Clinically used ZLHXTY capsules were obtained from the pharmacy department of the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, China. Standard compounds for HPLC quality control analysis including L-epicatechin, calycosin-7-O-β-glucoside, coumarin, ononin, calycosin, cinnamaldehyde, formononetin, and wogonin were purchased from Beijing Saibaicao Technology Co., Ltd. (Beijing, China). All the solvents such as ethanol, acetonitrile, formic acid and ammonium formate were of HPLC-grade and were purchased from Thermo Fisher Scientific (Massachusetts, USA). Hematoxylin & Eosin staining kit was purchased from Beyotime biotechnology, Shanghai, China (Cat. No. C01015-1). Nissl staining kit was purchased from Solarbio, Beijing, China (Cat. No. G1430). The list of primary antibodies used in this study are given in Table 2. Fluorescently labeled secondary antibodies for western blot i.e., goat anti-mouse IgG Alexa Fluor 790 (A11357), goat anti-rabbit IgG Alexa Fluor 680 (A21109); and for immunofluorescence i.e., goat anti-rabbit IgG Alexa Fluor 555 (A21429), goat anti-mouse IgG Alexa Fluor 488 (A11001) were purchased from Invitrogen Life technologies, United States.

For the ultra-high performance liquid chromatography coupled high resolution mass spectrometry (UPLC-HRMS) based chemical characterization of ZLHXTY capsule, firstly, the dried whole bodies of Hirudo nipponica Whitman and twigs of Cinnamomum cassia (L.) J. Presl (or Neolitsea cassia (L.) Kosterm.) were grinded together into a fine powder. The rest of the herbs, Pheretima aspergillum (E. Perrier), Astragalus membranaceus Fisch. ex Bunge (or Astragalus mongholicus Bunge), Sargentodoxa cuneata (Oliv.) Rehder and E.H.Wilson (or Holboellia cuneata Oliv.), were soaked in water to make decoction concentrate. Followed by drying, the herbs were grinded to make into the fine powder. The two powders were mixed together to prepare ZLHXTY mixture. Later, the ZLHXTY powder was extracted in ethanol (1:8 volume ratio) under sonication for 30 min to obtain the ethanolic extract or supernatant A. Then, the water was added in the filter residue (1:10 volume ratio) under sonication for 30 min to obtain the water extract or supernatant B. Finally, the two supernatants A and B were combined (1:1 volume ratio) together, concentrated by rotary evaporation and freeze vacuum drying to obtain extracts of ZLHXTY capsule for subsequent UPLC-HR-MS analysis.

The eight standard compounds L-epicatechin, calycosin-7-O-β-glucoside, coumarin, ononin, calycosin, cinnamaldehyde, formononetin, and wogonin, were firstly extracted with ethanol followed by high speed centrifugation to collect the supernatant that was later used for standard validation of ZLHXTY capsule under UPLC-HRMS technique.

The chemical characterization analysis of the ZLHXTY capsule extract was operated on Ultimate 3000 hyperbaric LC system coupled with high resolution Q-Exactive mass spectrometer via an electrospray ionization (ESI) interface (Thermo Fisher Scientific, Bremen, Germany), using BEH C18 column (1.7 μm, 2.1 mm ID × 100 mm, Waters) maintained at 35°C. Following are the optimized chromatographic parameters of our study: mobile phase was composed of water (0.1% formic acid, A) mixed in gradient mode with acetonitrile (0.1% formic acid, B), at a flow rate of 200 μL/min. The elution gradient was optimized as follows: 0–5 min, 2% B; 5–8 min, 2–20% B; 8–45 min, 20–55% B; 45–52 min, 55–100% B; 52–58 min, 100% B. The injection volume was 2.0 μL and the sampler was set at 4°C.

Positive full scan modes within the range of m/z (mass/charge ratio) 150-1500 at a resolution of 70,000 were used for acquisition of accurate molecular ion. The other parameters were set as follows: spray voltage, +3.5 kV; sheath gas flow rate, 35 arb; aux gas flow rate, 10 arb; capillary temperature, 320°C; vaporizer temperature, 250°C; RF lens, 50%. Xcalibur 3.0 software (Thermo Fisher) was used for UPLC-HRMS control and data handling.

The male C57BL/6 J mice (20–22 g body weight, 7–8 weeks old) were raised in regular and clean cages under maintained conditions of temperature at 22 ± 0.5°C, humidity 55 ± 5%, with 12-h alternate light-dark cycles. All animals were allowed free access to standard animal chow and water. The study was performed according to the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Research Committee of Southwest Medical University, Luzhou, China.

