SGI-1027

Effect of NaoluoXintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats
Lu Hong a, Weidong Chen a, c, Ling He b, d,*, Hui Tan b, d, Daiyin Peng a, c, Guodong Zhao a,
Xiaoqian Shi e, Lei Wang a, c, Mingming Liu a, Huihui Jiang a
a School of Pharmacy, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, China
b Key Laboratory of Xin′ an Medicine Ministry of Education, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, China
c Anhui Province Key Laboratory of Chinese Medicinal Formula, Hefei, Anhui, 230012, China
d School of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, China
e Department of Pharmacy, Huaibei People’s Hospital, Huaibei, Anhui, 235000, China

A R T I C L E I N F O

Keywords: NaoluoXintong (NLXT) Ischemic stroke
DNA methylation NogoA/RhoA/ROCK pathway

A B S T R C T

Ethnopharmacological relevance: NaoluoXintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear. Aim of the study: To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/ RhoA/Rock pathway in a rat model of IS by regulating DNA methylation.
Materials and methods: Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT + SGI-1027) and blocker group II (NLXT + Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow
(rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho- associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue.
Results: After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to
ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P < 0.01 or P < 0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P < 0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P < 0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P < 0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P < 0.01). Abbreviations: NaoluoXintong, (NLXT); Traditional Chinese Medicine, (TCM); Ischemic stroke, (IS); central nervous system, (CNS); neurite outgrowth inhibitor protein-A, (Nogo-A); Nogo receptor, (NgR); ras homolog gene family member A, (RhoA); rho-associated coiled-coil protein kinase 2, (ROCK2); middle cerebral artery occlusion, (MCAO); middle cerebral artery occlusion-reperfusion, (MCAO/R); Reverse transcription-polymerase chain reaction, (RT-PCR); Food and Drug Admin- istration, (FDA); recombinant tissue prothrombin activator, (rt-PA); neural stem cells, (NSCs); regional cerebral blood flow, (rCBF); National Institutes of Health Stroke Scale, (NIHSS); transient receptor potential channel 6, (TRPC6); phospho-CAMP-response element-binding, (p-CREB); glial fibrillary acidic protein, (GFAP); microtubule-associated protein 2, (MAP-2); sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE); PBS containing 0.05% Tween 20, (PBST); 5′-AMP-activated protein kinase, (AMPK); mitogen-activated protein kinase, (MAPK); Janus kinase/ signal transducer and activator of transcription, (JAK/STAT). * Corresponding author. Key Laboratory of Xin′ an Medicine Ministry of Education, School of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Meishan Avenue No.103, 230012, Hefei, Anhui, China. E-mail address: [email protected] (L. He). https://doi.org/10.1016/j.jep.2021.114559 Received 7 July 2021; Received in revised form 17 August 2021; Accepted 21 August 2021 Available online 28 August 2021 0378-8741/© 2021 Elsevier B.V. All rights reserved. Conclusion: NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect. 1. Introduction Stroke is a group of diseases characterized by symptoms of ischemic and hemorrhagic injury to the brain, of all stroke cases, 87% are ischemic (Roger et al., 2011). Leading causes of IS are complicated and various, whose mechanisms have been widely investigated in recent years. Among those mechanisms, potentially, DNA methylation appears a promising biomarker for stroke prevention (Zeng et al., 2020). The role of DNA methylation in cerebral ischemia keeps multifaceted, with genome-wide and gene-specific effects that influence the vulnerability of the central nervous system (CNS) to injury (Qureshi and Mehler, 2010). It might be its promoting effects on neuronal cell death, as is observed that mice who expressed lower levels of DNA methyltransferase, a catalyst of DNA methylation, were protected from cerebral ischemia (Endres et al., 2001). Meanwhile, down-regulating Nogo-A which ap- pears widely present in the mammalian CNS expression or blocking Nogo-A pathway contributes to nerve regeneration (Cafferty et al., 2008). What’s more, after binding with NgR, Nogo-A initiates the downstream RhoA/ROCK2 signaling pathway, thereby inhibiting the growth of axons and impeding nerve repair and regeneration (Peng et al., 2010; Schwab and Strittmatter, 2014). Thus, to remove the large obstacle inhibiting the process of nerve regeneration after IS, it is the downregulation of the whole pathway of NogoA/RhoA/ROCK that matters. Thrombolytic therapy has been the main treatment for IS. Food and Drug Administration (FDA) approved thrombolytic drugs, such as re- combinant tissue prothrombin activator (rt-PA) and aspirin, are widely used to prevent and treat IS. However, there exists an increase in the risk of mortality with alteplase treatment (Lees et al., 2010). As treatments for various stroke-related ailments, Chinese herbal therapies have been described in ancient medicine systems (Luo et al., 2019), which is derived from TCM. NLXT, originated from Xin’an Wang’s Medicine, which is one of the most significant academic schools of TCM and appeared