of the medium and high concentration group (P＜0.05); In the Bcl-2 mRNA transcription, Compare them to blank groups,Relexpression expression decreased in the other 6 groups, Statistics have differences between model group, Qingguangan II decoction group and low concentration groups (P＜0.05); The relative expression of Bcl-2 mRNA in high concentration group of effective component is higher than that of the model group, There are differences in statistics (P＜0.05). Conclusion:The high concentration of Qingguangan Ⅱ prescription probably attenuated Caspase-3 transcription in retinal ganglion cells by inhibiting the Rho / ROCK signaling pathway and activated Bcl-2 expression by inhibiting ROCK signaling, which attenuated apoptosis in retinal ganglion cells.

1. Introduction

Glaucoma as the first irreversible eye disease, the number of primary glaucoma patients worldwide exceeded 111.8 million in 2040[1]. At present, China has the largest number of glaucoma patients in the world. The overall prevalence of age 40 years and over in China was 2.3% to 3.6%, of which 54.9% to 82.0%of glaucoma patients are not diagnosed, and 6.3% to 14.1% of glaucoma patients are blindness in both eyes[2]. Studies have shown that 72% of glaucoma patients are advanced at the time of medical treatment[3]. Ocular pressure control is an important way to preserve vision in the clinic. However, after a stable IOP, visual acuity is still decreased,which is the optic nerve damage caused by glaucoma.Previous studies have shown that the green square may inhibit the Rho / ROCK signaling pathway to protect the retina[4]. However,its was validated only at the protein level. So, this study further used q-PCR to inhibit the Rho / ROCK signaling pathway to protect the retina of DBA / 2J mice.

2. Materials and Methods

2.1 Materials

2.1.1 Experimental animalsForty-eight healthy DBA / 2J mice were harvested at mass ranging from 18 to 22 g, (SPF grade, female, 10 weeks old, license number:SCXK (Jing) 2016-0006, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.); 8 C57BL / 6J mice, mass of 18 to 22 g, (SPF grade, female, 10 weeks old, license number: SCXK (Xiang)2016-0002, Hunan SJA Laboratory Animal Co., Ltd.). Ethical approval number: LL20191008021.

2.1.2 Experimental drugs

Qingguangan Ⅱ decoction: by Chinese wolfberry (production batch number: SL19112203), beef knee (production batch number:CK19101401) and other sterilized distilled water according to the specified proportion fried; Yimaikang tablet (Hunan Xiangya Pharmaceutical Co., LTD., lot No.: 1903119). Effective component of Qingguangan Ⅱ: it was extracted from the TCM components of Qingguangan formula based on TCM high-throughput screening system [5].

2.1.3 Main reagents and instrumentsRNA extract (Wuhan Servicebio Technology Co., Ltd.,cargo No.:G3013); Trichloromethane (Sinopharm Chemical Reagent Co.,Ltd.,cargo No.: 10006818); Isopropanol (Sinopharm Chemical Reagent Co., Ltd., cargo No.: 8010921); Anhydrous Ethanol(Sinopharm Chemical Reagent Co., Ltd.,cargo No.: 10009218);HyPureTMMolecular Biology Grade Water (HyClone, cargo No.: SH30538.02); Revert Aid First Strand cDNA Synthesis Kit(Thermo,cargo No.: # K1622); Fast Start Universal SYBR Green Master(Rox) (Wuhan Servicebio Technology Co., Ltd., cargo No.:4913850001).Homogenizer (Wuhan Servicebio Technology Co., Ltd., model No.:KZ-II); Desktop high-speed frozen type microcentrifuge (DLAB Scientific Inc.,model No.: D3024R); PCR instrument (Thermo Fisher Scientific, model No.: Stepone plus); Ultra net bench(Suzhou Antai Airtech CO.,LTD, model No.: SW-CJ-1FD); superluminosity(Thermo Fisher Scientific, model No.: NanoDrop2000); standard reagent pure water meter ( Qingdao Flom Technology Co. Ltd.,model No.: FBZ2001-up-p).

2.1.4 Primers

Primer synthesis company: Wuhan Servicebio Technology Co., Ltd.

