SCLM granules are derived from the classical Sancai decoction and Lianmei decoction with addition and subtraction in the “Detailed Analysis of Epidemic Warm Diseases,” which consists of Renshen (Panax ginseng C. A. Meyer), Tian-dong (Asparagus cochinchinensis (Lour.) Merr.), Dihuang (Rehmannia glutinosa (Gaert.) Libosch.), Wumei (Pru-nus mume (Sieb.) Sieb.), Rougui (Cinnamomum cassia Presl), and Huanglian (Coptis chinensis Franch.). SCLM was provided by the Hospital of Chengdu University of Traditional Chinese Medicine. (each gram of SCLM granules contains 1.475 g of raw drug).

Healthy male Sprague–Dawley rats (n = 60, 6–8 weeks old, weighing 180–220 g) were provided by Beijing HuaFukang Biosciences Co. (INC) (SYXK, 2019–0008, Beijing, China). All animals were maintained under specific pathogen-free conditions in a temperature-controlled room at a constant temperature of 24 ± 1°C with a 12-h light/dark cycle and had free access to a standard rodent diet and water. Following a series of experiments, the rats were anesthetized using an isoflurane inhalation anesthesia machine (3%-4% isoflurane in oxygen) and maintained using continuous mask inhalation (maintenance concentration: 2%). Then, the abdominal aorta was punctured, the circulating blood was drained, and the specimen was collected after perfusion with 4°C saline. All experiments were approved by the Ethical Committee for the Experimental Use of Animals at the Hospital of Chengdu University of Traditional Chinese Medicine (2021SDL-004).

Blood was collected via abdominal aorta, centrifuged (300 ×g, 10 min), and serum stored at −80°C. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr) and albumin (ALB) in the serum were detected via an automatic biochemical analyzer (URIT-8021A, Guilin Youlite Medical Electronic Co., Ltd.).

The brain tissues were fixed with 10% neutral formalin for 48 hours immediately after harvest and then rinsed with water for 30 min. Then, the brain tissues were dehydrated, cleared, penetrated in paraffin in an automatic dehydrator (EXCELSIOR AS, Thermos) and then embedded in paraffin (HISTOSTAR, Thermos). The continuous sliced histological sections were prepared using HM 340E (Thermos) with a thickness of 4 μm. A standard procedure for staining soft tissue with hematoxylin–eosin (H&E) and Nissl staining was performed to demonstrate the general histological structure of the tissues.

The sections were blocked with 10% bovine serum at room temperature for 1 hour and incubated overnight at 4°C with the following primary antibodies: rabbit monoclonal to Iba-1 (ab178846, 1:2000 dilution, Abcam, UK) and rabbit polyclonal to NeuN (ab104224, 1:250 dilution, Abcam, UK). Afterwards, the sections were incubated with horseradish peroxidase (HRP)-labeled secondary antiserum (DAKO EnVisonTM kit) at 37°C for 30 min. Immunoreactivity was identified with 0.05% diaminobenzidine, and the sections were visualized with an inverted fluorescence microscope (#IX83, Olympus, Tokyo, Japan). Three randomly selected microscopic fields were analyzed in the ischemic penumbra at the level of the bregma. The numbers of Iba-1-positive and NeuN-positive cells were calculated via ImageJ software.

PC12 and BV2 lines were purchased from Procell Life Science & Technology Co., Ltd. PC12 cells were incubated in RPMI 1640 medium supplemented with fetal bovine serum (FBS) (10%), and BV2 cells were incubated in DMEM supplemented with FBS (10%) at 37°C in a 5% CO₂ incubator and passaged according to the manufacturer’s instructions. The cells were seeded in plates at a density of ~10⁵ cells mL^-1 and cultured overnight prior to the experiments.

To evaluate the therapeutic effect of SCLM on HG+OGD-induced neuronal injury, PC12 cells were cultured in HG+OGD medium as a model group and incubated in complete RPMI 1640 medium supplemented with 10% normal serum as a control group. The HG+OGD group was treated with low, moderate, or high concentrations of SCLM drug-containing serum for 24 hours. FBS group cells were incubated in complete RPMI 1640 medium with 10% FBS as a negative control.

