Lansoprazole Improves Long-Term Neurological Function via Alleviating Intestinal Injury in a Mouse Model of Intracerebral Hemorrhage

Summary

A collagenase-induced mouse model of intracerebral hemorrhage (ICH) was treated with lansoprazole. Lansoprazole not only alleviated ICH-caused intestinal injury and reduced levels of lipopolysaccharide (LPS) and IL-1β, but it also inhibited brain inflammation. Lansoprazole enhanced learning and memory (long-term neurological function) of ICH mice.

Abstract

Intracerebral hemorrhage (ICH) can compromise the gastrointestinal tract and trigger intestinal injury, contributing to poor clinical outcomes. This study investigated the effects of lansoprazole on intestinal injury and neurological function in mice with ICH. Mice were stereotactically injected with collagenase to establish an ICH model. Neurological function, brain water content, blood-brain barrier permeability, intestinal injury, and intestinal permeability were evaluated. Levels of LPS and IL-1β in the blood and brain were also assayed. Lansoprazole treatment was associated with alleviated intestinal injury, reduced levels of LPS and IL-1β in blood and brain, decreased brain water content, and inhibited brain inflammation in ICH mice. While lansoprazole administration did not lead to improved performance in the Garcia, forelimb placing, and rotarod tests (short-term neurological function), it significantly enhanced learning and memory (long-term neurological function). These findings indicated that lansoprazole treatment was associated with improved long-term neurological outcomes and reduced intestinal injury in ICH mice.

Introduction

Hemorrhagic stroke has high rates of mortality and disability. Recently, the prevalence of stroke in China has increased, making it the leading cause of death¹. Although less common, intracerebral hemorrhage (ICH) is markedly more lethal than ischemic stroke. Studies report a 40% mortality rate within 1 month of ICH; furthermore, approximately 75% of survivors are unable to live independently 1 year later².

The brain communicates with the gastrointestinal tract through several pathways, including the vagus nerve, enteric nervous system, and gut microbial metabolism. The brain-gut axis is a well-recognized and clinically relevant regulatory system³. The intestinal mucosa regulates the absorption of water, electrolytes, and nutrients, while preventing the entry of pathogens and toxins into the bloodstream. ICH can compromise this intestinal barrier and trigger gastrointestinal injury. Barrier dysfunction, in turn, contributes to malnutrition, immunosuppression, and poor clinical outcomes⁴. ICH-induced increases in intestinal permeability allow harmful endogenous substances to enter the bloodstream, triggering systemic inflammatory responses and multiple organ dysfunction syndrome. Damage to the intestinal mucosa following ICH can even allow gut bacteria to translocate into the bloodstream, potentially leading to systemic infections⁵. Many patients develop intestinal dysmotility, microbial dysbiosis, mucosal bleeding, and sepsis. One previous study showed that ICH caused the migration of intestinal pathogens and toxins to the brain⁶.

Lansoprazole, a proton pump inhibitor (PPI), irreversibly inhibits the proton pump and thereby reduces gastric acid secretion. It is used in the treatment of gastric and duodenal ulcers, erosive esophagitis, gastroesophageal reflux disease, and nonsteroidal anti-inflammatory drug-associated gastric ulcers⁷. A previous study indicated that PPI prophylaxis negatively affected patient outcomes in ischemic stroke⁸. Other published studies suggested an association between PPI use and cognitive decline. An epidemiological study showed that patients administered PPIs had an increased risk of developing dementia and Alzheimer's disease⁹. A prospective cohort study comparing long-term PPI users with non-users found that PPI use was associated with an increased risk of dementia, and the authors advised against PPI use in at-risk patients as a preventative measure¹⁰. The present study was performed to investigate the effects of lansoprazole on intestinal injury as well as short- and long-term neurological function (specifically learning and memory) in mice with ICH. We hypothesize that lansoprazole may inhibit the permeation of LPS by virtue of its gut-barrier-protective effects, alleviating systemic and neurological inflammation, and therefore improving the neurological function in ICH mice.