2.2 Experimental Methods

2.2.1 Animals and GroupsForty-eight DBA / 2J mice were randomly divided into 6 groups according to random digital table method, which were set as model group, Yimaikang tablet group, Qingguangan II decoction group, and effective components of Qingguangan II are low, medium and high concentration groups, while 8 C57BL/6J mice were used as blank group.

2.2.2 Model establishment

According to the literature[6], DBA/2J mice fed to 38 weeks of age automatically form a glaucoma model.IOP and anterior segment changes were observed in DBA/2J mice to determine whether a glaucoma model was formed.

2.2.3 Drug method

After the model was established and stable, the lavage was started once a day for 4 weeks.Adult equivalent dose was calculated by the human / animal body surface area equivalent dose ratio table.The blank group and the model group were given an equivalent dose of distilled water; the Yimaikang tablet group was given an equivalent dose (0.31g / (Kg·d)) of suspension mixture of Yimaikang tablets; the Qingguangan II decoction group was given an equivalent dose(9.67g / (Kg·d)) of Qingguangan II decoction;the effective components of Qingguangan II are low, medium and high concentration groups were given 1/2 times, one time, and two times(0.85g/(Kg·d)、1.70g/(Kg d)、3.40g/(Kg·d)) of the equivalent dose of suspension mixture of effective components of Qingguangan II,respectively.

2.3 Materials extraction and testing

All groups of mice were sacrificed, the eyeballs were removed, the anterior segment and vitreous were removed, and the retinal tissue was quickly dissected out and cryopreserved for q-PCR detection.

2.3.1 IOP detection

Use the Tono-pen AVIA contact intraocular pressure pen, taking the average of 10 consecutive measurements (and the confidence indicators are displayed on the LCD not less than 95) as the IOP of the eye in that week, once every 4 weeks, from week 10, until DBA/2J mice had increased IOP and stop after stabilization.

2.3.2 Anterior segment detectionAll animals were examined for the anterior segment slit lamp after 3 days of adaptive feeding, once every 4 weeks.Three fixed experimenters completed the operation in the examination room;one was responsible for lifting the mice at the appropriate height of the slit lamp jaw frame device; one was responsible for adjusting the focal length, observing the mouse front segment through eyescopy,including cornea, iris, anterior chamber, pupil, lens, and one was responsible for computer photography.

2.3.3 Expression of RhoA mRNA, ROCK mRNA, Caspase-3 mRNA, and Bcl-2 mRNA transcription was determined by q-PCR

① RNA extraction: Add 1mL of Trizol Reagent in the homogenate tube, precold on ice, add 100mg tissue, fully grind using a homogenizer, centrifuged at 12000rpm for 10min, remove the supernatant, add 250 L trichomethane, mix well and stand still for 3min,centrifuged at 12000rpm for 10min, transfer the supernatant to a new centrifugal tube, add 0.8 x the volume of isopropanol,150 min, 12000 rpm centrifugation for 10min reversed, -20℃ for 15min and centrifuged at 12000rpm for 10min. The liquid was aspirated, the precipitation was washed with 1.5mL of 75% ethanol,centrifuged at 12000rpm for 5min, the liquid was aspirated, the centrifuge tube was placed on an ultra-net table for 3min, added 15 L of RNA enzyme free water dissolved RNA, incubated at 55℃ for 5min, and the RNA concentration and purity were determined by using Nanodrop 2000.Excessive concentrations of RNA were diluted in an appropriate proportion to achieve a final concentration of 100-500ng/μL.

② Reverse transcription: take a PCR tube, add with 10 μL RNA and 1 μL oligo (dT) 18, up to 12 L was replenished with ribonuclease-free deionized water,keep 65℃ insulation on the PCR instrument for 5min. Then, quickly the ice to cool, add 4 μL 5 Reaction Buffer,2μL10mM dNTP Mix,1μL RiboLock RNAase inhibitors and 1 μL RevertAi M-MuLV Reverse transcriptase (200U/μL), m. b, 42℃ for 60min, after that Retroslase was inactivated by 70℃ insulation for 5min.