Flow cytometric analysis was used to determine the extent of PC12 apoptosis induced by HG+OGD and the recovery ability of SCLM. Briefly, PC12 cells (1.5×10⁵/mL) were plated in 12-well plates, treated with SCLM for 4 h after adherence. Next, the cells were incubated with the ROS solution for another 20 min. After being removed from the medium and washed with cold phosphate-buffered saline (PBS), the cells were subsequently stained with annexin V and PI according to the experimental methods of the assay kit (FXP018, 4A Biotech) specification. Apoptosis was analyzed via a Beckman CytoFLEX S flow cytometer. According to the fluorescence intensity, the cell populations were assigned to four quadrants: live (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic (Q1) cells.

Immunoblot detection was carried out using the standard protocol. The antibodies used for immunoblotting were as follows: TLR4/Toll-like receptor 4 rabbit polyclonal antibody (cat.no. A8187, Beyotime), phospho-IκB alpha (Ser32) rabbit monoclonal antibody (cat.no. AF187, Beyotime), phospho-NF-κB p65 (Ser276), rabbit polyclonal antibody (cat.no. AF587, Beyotime), Bax rabbit monoclonal antibody (cat.no. AF1270, Beyotime), Bcl-2 rabbit polyclonal antibody (cat.no. AF6285, Beyotime), IL1B rabbit polyclonal antibody (cat.no. AF720, Beyotime), and anti-p53 (#C0041, Beyotime). β-Actin polyclonal antibody (cat. no. 20536-1-AP; Proteintech, USA), HRP-labeled goat anti-rabbit IgG (H+L) (cat. no. A0208, Beyotime) was used as a reference.

Briefly, PC12 cells (2.5×10⁵/mL) were seeded in 12-well plates, treated, and stained with DCFH-DA (20 min). Meanwhile, in the blank control group, no ROS solution was added, and the other experiments were the same as those in the experimental group. After washing with PBS, the ROS-positive cells were observed and recorded under an inverted fluorescence microscope (#IX83, Olympus, Tokyo, Japan), and the positive cell counts were calculated via ImageJ software.

TUNEL was conducted with a DMK500 in situ apoptosis kit (TAKARA BIO Inc., Shiga, Japan) according to the manufacturer’s instructions. In brief, 3 mm-thick coronal brain sections were immersed in a TdT-labeling reaction mixture for 90 min, followed by DAB staining for nearly 5 min and anti-FITC HRP counterstaining for 30 min at 37°C. TUNEL-positive cells in the ischemic penumbra of a coronal section were identified with an inverted fluorescence microscope (#IX83, Olympus, Tokyo, Japan). The number of TUNEL-positive cells in the ischemic penumbra from three randomly selected fields was determined via ImageJ software. The apoptosis index is expressed as the percentage of TUNEL-positive cells among the total number of cells per field of view.

GraphPad Prism version 8 (GraphPad Software Inc., CA, USA) was used for statistical analysis. To compare two groups, we used a two-tailed Student’s t test. To compare multiple groups, we used one-way analysis of variance (ANOVA). n.s., not significant, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The data are presented as the means ± standard deviations (SDs) if n ≥ 3.

We detected nine serum indicators in rats ( Figures 1e–m ) after MCAO. Compared with the control group, the DM/MCAO group presented significantly lower levels of ALB ( Figure 1i ).