ICH was induced by infusion of autologous blood. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg. Anesthetized mice were placed in stereotaxic frame with the head being carefully and firmly fixed in the apparatus. A small cut is made in the skin of the head followed by application of 30% H₂O₂ on the surface of skull to remove the periosteum and clarify the skull joints. A 1-mm burr hole was drilled at co-ordinates of 2 mm lateral and 0.2 mm anterior to the bregma in the right striatum of mice brain. A volume of 25 μL of autologous blood was collected in the Hamilton syringe from a tail cut and then injected in the brain through that burr hole with a needle insertion depth of 3 mm. Blood infusion flow was maintained at a rate of 5 μL/min. After completion of blood infusion, the needle was kept in place for 5 min to prevent backflow of blood, and after then the needle was withdrawn slowly and carefully. Then skin was sutured in aseptic conditions and the animals were allowed to recover and regain consciousness in a warm environment maintained at 37°C.

The animals were randomly divided into five groups (n = 18); 1) normal control, 2) ICH model group; and treatment groups 3) Low dose ZLHXTY-LD (0.35 g/kg) 4) Medium dose ZLHXTY-MD (0.7 g/kg), and 5) High dose ZLHXTY-HD (1.4 g/kg). The first ZLHXTY dose was administered orally within 2 h of ICH induction after mice regain consciousness, and continued as once daily dosing for 3 days. The mice in normal control group and ICH group received the same volume of normal saline orally. At 24 and 72 hrs the mice were killed to assess the neuroprotective effect of ZLHXTY at different time points. We observed the dose dependent effect of ZLHXTY capsules treatment in our experiments. The data for neurological scoring represent the effect of all three doses of ZLHXTY capsules treatment. For the subsequent experiments, the data presented here used the highest effective dose of ZLHXTY-HD 1.4 g/kg.

Behavioral tests were carried out 2 days before and after 6, 24, 48, and 72 h of ICH induction and the average score was obtained by observations form two independent observers, blinded to the experimental design. The tests included 28 point neurological scoring test (Clark et al., 1997), balance beam test (Feeney et al., 1982; Liu et al., 2020) and corner turn test (Krafft et al., 2014).

After completion of respective dosing regimen, the mice were terminally anesthetized with overdose of sodium pentobarbital then transcardially perfused with phosphate buffered saline followed by 4% paraformaldehyde. Later, the mice were decapitated and brains were collected and processed accordingly for histology and immunofluorescence studies. For qPCR and western blot experiments, the transcardial perfusion with 4% paraformaldehyde was omitted and the brain tissues were collected in RNase free eppendorfs and snap-frozen in liquid nitrogen and stored in −80°C for later use.

As described earlier, the perfusion fixed brain tissues were further underwent immersion fixation in 4% formaldehyde overnight. Then, the tissues were dehydrated in a series of graded alcohols 50, 60, 70, 80, 90, 100% and then in xylene for 30 min each. Later, the brain tissues were paraffin embedded and paraffin sections of 4 µm thickness at coronal plane were cut using microtome (Leica RM 2245, Wetzlar, Germany). For staining, the tissue slides were rehydrated using xylene, 100% alcohol, 90, 80, 70, 60, 50% alcohol, and then water. HE staining was carried out according to the standard procedure. Similarly, nissl staining was performed according to manufacturer’s instructions. The slides were observed under Leica DM500 microscope equipped with Leica ICC50W camera and images were captured using software Leica application suite X, at magnification 400 x .

The qRT-PCR was used to analyse the mRNA levels of NFкβ-P50, TNFα, IL1β, IL6, COX2 and iNOS at 24 and 72 h after ICH (n = 9 per group per time-point). Total RNA was extracted from hemorrhagic cortex using the Trizol reagent (Beyotime Biotechnology, China, Cat# R0016); reverse-transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co.,Ltd., China, Cat. No.R323-01) and qRT-PCR was performed on LightCycler^® 480 Instrument II (Roche, USA) in the presence of a fluorescent dye ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd., China, Cat. No.Q711-02/03). Absorbance was read at 260 and 280 nm using an UV spectrophotometer and the RNA samples with an OD260/OD280 value >1.8 were only considered appropriate for use. The mRNA level was normalized to the GAPDH and was calculated by the 2^−ΔΔCt method. The primer sequences are given in table 5.

Brain samples were homogenized with RIPA buffer containing 1 mM PMSF on ice. After centrifugation, the supernatants were collected and protein concentrations were measured using Bradford Coomassie brilliant blue method. From each sample, 40 µg protein was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skimmed milk for 1 h, the membranes were incubated with various primary antibodies mentioned in table 2, overnight at 4°C. Next day, the blots were washed with TBST 5 min x3 and then incubated with near infrared fluorescently labeled secondary antibodies at concentration 1:10,000, at room temperature for 1 h. Then, membranes were washed with TBST 5 min x3. Fluorescent detection was performed using the Amersham Typhoon™ laser scanner (Cytiva, USA). Protein levels were normalized with respect to GAPDH and quantified using the ImageJ software (NIH, Bethesda, MA, USA).