Protocol

The experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 8023, revised 1978). The protocol was approved by the Ethics Committee of Yantai University (Approval No. YTDX20240526). Hazardous materials were disposed of according to institutional chemical waste protocols. Figure 1 illustrates a schematic diagram of the experimental design. The reagents and the equipment used are listed in the Table of Materials.

1. Animal details

Male CD-1 mice weighing 22-25 g were housed in a room with a 12-/12-h light/dark cycle. The animals were free to get food and water.

2. Preparation of the ICH mouse model

The mouse model of ICH was established according to a previous report¹¹. Briefly, mice were anesthetized with xylazine (10 mg/kg) and ketamine (90 mg/kg), then positioned in a stereotaxic frame. A small burr hole was drilled at the right coronal suture (2.2 mm lateral to the midline, 0.2 mm posterior to the bregma). A microsyringe was inserted into the right brain at a depth of 3.5 mm from the skull surface. Using a microinfusion pump, bacterial collagenase (0.075 U in 0.5 µL phosphate-buffered saline [PBS]) was infused at a rate of 0.15 µL/min. The needle was gently withdrawn after 5 min. Mice in the sham group received an equivalent volume of PBS instead of collagenase. Sealed the burr hole with bone wax, and the scalp incision was sutured. The body temperature of animals was maintained at 37°C until awakening.

3. Experimental design

Male CD-1 mice were randomly assigned to three groups: sham, model, and lansoprazole. The ICH model was established as described above. Thirty minutes after ICH induction, mice in the lansoprazole group received lansoprazole at a dose of 30 mg/kg (the dose of lansoprazole was selected according to a previous report¹²), administered once daily for three consecutive days. Mice in the sham and model groups received intraperitoneal injections of PBS. At 3 days post-ICH, short-term neurological function was assessed using the Garcia test, forelimb placing test, and rotarod test. On day 29 after ICH induction, the novel object recognition and water maze tests were conducted to evaluate long-term neurological function.

4. Garcia test

Two researchers, blinded to the group, performed the Garcia test according to a previously reported protocol¹³. Neurological function was assessed using a scoring system ranging from 0 to 21. The evaluation consists of seven subtests, each scored from 0 to 3 (0 = worst, 3 = best).

5. Forelimb placing test

Following the Garcia test, the forelimb placing test was performed according to a previously described method¹⁴. The mice were held in a vertical position, parallel to the tabletop, allowing their vibrissae to brush along the surface. Forelimb placement was observed over 10 consecutive trials. The percentage of left forelimb placement was calculated as: Left forelimb placement / (Left forelimb placement + Right forelimb placement) × 100%.

6. Rotarod test

After the forelimb placing test, the mice were placed on a rotating rod at 5 rpm for 3 min for training. The rod was accelerated from 5 rpm to 30 rpm over 4 min. The duration each mouse remained on the rod was recorded over a 5-min period, and the escape latency was recorded¹⁵.

7. Intestinal permeability and brain edema assay

After the rotarod test, six mice from each group were randomly selected. According to the previous method¹⁶, FITC-dextran was intragastrically administered at a dose of 300 mg/kg. Three hours later, the mice were anesthetized with isoflurane, and blood was collected from the retro-orbital venous plexus, centrifuged at 4 °C and 11,000 × g for 5 min. FITC-dextran content was assayed using a fluorescence plate reader at 485-nm excitation and 528-nm emission wavelengths. The animals were then decapitated, and the right hemisphere of the brain was collected and weighed to obtain the wet weight. The tissue was dried at 100 °C for 24 h to determine the dry weight. Brain water content (%) was calculated as: [(Wet weight − Dry weight) / Wet weight] × 100%.

8. Blood - brain barrier (BBB) permeability evaluation

After the rotarod test, six mice from each group were randomly selected for evaluating BBB permeability. A 2% Evans blue solution was intraperitoneally administered at 4 mL/kg. Three hours later, the mice were anesthetized with isoflurane, and transcardial perfusion was performed using PBS. The right hemisphere of the brain was homogenized in 50% trichloroacetic acid at a ratio of 3:1 (brain tissue: trichloroacetic acid). The homogenate was centrifuged at 15,000 × g and 4 °C for 20 min. Then, 500 µL of the supernatant was mixed with 500 µL of 70% ethanol and incubated overnight at 4 °C in the dark. The concentration of Evans blue was measured at 610 nm and quantified using a standard curve. Data are presented as µg of Evans blue per mg of brain tissue.