from Song Dynasty of ancient Chinese (He et al., 2019). Clinical studies of NLXT explored that the improvement of National Institutes of Health Stroke Scale (NIHSS) score, Bathel index score, Fugl-Meyer Assessment score, TCM syndrome score as well as rCBF in the observation group patients were far superior to the control group (Dai et al., 2018; Deng, 2017; Ma et al., 2015). Thus, the neurological impairment, motor function, daily living ability, Qi deficiency syndrome as well as hemorheology of patients were ameliorated by NLXT. What’s more, in animal experiments, the level of transient receptor potential channel 6 (TRPC6) as well as expression of phospho-CAMP-response element-binding (p-CREB) and protein expressions of β-tubulinIII, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP-2) in NLXT group were extremely increased comparing to control group, suggesting that NLXT can reduce cell apoptosis and promote nerve proliferation and differentiation (He et al., 2019; Wu et al., 2020). NLXT consists of seven drugs: Astragali Radix, considered as the sover- eign drug, played a fundamental role in NLXT; Chuanxiong Rhizoma and Notoginseng Radix et Rhizoma were classified as the minister drugs; Carthami Flos and Angelicae Sinensis Radix belonged to assistant drugs; while guide drugs referred to Gastrodiae Rhizoma and Scolopendra. The components which played the role in treating IS had been explored previously (He et al., 2019), but the mechanism of NLXT remains unclear. In this study, the main research content was to regulate DNA methylation and interfere with the NogoA/RhoA/ROCK pathway using MCAO rat model with NLXT, and to select the DNA methylation trans- ferase blocker SGI-1027 and the ROCK pathway blocker Y27632 as the conditions for the blocking effect. We observed CpG expression in the CpG locus of NogoA, RhoA, NgR and ROCK2 genes in the brain tissue of rats with IS and observed NogoA changes in the RhoA/ROCK pathway related indicators to better understand the reaction of the brain at the same time. The purpose of this study is to discover whether NLXT plays a role in axonal regeneration through the NogoA/RhoA/ROCK signaling pathway by regulating its DNA methylation, and to provide experi- mental evidence for NLXT to facilitate the recovery of neurological function after stroke. 2. Experimental material 2.1. Animals Two hundred and forty healthy male Sprague-Dawley rats weighing 250–300 g and 8 weeks old were provided by the EXperimental Animal Center of Anhui Province [License No. 2011-002]. All rats were housed in individually ventilated cages at a temperature of (22 1) ◦C and 60% relative humidity, with 12 h of alternating light and 12 h of darkness. Food and water were available. 2.2. Materials 2.2.1. Crude drugs The names and ratios of the seven crude drugs of NLXT are listed in [Table 1]. All the crude drugs were purchased from Anhui Guangyintang Traditional Chinese Medicine Co. Ltd., China. All kinds of samples were authenticated by Prof. Nianjun Yu (School of Pharmacy, Anhui Uni- versity of Chinese Medicine, China). 2.2.2. Reagents SGI-1027 [s7276] and Y27632 [s1049] were obtained from Selleck, US; DEPC [20150802554] was obtained from Sigma-Aldrich company, America; EZ DNA Methylation-Gold Kit was gained from ZYMO, ZA, USA; The Trizol kit [90803] was purchased from Thermo Fisher Scien- tific, China; RIPA lysate [P002] and the BCA protein quantification kit [P0012S] were gained from Beyotime Biotechnology, China; Tween 20 [T8220] was obtained from Beijing Solaibao Technology Co., Ltd, China; The goat anti-mouse IgG-HRP [ZB-2305] and β-actin [TA-09] were gained from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd, China. 2.2.3. Consumable materials Chloroform [20151020], Anhydrous ethanol [20151125] and Iso- propanol [20151113] were all purchased in Shanghai Suyi Chemical Reagents Co., Ltd., China; The fiXation solution [4583], PBS buffer so- lution [G0002], and anti-fluorescence quenching sealing tablet [G1401] were all purchased in Google organisms. 3. Experimental methods 3.1. NLXT decoction preparation Astragali Radix 30 g, Chuanxiong Rhizoma 10 g, Notoginseng Radix et Rhizoma 6 g, Gastrodiae Rhizoma 10 g, Angelicae Sinensis Radix 10 g, Carthami Flos 10 g, and Scolopendra 4 g were weighed. Details of the preparation of the decoction are available in article of a project team (He et al., 2019). The concentration was 1 mL containing 1 g crude drugs. The fingerprint of NLXT was established using 3D-UPLC-DAD, and it was shown in S1 [Fig. S1]. 3.2. Model making and grouping Improved MCAO/R model (Jiang et al., 2004) with reference to Longa EZ method (Longa et al., 1989). The rCBF in the ischemic area was monitored by laser Doppler during surgery. Rats were divided into five groups by random number table method: normal group, model group, NLXT group, blocker group I (NLXT SGI-1027 group) and blocker group II (NLXT Group Y27632), 48 in each group. Each group created using the random number table method was divided into 1-, 3- and 7-day groups, with 16 nodes per time. The NLXT group and the blocker group I were intragastrically administered 8.54 g/kg/d of the crude drug according to clinical dosage(Tan et al., 2016). the SGI-1027 was treated with 11.2 mg/kg/d, same as the dosage commonly used (Datta et al., 2009). The intraperitoneal injection was performed 1 h before, and the blocker group II was intraperitoneally injected at 0.1 mg/kg/d (Narumiya et al., 2000) 1 h before surgery with Y27632. The model group and the normal group were given the same amount of physiological saline. 