The neurological scores and infarct areas of the stroke rats were analyzed after pretreatment with SCLM for 4 weeks ( Figure 1a ). As shown in Figure 2a , the stroke rats that were administered saline (DM+MCAO group) exhibited a significant area of infarction, which was reflected by the inability to stain with TTC solution, whereas pretreatment with SCLM in stroke rats significantly decreased the brain infarct area, and this effect was concentration dependent ( Figure 2b ). Furthermore, the neurological scores of stroke rats improved after pretreatment with SCLM ( Figure 2f ), and the therapeutic effect was maintained long-term ( Figure 2g ). We also elucidated the therapeutic effects of SCLM as a neuroprotective agent via pathological analysis of brain tissue. First, H&E staining of brain sections from stroke rats revealed that the saline-injected group exhibited a significant decrease in neurons with irregular morphology and disordered arrangement, indicating severe neuronal damage. In contrast, pretreatment with SCLM notably decreased the necrotic area ( Figure 2c ). We subsequently examined neuronal damage in the infarcted area of stroke rats via Nissl staining. As shown in Figure 2d , a noticeable reduction in Nissl bodies was observed in the infarcted area of stroke rats that received saline injection compared with the sham-operated control group. However, after MCAO rats were pretreated with SCLM, the number of Nissl bodies in ischemic brain tissue increased significantly. Additionally, compared with those in the control group, the number of NeuN⁺ cells in the model group was significantly lower, whereas the number of NeuN⁺ cells in SCLM-pretreated stroke rats was greater than that in saline-treated stroke rats in a dose-dependent manner ( Figures 2e,h and Supplementary Figure S1 ).

Oxidative stress plays an important role in the pathogenesis of ischemic stroke. Using both in vivo DM/MCAO rat models and in vitro experimental systems, we demonstrated that SCLM alleviates neuronal apoptosis by inhibiting oxidative stress. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels are important indicators for evaluating oxidative stress damage. Therefore, MDA and SOD levels of brain tissues were detected in this study to evaluate the antioxidant function of SLCM in DM/MCAO rats. Compared with that in the control group, the MAD content increased significantly ( Figure 3b ), whereas the activity of SOD decreased in the DM/MCAO group ( Figure 3c ). Compared with the DM/MCAO group, the SCLM group presented a significantly increased MDA clearance rate and SOD activity. These results indicate that SCLM inhibits lipid peroxidation in the brain tissues of DM/MCAO rats by exerting SOD enzyme-like activity. In addition, immunostaining via terminal deoxynucleotidyl TUNEL in brain sections revealed that the number of TUNEL⁺ cells in SCLM-treated rats was lower than that in saline-treated stroke mice, indicating that SCLM treatment likely contributed to protection against neuronal apoptosis ( Figures 3a, d ).

Oxidative damage, which is triggered by the overproduction of ROS, is critical in contributing to damage in ischemic brain tissues. Therefore, we investigated the effects and mechanisms of SCLM against ROS. The upregulation of proapoptotic p53 and bax and the downregulation of antiapoptotic Bcl-2 are associated with ischemia-induced cell death/apoptosis, whereas the upregulation of Bcl-2 can suppress the generation of ROS. We conducted Western blot studies with brain tissues to assess whether the regulation of the p53, Bax and Bcl-2 proteins is involved in the neuroprotective effect of SCLM in vivo. As shown in Figure 3e , in ischemic penumbra brain tissues, compared with the control group, the DM+MCAO group presented increased expression of proapoptotic p53 and Bax and decreased expression of antiapoptotic Bcl-2. Conversely, SCLM pretreatment explicitly downregulated the expression of proapoptotic p53 and bax proteins and upregulated the expression of antiapoptotic Bcl-2 proteins ( Figures 3f–h ).

PC12 cells have been used as a common in vitro neuronal model to explore the neuroprotective effects of novel pharmaceuticals (31). OGD has been extensively used as an available method to induce a hypoxic microenvironment and subsequently overproduce ROS in different cell lines and has been deemed a common in vitro model for exploring the mechanism of antioxidants against ischemia-associated disorders (32).