Following transcardial perfusion with PBS and 4% paraformaldehyde, the brain tissues were carefully removed out and kept in 30% sucrose solution overnight a 4°C. After the tissues were sank down in the sucrose solution, the brains were cryopreserved in Tissue-Tek^® O.C.T.medium (Sakura, Japan) and snap frozen in liquid nitrogen. Cryosections of 7 µm thickness at coronal plane were cut using a freezing microtome (Leica CM 1950; Wetzlar, Germany) and taken onto slides followed by 20 min perfusion with 0.3% Triton X-100 at room temperature. Later, the sections were washed with PBS 5 min x3 with subsequent blocking in 5% BSA for 1 h at room temperature. After blocking, the sections were incubated in a mixture of primary antibodies i.e., NeuN + TNFα, NeuN + NFкB, Iba1+TNFα and Iba1+NFкB, at a dilution of 1:1000 at 4°C overnight. Next day, the sections were washed with PBS 5 min x3 and then incubated in respective secondary antibody dilutions (1:1000) in dark for 1 h at room temperature. Next, the sections were incubated with DAPI for 10 min followed by washing in PBS 5 min x3 and coverslip mounting in glycerol. The slides were kept in dark and observed under Leica DM4 B fluorescence microcope equipped with Leica DMC6200 camera. The images were captured using Leica application suite X software at magnification 400x.

The data analysis was performed using two-way analysis of variance (ANOVA), followed by Tukey’s post-hoc tests using GraphPad Prism Version 8.0.1 software (GraphPad Software Inc., CA, USA). A value of p < 0.05 was considered statistically significant. Data are shown as the mean ± SD. All the experiments were repeated at least three independent times.

After optimization of chromatographic conditions, the UPLC-HRMS analysis for quality control of ZLHXTY capsules was conducted to obtain the chromatogram (Figure 1A). Eight standard compounds were confirmed in ZLHXTY capsules, including L-epicatechin, calycosin-7-O-β-glucoside, coumarin, ononin, calycosin, cinnamaldehyde, formononetin, and wogonin (Figure 1B). The analytical details of those identified compounds are summarized in table 6.

To investigate the neuroprotective role of ZLHXTY after ICH, we determined the neurological deficit score, corner turn test and balance beam test with neurological scoring, starting from 3 hrs of ICH induction followed by 24, 48 and 72 h observation time points. Modified 28 point neurological deficit scores are shown in Figure 2A. All the mice in normal control group were healthy with no obvious neuromotor dysfunction, while ICH group had demonstrated severe and significant (*p < 0.05) neurological impairment at all time points after ICH induction. However, ZLHXTY treated group had shown gradual improvement and significant (**p < 0.05) recovery.

We also determined the occurrence and severity of ICH damage by corner turn test. Since we induced the ICH in the right basal ganglia, motor and sensory functions of the left side of the body were paralyzed and affected, causing significant (*p < 0.05) increase in the frequency of right turns in ICH group (almost 100%) as compared with the normal group. However, the ZLHXTY treatment groups demonstrated gradual, progressive and significant recovery (**p < 0.05) than ICH group. The baseline of the corner turn test result from normal group was about 50%, as the probability of left or right turn was basically equal (Figure 2B).

Further, we also performed the balance beam test using a rating scale to measure the motor co-ordination and balance. The mice in ICH group showed increased fear and inability to move after 3 and 24 h. However, after 48 and 72 hrs, the animals in ICH group demonstrated slight ability to walk on the beam with severe difficulty and showed frequent paw slips as compared to the normal control (Figure 2C). The ZLHXTY treated mice showed increased fear, frequent paw slips and severe difficulty in crossing the beam until 24 h, however, the performance was improved significantly (**p < 0.05) after 48 hrs and further progressed after 72 h (Figure 1C). The balance beam motor deficit score is shown in Figure 2D.

Microscopy revealed that the brain sections of mice from normal control group had no pathological changes following HE staining. Neuropil was intact with normal texture; and healthy, nucleated pyramidal neurons were clearly observed (Figure 3A). In 24 h ICH model group, subtle pathologic changes were observed in perihematomal region of brain. The neuropil was found to be less intact as compared to normal brain with signs of vacuolation, parenchymal loss, granulovacuolar neuronal degeneration, neuronal shrinkage, reactive gliosis including relatively excessive number of oligodendrocytes, astrocytes and microglia (Figure 3B) which became more obvious after 72 h (Figure 3D) with evident edematous changes. ZLHXTY-HD capsule treatment for 24 h maintained the normal neuropil architecture with less neuronal degeneration and less reactive gliosis (Figure 3C) observed. These protective changes were more obvious after 72 h of ZLHXTY capsule treatment (Figure 3E) showing that the histopathological damage of brain was reduced after treatment with ZLHXTY capsule.