③ Quantitative PCR: 0.2mLPCR tube, add in 2× qPCR Mix(12.5μL), 7.5 μM gene primer (2.0 μL), reverse transcription products (2.5 μL), ddH2O (8.0 μL), 3 tubes.PCR amplification: predenaturation (95℃ 10min), cycle (40 times, 95℃ 15s→60℃ 60s),melting curve (60℃→95℃ heating 0.3℃ per 15s).

④ Result processing: 2-△△CtMethod: △ Ct test =Ct target-Ct internal reference, △Ct control =Ct target-Ct internal reference;△△Ct= △Ct test- △Ct control; relative expression =2-△△Ct.

2.4 Statistical Methods

Statistical data analysis was performed with SPSS26.0 software,two-sided tests were performed, and P <0.05 was considered statistically significant.Data are expressed as the mean ± standard deviation(x± SD).Tests for normality and homogeneity of variance were performed with or corrected analysis if the data were normally distributed.Multiple groups were compared using the Tukey method.If the normality requirements are not met, a non-parametric test is used.

3. Results

3.1 Ocular pressure results

C57BL/6J mice had sustained stable IOP, consistently fluctuating at 11–14mmHg, and DBA/2J mice increased continuously from 10 weeks old and reached 23.41±2.26mmHg at 38 weeks old[4].Meanwhile, a high-IOP mouse model was also formed.Then,from week 14, the blank group was different from the other groups (P<0.05), and greatly after 18 weeks (P <0.01).

3.2 Anterior segment detection

C57BL/6J mice showed no significant changes in the anterior segment, and DBA/2J mice showed corneal calcification at 10 weeks of age (Figure 2.3). At the same time, corneal calcification is aggravated, with the loss of iris pigment, local iris transmittance,iris matrix atrophy, and posterior pupil adhesion. By 38 weeks of age, iris pigmentation aggravation, pupil displacement and partially complicated by cataract (Figure 2.4).

Figure 1 IOP comparison at different time points between C57BL/6J mice and DBA /2J mice (±s)

Figure 2 Photo of different groups at different weeks of age

3.3 The 2.3 q-PCR test results

In the transcription of RhoA mRNA, the relative expression of the other 6 groups was higher compared with the blank group, but the model group, the Yimaikang tablet group and the low concentration of the effective component of Qingauangan II group were statistical differences (P <0.05). Compared with the model group, relative expression level of the other 6 groups was lower than that of the model group. There was only a statistical difference between the high concentration of the effective component of Qingauangan II group and the Qingguangan II decoction group (P <0.05). There was a statistical difference between the high concentration and the low concentration of the effective component of Qingauangan II groups(P <0.05), with no other statistical difference.In the transcription of ROCK mRNA, compared with the blank group,

the relative expression of the other 5 groups except the high concentration group increased significantly. Only the model group,the Yimaikang tablet group and the low concentration of the effective component of Qingauangan II group were statistical differences(P<0.05). The ROCK mRNA transcript group was higher than the other 6 groups and the medium concentration and the high concentration of the effective component of Qingauangan II groups were statistical differences(P <0.05). There was a statistical difference between the high concentration of the effective component of Qingauangan II group and the Yimaikang tablet group(P <0.05), with no other statistical differences.

In the transcription of Caspase-3 mRNA, the relative expression of the other 6 groups was higher compared with the blank group, but the model group, the Yimaikang tablet group and the low concentration of the effective component of Qingauangan II group were statistical differences (P <0.05). Compared with the model group, all of the other 6 groups decreased, and the medium concentration and the high concentration of the effective component of Qingauangan II groups were statistical differences(P<0.05).The Yimaikang tablet group, the low concentration and high concentration of the effective component of Qingauangan II group were statistical differences(P<0.05), with no other statistical differences.In the transcription of Bcl-2 mRNA, the relative expression of other 6 groups decreased compared with the blank group.The model group,the Qingguangan II decoction group and the low concentration of the effective component of Qingauangan II group were statistical differences(P<0.05). The relative expression of Bcl-2 mRNA in the medium concentration and the high concentration of the effective component of Qingauangan II groups was higher than in the model group,which was statistically different(P <0.05). There was a statistical difference between the high concentration and low concentration of the effective component of Qingauangan II groups(P <0.05), with no other statistical differences.