3.3. Neurological deficit tests Neurological evaluation (n 16 in each group) was performed by the same examiner, who was blinded to the group assignment at 24 h after reperfusion. The Longa test was used for this evaluation (Ye et al., 2010): grade 0, symptoms without neurological impairment (normal); grade 1, inextensibility of its left forepaw when lifting the rats’ tail (mild); grade 2, circling to the left side while walking (moderate); grade 3, walking hard and leaning to the left (severe); and grade 4, cannot walk spontaneously (very severe). 3.4. Determination of rCBF (Du et al., 2011) Rats with ischemic side lesions and surrounding tissues were selected as monitoring areas using laser Doppler. The rCBF of each group was monitored 2 h after the insertion and 1, 3 and 7 days after the operation. 3.5. Determination of CpG site methylation level of NogoA, RhoA, NgR and ROCK2 genes in rat brain using the bisulfite method Primer design and synthesis: NogoA, RhoA, NgR and ROCK2 gene primer sequences are showed down below. Bisulfite treatment: sample treatment was performed using the EZ DNA Methylation-Gold Kit. Sample target fragment multiplex PCR reaction. Multiplex PCR ampli- fication was performed using the transformed sample genome as a template. After quality control, the amplification products of all multi- plex PCR primer panels using the same sample genomic DNA as a tem- plate were miXed, and the amount of amplification product per primer was ensured to be equivalent. Sequencing after sequencing: all samples were amplified with equal amounts of index PCR amplification products and recovered by tapping to obtain the final Methyl Target sequencing library. The library’s fragment length distribution was verified using an Agilent 2100 Bioanalyzer. After accurate quantification of the library molar concentration, high-throughput sequencing was performed on the Illumina Hiseq/Miseq platform [Illumina, San Diego, CA, USA] in a 2 × 150 bp/2 × 250 bp double-end sequencing mode to obtain FastQ data. Table 1 Names and ratios of seven crude drugs in NaoluoXintong (NLXT). 3.6. RT-PCR to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain Primer design and synthesis: primers were designed and analyzed using Primer Premier 5 software [Shanghai Sangon Bioengineering Technology Services Co., Ltd]. NogoA(123bp): F:5′-GAGCCTGTGATACCCTCCTC-3′, R:5′-AGGAA- GAGAGGCAGCAGTTT-3’. RhoA(156bp): F: 5′- GGCAAACAGGATTGGCGCTT-3′, R:5′- CGCGTCACAAGGCTTCACAA-3’. NgR1(103bp): F: 5′-GCAGCTCTGCAGTACCTCTA-3′, R:5′- TGCCATGCAGAAAGAGATGC-3’. NgR2(127bp): F: 5′-CCTATGCTCTGCACCTGCTA-3′, R:5′-AGC- GAATGAGGTTGTTCTGC-3’. ROCK2(130bp): F: 5′-CTGCAGATGACCCTGGACAGT-3′, R: 5′- ATCCGTCATCAGGCTCAGCA-3’. β-actin(150bp): F: 5′-CCCATCTATGAGGGTTACGC -3′, R: 5′- TTTAATGTCACGCACGATTTC -3’. RT-PCR procedure: total RNA was extracted according to the in- structions of the Trizol kit. Reverse transcription was conducted ac- cording to the method provided in the M-Mulv Reverse Transcription Kit. The real-time PCR reaction system was 20 μL, including 10 μL of 2 × Goldstar Taqman miXture premiXed system, 2 μL of reverse transcription product, 0.4 μL of 0.4 μL probe for each of the positive and negative primers, and water supplemented to 20 μL. The reaction conditions were pre-denaturation (95 ◦C) for 10 min followed by 40 cycles of reaction, with each cycle including denaturation (95 ◦C) for 15 s, annealing for 40 s, extension (60 ◦C) for 60 s and termination at 4 ◦C. The analytical method used in this experiment was a relative quantification study; the index used for the analysis was: 2-△△Ct. 3.7. Western blotting detection of NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression Total protein extraction: 100 mg of rat hippocampal brain tissue was taken. 1.5 mL of RIPA lysate was added to fully lyse and the supernatant was taken using low-temperature centrifugation (4 ◦C, 12000 g) for 3 min. The BCA protein quantification kit was used to measure and calculate the concentration and adjust the protein concentration of each sample to 2 μg/μL. Electrophoresis: 16 μL of the extracted protein solution was taken and added to 4 μL of the loading buffer (5 ) for sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Transfer film: transferred at 120 mA for 3 h, then stained the cellu- lose film in Ponceau for a while and marked the order of loading and the position of the standard protein on the upper and side edges of the membrane. Blocked: 5% skimmed milk powder/PBS blocked overnight. Reaction: RIPA were miXed at 1:500 dilution, shaken on a shaker for 2 h, washed 3 times with PBS containing 0.05% Tween 20 (PBST) for 10 min. Added 1:1000 dilution of goat anti-mouse IgG-HRP, shook for 1 h on a shaker, washed 3 times with PBST for 10 min each time, then washed once with PBS without Tween 20 for 10 min. Color develop- ment, development, and fiXing. Photography: the results were scanned using a gel imaging system; Chinese name Genus Family Botanical names Weight (g) Part used Batch number Region Huang Qi Astragalus membranaceus(Fisch.)Bunge Astragali Radix 30 Dried root 160902 Gansu San Qi Panax notoginseng(Burk.)F.H.Chen Notoginseng Radix et Rhizoma 6 Dried rhizome 160901 Yunnan Chuan Xiong Ligusticum chuanxiong Hort. Chuanxiong Rhizoma 10 Dried rhizome 160901 Sichuan Tian Ma Gastrodia elata Bl. Gastrodiae Rhizoma 10 Dried rhizome 160801 Anhui Hong Hua Carthamus tinctorius L. Carthami Flos 10 Dried flower 160801 Xinjiang Dang Gui Angelica sinensis(Oliv.)Diels Angelicae Sinensis Radix 10 Dried root 160803 Gansu Wu Gong Scolopendra subspinipes mutilans L. Koch Scolopendra 4 Dried whole herb 160801 Hubei the BANDSCAN software was used to analyze the bands and calculate the gray value of the bands to compare with the β-actin gray value as the relative protein expression. 