Nissl staining was also used to identify the ICH induced neuronal injury since the loss of nissl substance indicate the damage to neurons. Figure 3F showed the presence of several blue coloured nissl bodies in pyramidal neurons in normal brain. After 24 h of ICH induction the number of nissl bodies were decreased (Figure 3G), that were further reduced after 72 h of ICH (Figure 3I). ZLHXTY-HD capsule treatment after 24 and 72 h as shown in Figures 3H,J, revealed the increased number of nissl stained neurons as compared to 24 and 72 h ICH model groups, indicating its neuroprotective effect.

The mRNA expression levels of transcription factor, NFкB-P50, and its downstream target genes for inflammatory cytokines such as TNFα, IL6, IL1β, iNOS, and COX2 were assayed after 24 and 72 h of ZLHXTY treatment post-ICH. After 24 h of ICH, the values of all the measured inflammatory cytokines and NFкB were significantly higher than that in the normal control group (*p < 0.05), but significantly reduced with ZLHXTY-HD 24 h treatment (**p < 0.05 as compared to ICH group). After 72 h of ICH, the mRNA levels of NFкB and inflammatory cytokines were even higher than after 24 h of ICH, however, ZLHXTY-HD 72 h treatment significantly reduced the mRNA levels of NFкB and inflammatory cytokines (Figures 4A–F).

We further analysed the protein expressions of NFκB-p65, P-NFκB-p65, IKKβ, P-IKKβ, IкBα, P-IкBα, TNFα, IL1β, IL6, iNOS, and COX2 with western blotting after 24 and 72 h of ICH induction and ZLHXTY treatment. Figure 5A represent all the protein blotting results of our experiment. The total and phorphorylated protein levels of NFκB-p65 and IKKβ were significantly (*p < 0.05) upregulated after 24 h of ICH induction. Complementarily, the expression of total IKβα was reduced with increase in phosphorylated IKβα expression after 24 h of ICH. Concordantly, the protein levels of the inflammatory cytokines TNFα, IL6, IL1β, iNOS, and COX2 were also significantly (*p < 0.05) upregulated as compared to normal control group after 24 h of ICH. All these effects were significantly (*p < 0.05) augmented after 72 h of ICH. ZLHXTY-HD capsules induced significant (**p < 0.05) down-expression of NFκB-p65, P-NFκB-p65, IKKβ, P-IKKβ, TNFα, IL1β, IL6, iNOS and COX2. Whereas, the total protein expression of IKβα was upregulated and phosphorylated IKβα was downregulated after ZLHXTY treatment for 24 and 72 h (**p < 0.05) (Figures 5B–I).

We further examined the expressions of TNFα and NFκB-p65 in neurons and microglia. In the normal control group, the uniformly distributed NeuN staining was observed throughout the brain tissue. The co-expression of TNFα and NeuN was not detectable in normal neurons whereas subtle expression of NFκB-p65 was noticeable in some neurons with very weak fluorescence (Figures 6A–D and Figures 7A–D). In the 24 h ICH group, the number and expression of NeuN was decreased compared to normal control, whereas, the expression of both the TNFα and NFκB-p65 was upregulated (Figures 6E–H and Figures 7E–H). This effect was further increased after 72 h of ICH (Figures 6M–P and Figures 7M–P). Interestingly, we found that positive expression of neuronal TNFα and NFκB-p65 was associated with very weak staining for NeuN. After 24 h of ZLHXTY-HD capsule treatment, the number of NeuN was increased and the fluorescence for TNFα and NFκB-p65 was reduced as compared to ICH 24 h group (Figures 6I–L and Figures 7I–L) which was markedly obvious after 72 h of ZLHXTY-HD capsule treatment (Figures 6Q–T and Figures 7Q–T).

The co-expression of TNFα and NFκB-p65 with Iba1 was also detected (Figures 8, 9). In normal brain sections, Iba1 expression was detected as few, small, compact soma bearing long, thin, ramified processes indicating inactivated form of microglia (Figures 8A–D and Figures 9A–D). After 24 h of ICH, the number of activated Iba1 positive cells were increased (Figures 8E–H and Figures 9E–H) with morphological changes i.e., cellular hypertrophy, membrane ruffling and retraction of processes. The co-expression of TNFα and NFκB-p65 with Iba1 was also increased as shown in Figures 8E–H and Figures 9E–H. These effects were further pronounced after 72 h of ICH (Figures 8M–P and Figures 9M–P). Conversely, after ZLHXTY treatment the number of Iba1 stained cells was controlled and also the expression of TNFα and NFκB-p65 was attenuated after 24 h (Figures 8I–L and Figures 9I–L) and more evident after 72 h (Figures 8Q–T and Figures 9Q–T).