9. Histopathological examination of the intestine and brain

After the rotarod test, three mice from each group were randomly selected. The mice were anesthetized with isoflurane. The jejunum, ileum, and brain were collected and fixed in 10% formaldehyde. Tissues were sectioned at 5 µm thickness, and hematoxylin-eosin (HE) staining was performed. Histopathological examination of the intestine and brain was carried out using a microscope by two experimenters who were blinded to the group assignments. The number of microglia surrounding the hematoma was counted in representative sections. Cell counts were performed in five arbitrary visual fields across three sections per animal.

10. Measurement of LPS and IL-1β

After the rotarod test, six mice from each group were randomly selected. The mice were anesthetized with isoflurane, and blood was collected. Blood samples were centrifuged at 5,000 × g and 4 °C for 10 min, and serum was collected. The right hemisphere of the brain was harvested, and brain homogenates were prepared at a ratio of 1:3 (brain weight in grams to PBS volume in milliliters). The homogenates were centrifuged at 12,000 × g and 4 °C for 10 min. Protein concentrations in the supernatant were determined using a BCA kit. Levels of LPS and IL-1β in both serum and brain homogenate were measured using ELISA kits, following the manufacturer's instructions.

11. Transmission electron microscopy

For electron microscopic examination, three animals from each group were randomly selected after the rotarod test. The mice were anesthetized with isoflurane and underwent transcardiac perfusion with 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M PBS (following institutionally approved protocols). The brain, jejunum, and ileum were then harvested and placed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4 °C overnight. The samples were subsequently transferred to 1% osmium tetroxide for 1 h at room temperature. Tissues were cut into ultrathin sections (50 nm). After dehydration, the sections were stained with uranyl acetate and lead citrate for observation under a transmission electron microscope. All sections were evaluated in a blinded manner and photographed using a transmission electron microscope.

12. Novel object recognition

At 29 days after ICH modeling, the novel object recognition test was performed to assess the learning and memory function of mice. On the first day, no objects were placed in the apparatus (length: 40 cm, width: 40 cm, height: 40 cm), allowing the mice to explore freely for 5 min. On the second day, two identical objects were placed in the apparatus, and the mice were allowed to explore them for 5 min. Twenty-four hours later, one of the objects was replaced with a novel object. The mice explored the two objects for 5 min again. Exploration time was recorded when the mouse's nose touched an object or when its head was oriented toward the object within a 1-cm distance. The time spent exploring the novel object was measured and calculated. A longer exploration time of the novel object indicates better learning and memory ability.

13. Morris water maze

At 32 days after ICH modeling, learning and memory functions were assessed using the Morris water maze. The apparatus consisted of a circular pool (diameter: 100 cm, height: 40 cm) filled with water (depth: 25 cm) maintained at 22 °C ± 1 °C. An escape platform (diameter: 9 cm) was submerged 1 cm below the surface of the water. In the navigation test, mice underwent four trials per day. The time taken to find the platform was recorded as the escape latency. Mice that failed to locate the platform within 60 s were gently guided to it. On the fifth day, a 60-s probe test was conducted. Memory function was evaluated by recording the escape latency, number of crossings of the platform, and time spent in the target quadrant. Data were analyzed using Video Tracking Software.

14. Statistical analysis

Data were analyzed using GraphPad Prism software version 7.0. Results are presented as mean ± standard deviation. The Shapiro-Wilk test was used to assess whether data from each group followed a normal distribution. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. For non-parametric comparisons, the Kruskal-Wallis test followed by Dunn's test was used. A p-value of <0.05 was considered statistically significant.