3.8. Statistical processing IBM SPSS Statistics for Windows, version 20.0 (IBM Corp., Armonk, N.Y., USA) was used for data processing; the measurement data results were all expressed as means standard deviation (mean Std. D). One- way analysis of variance was adopted, and normalization and homo- geneity of variance were tested. Since the variance was the smallest, the pairwise comparison was conducted by the significance method. When the variance was not uniform, the Games–Howell method was used. A P- value < 0.05 was considered statistically significant. The statistical chart was drawn using EXcel 2007 software. 4. Result 4.1. 1Neurological scores After the MCAO modeling, all rats exhibited significant neurological deficit except normal group, suggesting that the model was successful. Fig. 1. 4.2. Effect of NLXT on rCBF in rats with IS At each time point after the operation, the rCBF levels of the model and treatment groups decreased compared with the normal group (P < 0.01 or P < 0.05). In comparison with the model group, the rCBF levels of the NLXT group increased prominently after 1, 3 and 7 days (P < 0.01 or P < 0.05). Meanwhile, rCBF increased in blocker group I and blocker group II after 3 days compared to the model group (P < 0.05). Fig. 2. 4.3. Effects of NLXT on CpG site methylation of NogoA, RhoA, NgR and ROCK2 genes in IS rats 4.3.1. NogoA methylation level in experimental rats and the effect of drugs Different DNA methylation sites of NogoA gene in normal group, model group and NLXT group were analyzed. Compared with the Fig. 2. Results of rCBF in each group rats (x ± s, PU, n = 16). Blocker I group represents NLXT + SGI-1027; Blocker II group represents NLXT + Y27632. At each time point after the operation, the rCBF levels of the model and treatment groups decreased compared with the normal group (P < 0.01 or P < 0.05). In comparison with the model group, the rCBF levels of the NLXT group increased prominently after 1, 3 and 7 days (P < 0.01 or P < 0.05). Meanwhile, rCBF increased in blocker group I and blocker group II after 3 days compared to the model group (P < 0.05). Note: Compared with the normal group, △P < 0.05, △△P < 0.01; compared with the model group, *P < 0.05, **P < 0.01. normal group, there were statistically significant enhancements in methylation levels at CpG44, CpG61, CpG100, CpG102, CpG141 and CpG150 sites in the model group (P < 0.05), and there were statistically significant decreases in the same sites in the NLXT group (P < 0.05). There was no significant difference in the methylation of other CpG sites between the two groups (P > 0.05). Table 2, Fig. 3a.
4.3.2. RhoA methylation level in experimental rats and the effect of drugs The RhoA gene DNA methylation sites in the normal, model and NLXT groups were analyzed. Compared with the normal group, the model group contained CpG33, CpG43, CpG45, CpG81, CpG146, CpG166, CpG168, CpG182, CpG217, CpG231, CpG238. The difference
in site methylation levels was observably increased (P < 0.05). In comparison with the model group, the difference in the above- mentioned sites in the NLXT group was statistically significant Table 2 Methylation of NogoA in each group rats(n = 5). gene time locus group P-value model NLXT NogoA 1d 44 0.01208 ± 0.00010 0.00823 ± 0.00101 0.00028 102 0.00966 ± 0.00174 0.00698 ± 0.00121 0.02203 150 0.01193 ± 0.00227 0.00867 ± 0.00109 0.02012 3d 44 0.01338 ± 0.00202 0.00837 ± 0.00152 0.00698 61 0.01052 ± 0.00280 0.00646 ± 0.00194 0.04960 100 0.01182 ± 0.00119 0.00840 ± 0.00079 0.00250 120 0.01160 ± 0.00236 0.00839 ± 0.00125 0.04140 7d 141 0.01107 ± 0.00142 0.00809 ± 0.00115 0.00652 147 0.01109 ± 0.00162 0.00897 ± 0.00090 0.03376 150 0.01363 ± 0.00187 0.00965 ± 0.00131 0.00456 time locus group P-value model normal 1d 44 0.01208 ± 0.00010 0.00810 ± 0.00122 0.00046 Fig. 1. Comparison of neurological deficit score in different groups (n = 16). Data are presented as the means ± SD. *P < 0.05 versus normal group, △P < 102 0.00967 ± 0.00174 0.00660 ± 0.00162 0.02040 3d 44 0.01338 ± 0.00202 0.00810 ± 0.00122 0.00330 100 0.01182 ± 0.00119 0.00783 ± 0.00181 0.01536 7d 44 0.01046 ± 0.00059 0.00810 ± 0.00122 0.00046 0.05 versus model group. 150 0.01363 ± 0.00187 0.01084 ± 0.00111 0.02084 Fig. 3a. Methylation PCA plots of Nogo-A in various groups of rats. MX means model group; YW means NLXT group; ZC means normal group. Different DNA methylation sites of NogoA gene in normal group, model group and NLXT group were analyzed. Compared with the normal group, there were statistically sig- nificant enhancements in methylation levels at CpG44, CpG61, CpG100, CpG102, CpG141 and CpG150 sites in the model group (P < 0.05), and there were statistically significant decreases in the same sites in the NLXT group (P < 0.05). There was no significant difference in the methylation of other CpG sites between the two groups (P > 0.05).