Table 1 Comparison of RhoA mRNA and ROCK mRNA relative expression in different groups (±s,n=3)

Table 1 Comparison of RhoA mRNA and ROCK mRNA relative expression in different groups (±s,n=3)

*P <0.05, which was different when compared with the blank group;#P <0.05, which was different when compared with the model group;△P <0.05, which was different when compared with the low concentration of the effective component of Qingauangan II group;△△△P <0.05, which was different when compared with the high concentration of the effective component of Qingauangan II group;

Group RhoA mRNA ROCK mRNA Blank group 1 1 Model group 7.30±0.83* 125.20±13.13*Yimaikang tablet group 4.21±0.25* 62.79±1.30*△△△Qingguangan II decoction group 3.71±0.30# 47.54±1.88 Low concentration of the effective component of Qingauangan II group 4.83±0.12* 52.10±2.85*Medium concentration of the effective component of Qingauangan II group 4.06±0.70 25.35±6.12#High concentration of the effective component of Qingauangan II group 1.97±0.12#△ 9.03±0.76#H 18.333 19.549 P 0.005 0.003

Table 2 Comparison of relative Caspase-3 mRNA and Bcl-2 mRNA expression of different groups (`±s,n=3)

Table 2 Comparison of relative Caspase-3 mRNA and Bcl-2 mRNA expression of different groups (`±s,n=3)

*P <0.05, which was different when compared with the blank group;#P <0.05, which was different when compared with the model group;△P <0.05, which was different when compared with the low concentration of the effective component of Qingauangan II group;△△△P <0.05, which was different when compared with the high concentration of the effective component of Qingauangan II group;

Group Caspase-3 mRNA Bcl-2 mRNA Blank group 1 1 Model group 6.90±1.26* 0.12±0.01*Yimaikang tablet group 4.49±0.34*△△△ 0.44±0.01 Qingguangan II decoction group 3.59±0.37 0.43±0.05*Low concentration of the effectivecomponent of Qingauangan II group 4.45±0.13*△△△ 0.40±0.00*Medium concentration of the effectivecomponent of Qingauangan II group 3.25±0.70# 0.47±0.06#High concentration of the effectivecomponent of Qingauangan II group 1.86±0.09# 0.62±0.01#△H 19.132 18.068 P 0.004 0.006

4. Discussion

The slow and gradual loss of irreversible retinal ganglion cells(RGCs) and their axons in the middle and late stages of glaucoma is inevitable, and it still cannot be relieved in the case of IOP control.So, how to protect the optic nerve and reduce the optic nerve apoptosis is the focus of the current research.In a previous study,we found that the has an advantages in optic nerve protection.We previously found that one of the mechanisms of action of green is to inhibit the K-3βmRNA expression of GSK, subsequently promote β-catenin mRNA expression and activate Wnt/β-catenin signaling pathway[7]. Later, the expression level of PAX6, Ngn1, and Ngn2 mRNA were up-regulation[8], which to play a role of protecting the optic nerve. Another mechanism is the alteration of NF-B / HIF-1 pathway by Qingauangan II, inhibiting BNIP-3 and SOD, which the former attenuated mitochondrial autophagy of RGCs, while the latter reduced of MDA[9]. It is also possible that by promoting expression of HSP[10], it subsequently protects mitochondrial function and thus attenuates mitophagy in optic neurolia cells.