Results

Effects of lansoprazole on short-term neurological function in ICH mice

Compared with the sham group, mice in the ICH group showed significantly lower scores in the Garcia test (p < 0.01). However, mice in the lansoprazole group did not show improved Garcia scores compared with the ICH group (p > 0.05) (Figure 2A). Similarly, the percentage of left forelimb placement was significantly reduced in the ICH group compared with the sham group (p < 0.01). Consistent with the Garcia test results, lansoprazole treatment did not increase the percentage of left forelimb placement (p > 0.05) (Figure 2B). In the rotarod test, the time spent on the rod was markedly decreased in the ICH group compared with the sham group (p < 0.01). Lansoprazole treatment did not improve performance in this test, as no significant increase in time on the rod was observed (p > 0.05) (Figure 2C). These results indicated that lansoprazole administration did not enhance the short-term neurological function.

Effects of lansoprazole on serum FITC-dextran, LPS, and IL-1β levels in ICH mice

Compared with the sham group, the serum levels of FITC-dextran, LPS, and IL-1β were significantly elevated following ICH (p < 0.01). However, treatment with lansoprazole reduced the levels of serum FITC-dextran (Figure 3A), LPS (Figure 3B), and IL-1β (Figure 3C) in ICH mice (p < 0.05 or p < 0.01).

Effects of lansoprazole on intestinal injury in ICH mice

In HE staining, the intestinal villi of mice in the sham group appeared normal, with no signs of necrolysis. By contrast, the ICH group exhibited necrolysis and dissolution at the tips of the intestinal villi. Lansoprazole administration significantly ameliorated these ICH-induced changes, reducing necrolysis and villus damage (Figure 4A-F). Transmission electron microscopy revealed clear and intact epithelial tight junctions (TJs) in the sham group, with no abnormalities observed. In the ICH group, the structure of TJs appeared blurred and disrupted. Lansoprazole treatment attenuated these abnormalities and improved the structural integrity of TJs in ICH mice (Figure 4G-L). It suggested that lansoprazole treatment reduced LPS-induced systemic inflammation by protecting the intestine.

Effects of lansoprazole on brain water content, Evans blue extravasation, LPS, and IL-1β in brain tissue of ICH mice

Compared with the sham group, the brain water content and Evans blue extravasation were significantly increased in the ICH group (p < 0.01). However, lansoprazole treatment did not significantly reduce the brain water content or Evans blue extravasation compared with the ICH group (p > 0.05) (Figure 5A,B). The LPS and IL-1β levels in brain tissue were also significantly elevated in the ICH group compared with the sham group (p < 0.01). By contrast, treatment with lansoprazole significantly reduced brain levels of LPS and IL-1β compared with the ICH group (p < 0.05 or p < 0.01) (Figure 5C,D).

Effects of lansoprazole on the number of microglia and the BBB in ICH mice

HE staining revealed that the number of microglia around the hematoma was significantly increased in the ICH group compared with the sham group (p < 0.01). However, the number of microglia was significantly reduced in the lansoprazole group compared with the ICH group (p < 0.01) (Figure 6A-G). Transmission electron microscopy showed a normal structure of endothelial cells and TJs in the BBB of sham mice. By contrast, endothelial cell edema and TJ deformation were observed in the ICH group. These structural abnormalities were ameliorated in the lansoprazole group (Figure 6H,I). Several factors are associated with rain edema and BBB disruption, including the clot, local ischemia, and the release of toxic substances from the clot. Although lansoprazole treatment inhibited the penetration of LPS from the gut into circulation, partially reducing neuroinflammation, it still cannot preserve the BBB, decrease brain edema, and therefore improve short-term neurological function.

Effects of lansoprazole on time spent in the zone of a new object in ICH mice

In the novel object recognition test, there was no significant difference in mean speed among the sham, ICH, and lansoprazole groups (p > 0.05) (Figure 7A). However, compared with the sham group, the ICH group showed a significant decrease in time spent in the zone of the new object (p < 0.01). Lansoprazole treatment significantly increased the time spent in the zone of the new object compared with the ICH group (p < 0.01) (Figure 7B).