decreased (P < 0.05). There was no significant difference in the methylation of the remaining CpG sites between the groups (P > 0.05). Table 3, Fig. 3b.

Table 3
Methylation of RhoA in each group rats(n = 5).
gene time locus group P-value

model NLXT

RhoA 1d 33 0.10257 ± 0.02109 0.06870 ± 0.01669 0.02264
81 0.00329 ± 0.00319 0.01046 ± 0.00482 0.02414
162 0.00190 ± 0.00200 0.00474 ± 0.00184 0.04583
168 0.00964 ± 0.00185 0.00591 ± 0.00261 0.03132
182 0.00709 ± 0.00683 0.01895 ± 0.00694 0.02616

Fig. 3b. Methylation PCA plots of NgR in various groups of rats. MX means model group; YW means NLXT group; ZC means normal group. The RhoA gene DNA methylation sites in the normal, model and NLXT groups were analyzed. Compared with the normal group, the model group contained CpG33, CpG43, CpG45, CpG81, CpG146, CpG166, CpG168, CpG182, CpG217, CpG231,
CpG238. The difference in site methylation levels was observably increased (P
< 0.05). In comparison with the model group, the difference in the above- mentioned sites in the NLXT group was statistically significant decreased (P < 0.05). There was no significant difference in the methylation of the remaining CpG sites between the groups (P > 0.05).

4.3.3. NgR gene methylation level in experimental rats and the effect of drugs
The different NgR gene DNA methylation groups in the normal, model and NLXT groups were analyzed. Compared with the normal group, the methylation levels of CpG38, CpG77, CpG122, CpG144,
CpG160, CpG172 and CpG175 in the model group differed prominently (P < 0.05). In comparison with the model group, the difference in the above-mentioned sites in the NLXT group was statistically significant (P < 0.05). There was no significant difference in the methylation of the remaining CpG sites between the groups (P > 0.05). Table 4, Fig. 3c.
4.3.4. ROCK2 gene methylation level in experimental rats and the effect of drugs

231 0.01300 ± 0.00311 0.00753 ± 0.00327 0.02686
3d 112 0.00985 ± 0.00113 0.00774 ± 0.00077 0.00874
152 0.01559 ± 0.00226 0.01120 ± 0.00287 0.02762
162 0.00182 ± 0.00407 0.01325 ± 0.01200 0.07828
7d 149 0.00093 ± 0.00207 0.01024 ± 0.00494 0.00459
217 0.00465 ± 0.00442 0.01111 ± 0.00388 0.03954
time locus group P-value
model normal

1d 98 0.01015 ± 0.00238 0.01318 ± 0.00214 0.01534
3d 43 0.03549 ± 0.01629 0.01890 ± 0.00693 0.09146
45 0.04458 ± 0.02277 0.01779 ± 0.00849 0.06182
121 0.01740 ± 0.00183 0.01476 ± 0.00290 0.07450
146 0.01794 ± 0.00572 0.00826 ± 0.00764 0.01870
166 0.00781 ± 0.00559 0.01910 ± 0.01776 0.04520
192 0.04534 ± 0.00488 0.03953 ± 0.00429 0.02039
231 0.00566 ± 0.00675 0.01791 ± 0.01206 0.04678

The ROCK2 gene methylation levels in the normal, model and NLXT groups were compared. Compared with the normal group, the model group had CpG29, CpG42, CpG50, CpG69, CpG97, CpG106, CpG114
and CpG118 methylation levels. The difference was statistically signif- icant (P < 0.05). In comparison with the model group, the difference in the above-mentioned sites in the two groups was statistically significant (P < 0.05). There was no significant difference in the methylation of the remaining CpG sites between the groups (P > 0.05). Table 5, Fig. 3d.

4.4. Effects of NLXT on NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rats with IS
4.4.1. Changes of NogoA gene levels in rats in each group and the effects of drugs

238 0.00515 ± 0.00777 0.01562 ± 0.00757 0.01586
7d 81 0.02011 ± 0.01057 0.00897 ± 0.00721 0.01562
149 0.00093 ± 0.00207 0.00821 ± 0.00876 0.00842

Compared with the normal group, NogoA gene expression in the model group increased obviously (P < 0.01). In comparison with the 166 0.00675 ± 0.00655 0.01910 ± 0.01776 0.15162 model group, NogoA gene expression in the blocker group II reduced Table 4 Methylation of NgR in each group rats(n = 5). gene time locus group P-value model NLXT NgR 1D 77 0.01614 ± 0.00197 0.01079 ± 0.00303 0.01080 3D 39 0.01092 ± 0.00145 0.00838 ± 0.00128 0.01872 69 0.01702 ± 0.00254 0.01370 ± 0.00105 0.02702 144 0.02353 ± 0.00225 0.01816 ± 0.00411 0.03354 Table 5 Methylation of ROCK2 in each group rats(n = 5). gene time locus group P-value model NLXT ROCK2 3d 50 0.01198 ± 0.01259 0.03980 ± 0.01645 0.01698 7d 97 0.00781 ± 0.00444 0.03673 ± 0.02211 0.04558 time locus group P-value model normal 161 0.01774 ± 0.00201 0.01451 ± 0.00196 0.03248 7D 175 0.02753 ± 0.00838 0.01826 ± 0.00268 0.07796 1d 69 0.02056 ± 0.01250 0.00991 ± 0.00794 0.03706 175 0.01780 ± 0.00654 0.03446 ± 0.01522 0.05466 time locus group P-value model normal 3D 39 0.01092 0.00145 0.00817 0.00115 0.00038 106 0.00584 ± 0.00445 0.01615 ± 0.01559 0.03464 3d 114 0.07053 ± 0.04430 0.03695 ± 0.02481 0.04544 118 0.08017 ± 0.04534 0.04389 ± 0.02511 0.03482 42 0.05886 ± 0.02103 0.03535 ± 0.01979 0.03584 83 0.01711 ± 0.00350 0.01372 ± 0.00193 0.01268 160 0.01612 ± 0.00182 0.01911 ± 0.00341 0.08054 161 0.01774 ± 0.00201 0.01488 ± 0.00276 0.04768 172 0.02448 ± 0.00232 0.01968 ± 0.00291 0.00372 175 0.02753 ± 0.00838 0.02040 ± 0.00333 0.13722 7D 38 0.01027 ± 0.00377 0.00653 ± 0.00227 0.01458 83 0.01596 ± 0.00142 0.01372 ± 0.00193 0.02924 98 0.01256 ± 0.00135 0.01010 ± 0.00220 0.03146 122 0.01445 ± 0.00214 0.01197 ± 0.00213 0.03784 Fig. 3c. Methylation PCA plots of RhoA in various groups of rats. MX means model group; YW means NLXT group; ZC means normal group. The different NgR gene DNA methylation groups in the normal, model and NLXT groups were analyzed. Compared with the normal group, the methylation levels of CpG38, CpG77, CpG122, CpG144, CpG160, CpG172 and CpG175 in the model group differed prominently (P < 0.05). In comparison with the model group, the difference in the above-mentioned sites in the NLXT group was statistically significant (P < 0.05). There was no significant difference in the methylation of the remaining CpG sites between the groups (P > 0.05).