At present, the active components of Qinguangan II that we studied were screened by high-throughput screening system based on Wnt/β-Catenin/Pax6. We also verified that it is protective against glaucomatous optic ganglion cells through the Wnt/β-Catenin signaling pathway[8]. Therefore, we believe that the Wnt/β-Catenin signaling pathway and the Rho/ROCK signaling pathway can cooperate in protecting the glaucomatous optic nerve effect. Studies have shown that intravitreal injection of the Rho-GTPase inhibitor C3 transferase (BA-210) improves the survival rate of RGC and axon regeneration[11]. Meanwhile, transient retinal ischemia and reperfusion, which mainly infiltrates leukocytes into the neural tissue through vascular endothelial cells, resulting in the loss of neuronal cells in the inner retinal layer[12]. However, the Rho/ROCK signaling pathway mainly connects white fine cells and endothelial cells tightly, allowing them to reduce infiltration[13]. The ROCK inhibitor Y-27632 promoted the activity of primary RGCs-5 cell lines, and local application of Rock/Net inhibitor promoted the survival and regeneration of RGC after optic nerve injury[14]. Treatment with the ROCK inhibitor E212 inhibited the oxidative stress and antiinflammatory effects, and reduced the loss of RGCs[15]. Inactivation of the Rho/ROCK pathway promotes neurite regeneration[16,17]。

Chinese traditional medicine believes that glaucoma is blood stasis due to qi deficiency, blockage of the vessel, cause no nutrition in the eyes, the aqua channel is closed and make the aqueous humor blood stasis[18]. Therefore, in the treatment of using enhance the circulation of vitality, blood and aqueous humor treatment[19]. Qingguangan II focuses on treatment deficiency of liver-yin and kidney-yin. At the same time, on the basis of tonic, promote vitality, blood and aqueous humor circulation. When the circulation of vitality is well, the circulation of blood is well. The smoothly circulation of vitality is same as the smoothly circulation of aqueous humor. The whole side complement both, so that the eyes unobstructed, the circulation of vitality and blood harmony.

Experimental results are similar to the previous WB results[4],which finding an elevated relative expression of RhoA mRNA,ROCK mRNA, and Caspase-3 mRNA when these transcribed in the retina. The model group was significantly elevated. Compared with the model group, the Qingguangan II decoction group and the high concentration of the effective component of Qingauangan II group both have obvious differences with model group. It shows that the effective group of Qingguangan II decoction group and the high concentration of the effective component of Qingauangan II group had an obvious effect. However, among the three groups of the effective component of the Qingauangan II, there were significant differences between the the high concentration and low concentration of the effective component of Qingauangan II groups. There was no significant difference with the Qingguangan II decoction group. This indicates that the high concentration of the effective component of Qingauangan II group had significant inhibition on the transcription of RhoA mRNA and Caspase-3 mRNA. The relative expression of Bcl-2 mRNA was reduced, and the model group was the lowest,while the medium concentration and high concentration of the effective component of Qingauangan II group both had differences with model group, and had no differences with blank group. It indicating that the two groups had an obvious effect. In the three different concentrations of the effective component of Qingauangan II groups, the high concentration of the effective component of Qingauangan II groups had an obvious different with its low concentration group, and no obvious difference with Qingguangan II decoction group. It shows that it can improve the transcription of Bcl-2 mRNA, thus enhancing the expression of Bcl-2, to inhibit the transcription of Caspase-3 mRNA and then inhibit Caspase-3.

However, we found that the expression of ROCK mRNA in the effective component of Qingauangan II group dosage (25.35 ± 6.12)had a certain difference with the Qingguangan II decoction group(47.54 ± 1.88), but there was no significant difference (P=0.51). It may be because the active ingredient has a specific inhibitory effect on ROCK mRNA, while the decoction may be because the unknown ingredient has a certain inhibitory effect on the active ingredient.Therefore, according to its expression of related factors, it can be found that the active ingredient of Qingguangan II is better, which can provide guidance for clinical medication in the future, and achieve better curative effect through improved dosage form.

In conclusion, this experiment result is the same as the previous experiment, indicating that the high concentration of Qingguangan II may reduce the apoptosis of retinal cells by inhibiting the Rho/ROCK signaling pathway.

Conflict of interest: This article has no conflict of interest with any other interests.

Division of labor: Shi Jian: experiment, writing the paper; Peng Jun: data analysis; Yao Xiaolei: guide the experiment; Sun Jiahui:experiment; Li Yinxin: experiment; Peng Qinghua: guidance and supervision.