Effects of lansoprazole on escape latency, crossing platform, and time in the target quadrant in ICH mice

The mice in the ICH group exhibited longer escape latency than those in the sham group on days 3, 4, and 5 (p < 0.05 or p < 0.01). Lansoprazole treatment significantly shortened escape latency on days 4 and 5 compared with the ICH group (p < 0.05 or p < 0.01) (Figure 8A). In the probe test, the number of crossing platforms and the time spent in the target quadrant were significantly reduced in the ICH group compared with the sham group (p < 0.01). Lansoprazole administration significantly increased both the number of crossing platforms and the time in the target quadrant compared with the ICH group (p < 0.05 or p < 0.01) (Figure 8B,C). The results suggested that lansoprazole treatment was associated with improved long-term neurological outcomes by alleviating intestinal injury, reducing levels of LPS and IL-1β, and inhibiting brain inflammation in ICH mice.

DATA AVAILABILITY:

The data that support the findings of this study are available at https://data.mendeley.com/preview/njf8b7x3nx?a=96c33d68-a155-44ab-8d8c-bf1a778b8ac4.

Figure 1: The schematic diagram of the experimental design. Please click here to view a larger version of this figure.

Figure 2: Effects of lansoprazole on the short-term neurological function in ICH mice. Data were analyzed using GraphPad Prism software version 7.0. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test or the Kruskal-Wallis test followed by Dunn's test. (A) Garcia test for evaluating short-term neurological function; (B) Forelimb placing test for evaluating short-term neurological function; (C) Rotarod test for evaluating short-term neurological function. n = 35 or 36. ^##p < 0.01 compared with the sham group. Please click here to view a larger version of this figure.

Figure 3: Effects of lansoprazole on the serum FITC-dextran, LPS, and IL-1β levels in ICH mice. Data were analyzed using GraphPad Prism software version 7.0. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. (A) FITC-dextran level for evaluating intestinal permeability; (B) LPS level in serum; (C) IL-1β level in serum. n = 6. ^##p < 0.01 compared with the sham group. ^*p < 0.05, ^**p < 0.01 compared with the ICH group. Please click here to view a larger version of this figure.

Figure 4: Effects of lansoprazole on the intestinal injury in ICH mice. (A) Sham group; (B) ICH group; (C) Lansoprazole group. Scale bars = 200 μm. (D) Sham group; (E) ICH group; (F) Lansoprazole group. Scale bars = 50 μm. HE staining, n = 3. (G) Sham group; (H) ICH group; (I) Lansoprazole group. Scale bars = 1 μm. (J) Sham group; (K) ICH group; (L) Lansoprazole group. Scale bars = 500 nm. Transmission electron microscopy, n = 3. The tight junctions were indicated by the red arrows. Please click here to view a larger version of this figure.

Figure 5: Effects of lansoprazole on the brain water content, Evans blue extravasation, LPS, and IL-1β in the brain of ICH mice. Data were analyzed using GraphPad Prism software version 7.0. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. (A) Brain water content; (B) Evans blue extravasation; (C) LPS level; (D) IL-1β level. n = 6. ^##p < 0.01 compared with the sham group. ^*p < 0.05, ^**p < 0.01 compared with the ICH group. Please click here to view a larger version of this figure.

Figure 6: Effects of lansoprazole on the number of microglia and BBB in ICH mice. Data were analyzed using GraphPad Prism software version 7.0. Differences among groups were analyzed using the Kruskal-Wallis test followed by Dunn's test. (A) Sham group; (B) ICH group; (C) Lansoprazole group. Scale bars = 200 μm. (D) Sham group; (E) ICH group; (F) Lansoprazole group. Scale bars = 50 μm. (G) Bar graphs of quantitative analysis of the number of microglia in the loci around the hematoma of ICH mice. HE staining, n = 3. The microglia were indicated by the black arrows. (H) Sham group; (I) ICH group; (J) Lansoprazole group. Scale bars = 1 μm. Transmission electron microscopy, n = 3. The tight junctions were indicated by the red arrows. Please click here to view a larger version of this figure.