remarkably on the 1st day (P < 0.01). After 7 days of observation, NogoA gene expression decreased obviously in the other groups in time- dependent manner (P < 0.01). Fig. 4. 4.4.2. RhoA gene levels and drug effects in each group Similarly, on days 1st, 3rd and 7th, RhoA gene expression in the model group was remarkably higher than that in the normal group (P < 0.01). Compared with the model group, RhoA gene expression in the 69 0.02703 ± 0.01605 0.00991 ± 0.00794 0.08362 97 0.00781 ± 0.00444 0.02629 ± 0.01602 0.00088 Fig. 3d. Methylation PCA plots of ROCK2 in various groups of rats. MX means model group; YW means NLXT group; ZC means normal group. The ROCK2 gene methylation levels in the normal, model and NLXT groups were compared. Compared with the normal group, the model group had CpG29, CpG42, CpG50, CpG69, CpG97, CpG106, CpG114 and CpG118 methylation levels. The differ- ence was statistically significant (P < 0.05). In comparison with the model group, the difference in the above-mentioned sites in the two groups was sta- tistically significant (P < 0.05). There was no significant difference in the methylation of the remaining CpG sites between the groups (P > 0.05).

other groups was prominently lower (P < 0.01). Fig. 5. 4.4.3. NgR1 gene levels and drug effects in rats Compared with the normal group, NgR1 gene expression in the model group increased significantly (P < 0.01). In comparison with the model group, NgR1 gene expression in blocker group II remarkably declined on the 1st day (P < 0.01). On the 3rd day, NgR1 gene expres- sion was prominently lower in the other groups compared with day 7 (P < 0.01). Fig. 6. 4.4.4. NgR2 gene levels and drug effects in rats in each group Compared with the normal group, NgR2 gene expression in the model group increased significantly (P < 0.01). In comparison with the Fig. 4. Comparison of levels of mRNA expression of NogoA in hippocampal of rats of all groups. Compared with the normal group, NogoA gene expression in the model group obviously increased (P < 0.01). In comparison with the model group, NogoA gene expression in the blocker group II reduced remarkably on the 1st day (P < 0.01). After 7 days of observation, NogoA gene expression decreased obviously in the other groups in time-dependent manner (P < 0.01). Compared with the normal group, △△P < 0.01; compared with the model group, **P < 0.01. Fig. 5. Comparison of levels of mRNA expression of RhoA in hippocampal of rats of all groups. On days 1st, 3rd and 7th, RhoA gene expression in the model group was remarkably higher than that in the normal group (P < 0.01). Compared with the model group, RhoA gene expression in the other groups was prominently lower (P < 0.01). Compared with the normal group, △△P < 0.01; compared with the model group, **P < 0.01. model group, NgR2 gene expression decreased observably on the 3rd and 7th days (P < 0.01). Fig. 7. 4.4.5. ROCK2 gene levels and drug effects in each group Compared with the normal group, ROCK2 gene expression in the model group increased remarkably (P < 0.01). In comparison with the model group, ROCK2 gene expression in the other groups declined significantly (P < 0.01). Fig. 8. 4.5. Effects of NLXT on NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins in brain tissue of rats with IS 4.5.1. NogoA protein expression and drug effects in rats of each group Compared with the normal group, NogoA protein expression in the model group increased obviously (P < 0.01); In comparison with the model group, its expression decreased significantly in the other groups (P < 0.01). Fig. 9A. 4.5.2. RhoA protein expression and drug effects in rats Compared with the normal group, RhoA protein expression in the Fig. 6. Comparison of levels of mRNA expression of NgR1 in hippocampal of rats of all groups. Compared with the normal group, NgR1 gene expression in the model group increased significantly (P < 0.01). In comparison with the model group, NgR1 gene expression in blocker group II remarkably declined on the 1st day (P < 0.01). On the 3rd day, NgR1 gene expression was prominently lower in the other groups compared with day 7 (P < 0.01). Compared with the normal group, △△P < 0.01; compared with the model group, **P < 0.01. Fig. 7. Comparison of levels of mRNA expression of NgR2 in hippocampal of rats of all groups. Compared with the normal group, NgR2 gene expression in the model group increased significantly (P < 0.01). In comparison with the model group, NgR2 gene expression decreased observably on the 3rd and 7th days (P < 0.01). Compared with the normal group, △△P < 0.01; compared with the model group, **P < 0.01. Fig. 8. Comparison of levels of mRNA expression of ROCK2 in hippocampal of rats of all groups. Compared with the normal group, ROCK2 gene expression in the model group increased remarkably (P < 0.01). In comparison with the model group, ROCK2 gene expression in the other groups declined significantly (P < 0.01). Compared with the normal group, △△P < 0.01; compared with the model group, **P < 0.01. Fig. 9. Comparison of levels of expression of NogoA, RhoA, NgR1, NgR2 and ROCK2 in hippocampal of rats of all groups. Western blotting detection was used to examine NogoA, RhoA, NgR1, NgR2 and ROCK2 expression. Number 1 represents the normal group. Number 2 represents the model group. Number 3 represents NLXT group. Number 4 represents blocker I group, and number 5 represents blocker II groups. A-E are the expression of NogoA, RhoA, NgR1, NgR2 and ROCK2 respectively. Compared with the normal group, △△P < 0.01; compared with the model group, **P < 0.01. A. Comparison of levels of protein expression of NogoA in hippocampal of rats of all groups. Compared with the normal group, NogoA protein expression in the model group increased obviously (P < 0.01); In comparison with the model group, its expression decreased significantly in the other groups (P < 0.01) B. Comparison of levels of protein expression of RhoA in hippocampal of rats of all groups. Compared with the normal group, RhoA protein expression in the model group increased observably (P < 0.01). In comparison with the model group, its expression declined prominently in the other groups (P < 0.01) C. Comparison of levels of protein expression of NgR1 in hippocampal of rats of all groups. Compared with the normal group, NgR1 protein expression in the model group increased significantly (P < 0.01); in comparison with the model group, its expression reduced remarkably in the other groups (P < 0.01) D. Comparison of levels of protein expression of NgR2 in hippocampal of rats of all groups. Compared with the normal group, NgR2 protein expression in the model group increased obviously (P < 0.01); in comparison with the model group, its expression decreased prominently in the other groups (P < 0.01) E. Comparison of levels of protein expression of ROCK2 in hippocampal of rats of all groups. Compared with the normal group, ROCK2 protein expression in the model group increased observably (P < 0.01). In comparison with the model group, its expression declined significantly in the other groups (P < 0.01). Fig. 9. (continued). model group increased observably (P < 0.01). In comparison with the model group, its expression declined prominently in the other groups (P < 0.01). Fig. 9B. 4.5.3. NgR1 protein expression and drug effects in rats Compared with the normal group, NgR1 protein expression in the model group increased significantly (P < 0.01); in comparison with the model group, its expression reduced remarkably in the other groups (P < 0.01). Fig. 9C. 4.5.4. NgR2 protein expression and drug effects in rats Compared with the normal group, NgR2 protein expression in the model group increased obviously (P < 0.01); in comparison with the model group, its expression decreased prominently in the other groups (P < 0.01). Fig. 9D. 4.5.5. ROCK2 protein expression and drug effects in each group Compared with the normal group, ROCK2 protein expression in the model group increased observably (P < 0.01). In comparison with the model group, its expression declined significantly in the other groups (P < 0.01). Fig. 9E. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expressions in the model group increased remarkably (P < 0.01) at different time points from day 1 to day 7; However, in comparison with the model group, their expressions reduced greatly in the other groups (P < 0.01). Fig. 9A–E. 5. Discussion Although stroke mortality has declined in recent decades, it remains the leading cause of disability worldwide (Lee et al., 2019). Xin’an Medicine has always been focusing on the treatment of IS, using Qi to invigorate Blood to provide a good environment for Qi and Blood infiltration in the brain. NLXT prescription is a clinical prescription for the treatment of stroke issued by Mr. Wang Letao, who was the 5th generation heritage of Xin’an Wang’s Medicine. On the one hand, the vitality of Qi can generate new blood, so that there is a source of transformation, treating the principal aspect. On the other hand, because Qi moves, Blood moves, eliminating blood stasis in the veins and treating the secondary symptoms. As previous discoveries had shown, NLXT appeared effective for patients in both the acute and re- covery phases of IS (Chen et al., 2018; Yang et al., 2013). Firstly, rCBF can directly determine whether ischemia exists and indirectly reflects the degree of neurological deficit (Nakazaki et al., 2017). In this paper, laser Doppler was used to monitor rCBF to reflect local cerebral blood flow in real-time and intuitively. The experimental results showed that after reperfusion of the rat MCAO model, the cere- bral blood flow decreased prominently (P < 0.01 or P < 0.05) [Fig. 2], which proved that the model was successful. After drug intervention, model rats in the NLXT group, blocker group I, and blocker group II all showed improved neurological function. With longer treatment times, the recovery of rCBF in the model rats was increasingly clear. The effect of blocker II was better than that of NLXT. The blocker II and NLXT groups showed superior results to blocker I, which was consistent with other indexes. NLXT can restore rat rCBF to different degrees and exert neurovascular homeostasis remodeling, which provides a good basic environment for the recovery of nerve regeneration and damaged nerve function after IS. An obvious and interesting phenomenon that the efficacy of some blockers in treating IS is better than that of TCM has been observed. In this paper, we found that the effect of blocker II was better than that of NLXT in treating IS. Does it necessarily mean that TCM has no efficacy advantages in treating IS? In fact, TCM has an excellent therapeutic performance in the treating process of IS for TCM has the multi-target, multi-pathway characteristics. For instance, TuoXinluo has been exten- sively used to treat IS (Chang et al., 2020; Yu et al., 2018); Chen et al. reported that Buyanghuanwu Decoction exhibits protective effects against cerebral I/R injury in MCAO rat, demonstrated by relieving the cerebral edema and improving neurological function score (Chen et al., 2019). And NLXT, particularly, promotes the differentiation of NSCs into neurons by inhibiting Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (Han and Wang, 2017) to inhibit neuronal apoptosis and activates Wnt pathway (Tan et al., 2020). With regard to stroke recovery, potential therapeutic targets such as 5′-AMP-activated protein kinase (AMPK) (Jiang et al., 2018), the mitogen-activated protein kinase (MAPK) (Sun and Nan, 2016), Nogo-A (Kumar and Moon, 2013), ROCK (Sladojevic et al., 2017) as well as their blockers have been widely explored. The selective inhibitors have an excellent performance in interdicting targets as well as pathways so to produce the anti-IS effect, however, whether they have further thera- peutic benefit remains to be verified. In this paper, only one signaling pathway was studied. In terms of the degree of pathway inhibition, the data of some targets such as RhoA [Fig. 7] as well as ROCK2 [Fig. 8] showed that the blocker I (NLXT SGI-1027) and blocker II (NLXT Y27632) group had a little better effect than the NLXT group, but whether the blockers all have such as the recovery of neurological function have not been confirmed. Then, DNA methylation plays a pivotal role in the pathogenesis of neurodegenerative diseases and cognitive impairment (Hu et al., 2012; Xu, 2015), its process is a covalent bond change of a latent sequence of nucleic acid molecules (Hu et al., 2015), which can participate in gene transcription and regulate the development and differentiation of the body. DNA methylation can regulate the proliferation of NSCs and their differentiation into neurons and glial cells (Al-Mahdawi et al., 2014; Tao et al., 2018; Wang et al., 2014), which affects neural plasticity and synaptic connections (Halder et al., 2016; Huat et al., 2015; Sagarkar et al., 2017; Wang et al., 2013). CpG-binding structural domain proteins can regulate the expression of related factors, which in turn facilitate the regeneration of NSCs (Hu et al., 2012). DNMT3a and DNMT3b mainly mediate re-methylation, while DNMT1 mediates maintenance methyl- ation reaction, maintaining the methylation state of CpG island through DNA replication and providing a stable environment for DNA methyl- ation of maternal cells (O’Doherty et al., 2015). DNA methylation generally occurs at the position of dinucleotide of CpG island. CpG located in the promoter region of genes appears highly conserved in humans and rodents, and under normal conditions exists mostly non-methylated, and CpG islands are usually located near the tran- scriptional regulatory region and are associated with 56% of the coding genes in the human genome (Blackledge and Klose, 2011; Illingworth et al., 2010). Therefore, in the study of neurological function recovery after IS, it is very important to study the mechanism of DNA methylation regulating neural stem cell differentiation (Hartley et al., 2013). Results of Western blotting detection showed that there was no significant change in DNA methylation levels on day 1 in the model or drug groups [Tables 2–5] (P < 0.01). On days 3rd and 7th, compared with the normal group, the DNA methylation level of each gene in the model group was significantly higher at different sites [Tables 2–5] (P < 0.01). In com- parison with the model group, the DNA methylation levels of each gene in the NLXT group were remarkably lower at different sites [Tables 2–5] (P < 0.01). There was no significant difference between the normal group and the model group (P > 0.05). PCA statistical graph showed that the normal group and the model group were in the same quadrant
[Fig. 3a, 3b, 3c and 3d]. On this basis, we selected DNMT blocker SGI-1027 as blocker I to explore how NLXT promotes neural stem cell regeneration in rats with IS.
As is well-known, after IS, neural stem cell’s ability to regenerate
neurons and axons of the CNS is limited because of inhibition of NogoA and its receptor NgR and downstream RhoA (Kan et al., 2017). The RhoA/ROCK signaling pathway seems an important signaling pathway that regulates neuronal regeneration and keeps involved in cytoskeletal