First, a CCK-8 assay was used to investigate the toxicity of SCLM to PC12 cells. ( Supplementary Figure S2 ). Different concentrations of glucose were subsequently used to establish HG models, and mannitol (at the same concentration as HG) was used to eliminate the effect of osmotic pressure on the cells ( Supplementary Figure S3 ). Finally, the duration of hypoxia was determined. We found that additional glucose did not significantly affect cell viability, whereas 3 hours of hypoxia resulted in a significant increase in cell viability ( Supplementary Figure S4 ). Therefore, this condition (HG: 50 mmol/L, OGD: 3 h) was used in the subsequent experiments.

The free radical scavenging ability of SCLMs in PC12 cells was first measured by DCFH-DA. PC12 cells treated with HG+OGD were used as a free radical positive control. As shown in Figure 4a , compared with those in the HG+OGD group, the free radical levels in the SCLM groups significantly decreased in a dose-dependent manner. Furthermore, we used flow cytometry to verify the fluorescence intensity of the ROS levels in PC12 cells after treatment with SCLM ( Figure 4b ).

ROS play important roles in the induction of apoptosis. Therefore, we performed an Annexin V and propidium iodide costaining assay to examine the reversal of ROS-induced apoptosis by SCLM. Similarly, SCLM decreased apoptosis in HG+ODG-stimulated PC12 cells in a dose-dependent manner ( Figure 4c ).

We also performed Western blot analyses on PC12 cells to investigate whether the regulation of the p53, Bax and Bcl-2 proteins contributes to the neuroprotective effects of SCLM in vitro. As shown in Figures 4d–g , PC12 cells subjected to high glucose and hypoxic conditions presented elevated levels of the proapoptotic proteins p53 and Bax, which are long-term proteins that reduce the level of the antiapoptotic protein Bcl-2, compared with those in cells not exposed to HG+OGD. In contrast, pretreatment with SCLM significantly downregulated the expression of the proapoptotic proteins p53 and Bax while increasing the expression of the anti-apoptotic protein Bcl-2.

These findings indicate that SCLM can exert antiapoptotic effects to protect cells from ischemia-induced injury by regulating the expression of anti- and proapoptotic proteins.

The inflammatory response is recognized as another crucial contributor to cell death after ischemic stroke. Immune cells in brain, microglia, can be dramatically activated following ischemia, which leads to secretion of inflammatory mediators and further contributes to the invasion of peripheral inflammatory cells (33). These, in turn, can further exacerbate neuronal injury in the cerebral ischemic penumbra and exaggerate damage to the surrounding regions. As observed in this study, DM+MCAO markedly increased the number of Iba-1-positive cells in the ischemic penumbra ( Figures 5a–c ). However, these alterations were significantly attenuated in SCLM-pretreated DM+MCAO rats.

NF-κB proteins are a family of transcription factors that are of central importance in inflammation (21). ROS plays a vital role in NF-κB signaling (34). In addition, activation of microglia has been reported to contribute to the generation of ROS (35). To determine whether SCLM confers cellular protection through its anti-inflammatory properties, we examined the expression levels of proinflammatory mediators and cytokines in brain tissues. As illustrated in Figures 5d–h , SCLM significantly inhibited the expression of TLR4, p-IκB, p-p65, and IL-1β in the brain tissues of DM+MCAO rats. For in vitro experiments, we utilized LPS-stimulated BV2 cells, a widely recognized in vitro inflammatory model derived from C57BL/6 mouse microglia. First, the toxicity of SCLM in BV2 cells was evaluated, and similarly, no obvious cytotoxicity was detected ( Supplementary Figure S5 ). As shown in Figures 6a, b , compared with those in the LPS group, the free radical levels in the SCLM group significantly decreased in a dose-dependent manner. Then, we detected the expression levels of proinflammatory mediators and cytokines in LPS-stimulated BV2 cells using western-blot. The results showed that SCLM inhibited the protein expression of TLR4, p-IκB, p-p65 and IL-1β in LPS-stimulated BV2 cells ( Figures 6c–g ). Similarly, these findings suggest that SCLM pretreatment may reduce inflammation by inhibiting NF-κB activation, thereby decreasing the release of proinflammatory mediators and cytokines after ischemic injury.