reorganization, cell migration and stress fiber formation (Galindo et al., 2018; Hemphill et al., 2015; Park et al., 2018). What’s more, ROCK appears a downstream target effector molecule of Rho, which has many
functions including regulating cell contraction and proliferation (Amano et al., 2010). In nerve injury, Rho activation induces atrophy of growth cones, which leads to axonal regeneration disorder, this induces chon- droitin sulfate proteoglycan inhibition on neuronal growth (Forgione and Fehlings, 2014; Mulherkar et al., 2017). This study showed that the NogoA/RhoA/ROCK pathway-related indicators NogoA [Fig. 4], RhoA [Fig. 5], NgR1 [Fig. 6], NgR2 [Fig. 7], and ROCK2 [Fig. 8] mRNA levels
and protein levels [Fig. 9A–E] were observably increased in the model group compared with the normal group (P < 0.01). In comparison with the model group, the levels of NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA and protein in the NLXT group, the blocker group I and the blocker group II were reduced. This indicates that after IS, NLXT can facilitate NSCs regeneration by reducing the inhibition of neuronal growth by the NogoA/RhoA/ROCK pathway and exert neuroprotective effects. 6. Conclusion NLXT can reduce the DNA methylation level of the NogoA pathway of MCAO rats, thus inhibit the expression of the NogoA/RhoA/ROCK pathway to treat IS. It is through the inhibition of pathway by NLXT that has played a therapeutic role. Consequently, based on this study, our extension researches of the mechanism of NLXT on more pathways will be conducted to fulfill its pharmacologic actions. Its mechanism in treating IS in vitro as well as in vivo will be further investigated. Authors’ contributions Ling He and Weidong Chen conceived as well as designed this study; Lu Hong, Xiaoqian Shi and Guodong Zhao performed the experiments; Lu Hong and Mingming Liu drafted the manuscript; Lei Wang, Daiyin Peng as well as Huihui Jiang modified the manuscript; Guodong Zhao did data processing; and Hui Tan assisted in performing the experiments. All authors have read this manuscript and would like to have it considered exclusively for publication in Journal of ethno- pharmacology. None of the material related to this manuscript has been published or is under consideration for publication elsewhere, including the Internet. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the Key Programs of Natural Science Research in Colleges of Anhui Province (KJ2019A0452). Appendix A. 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