Figure 7: Effects of lansoprazole on the time in the zone of the new object in ICH mice. Data were analyzed using GraphPad Prism software version 7.0. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. (A) Mean speed; (B) Time in zone of new object. n = 11 or 12. ^##p < 0.01 compared with the sham group. ^**p < 0.01 compared with the ICH group. Please click here to view a larger version of this figure.

Figure 8: Effects of lansoprazole on the escape latency, number of crossings of the platform, and time in the target quadrant in ICH mice. Data were analyzed using GraphPad Prism software version 7.0. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test or the Kruskal-Wallis test followed by Dunn's test. (A) Escape latency; (B) Number of crossing platforms; (C) Time in target quadrant. n = 11 or 12. ^#p < 0.05 or ^##p < 0.01 compared with the sham group. ^*p < 0.05 or ^**p < 0.01 compared with the ICH group. Please click here to view a larger version of this figure.

Discussion

Intestinal injury is a common complication in patients with ICH. ICH-induced intestinal damage can lead to malnutrition, prolonged hospitalization, poor outcomes, and even mortality¹⁷. Using an ICH animal model, this study demonstrated for the first time that lansoprazole attenuates intestinal injury in ICH mice. Although lansoprazole did not show beneficial effects on short-term neurological function, it significantly improved long-term neurological outcomes.

Stroke, whether ischemic or hemorrhagic, is a leading cause of death globally. Following a stroke, the body undergoes a systemic stress response, which can trigger pathological changes in organs beyond the brain. The intestine is one of the primary targets of this systemic response. Clinically, stroke-induced digestive symptoms such as mucosal damage, abdominal distension, and constipation are commonly observed^18,19. In ischemic stroke models, animals exhibit dysfunction of the intestinal barrier, which can result in the translocation and dissemination of gut bacteria to other organs, such as the lungs⁵. Previous studies have shown that ICH induces intestinal barrier dysfunction, mucosal destruction, impaired motility, inflammatory responses, and oxidative stress. These pathological processes can begin as early as 2 h after ICH onset and persist for up to 7 days²⁰. The gut microbiota plays a critical role in neuroinflammation following ICH. For instance, metformin has been reported to improve neurological deficits and reduce neuroinflammation in ICH mice by decreasing levels of LPS-binding protein, a biomarker of intestinal barrier damage. These effects were associated with enhanced intestinal barrier function²¹. Another study found that ICH caused persistent gut dysbiosis, which was associated with inflammation, barrier dysfunction, and neurological deficits. Oxymatrine, a compound with anti-inflammatory properties, improved both systemic inflammation and intestinal barrier integrity, thereby promoting recovery of long-term neurological function²². Ghrelin, known for its protective effects in models of gastrointestinal injury, also reduced mucosal damage and improved intestinal permeability in an intrastriatal autologous blood infusion ICH model. It upregulated TJ-related proteins such as ZO-1 and claudin-5, inhibited endotoxin translocation, and improved survival rates in ICH mice²³. Astragaloside IV, a compound extracted from traditional Chinese medicine, has also shown protective effects. In ICH models, it reduced intestinal barrier damage and LPS leakage, while mitigating blood-brain barrier disruption and neurological deficits²⁴.

In the present study, ICH caused intestinal injury, LPS translocation and dissemination, systemic inflammation, BBB impairment, and neuroinflammation. Lansoprazole not only ameliorated ICH-induced pathological injury in the intestine but also improved intestinal barrier function. As a result, it inhibited the translocation and dissemination of LPS from the host intestine. Subsequently, lansoprazole mitigated systemic inflammation, BBB disruption, and neuroinflammation. Although lansoprazole showed beneficial effects on ICH-induced intestinal complications and reduced neuroinflammation, it did not restore short-term neurological function in mice with ICH. Several possible explanations may account for this seemingly illogical outcome. First, short-term neurological impairment is primarily caused by the presence of blood in the brain parenchyma, which exerts mechanical pressure on surrounding tissue and releases neurotoxic blood components and degradation products²⁵. Therefore, treatment with lansoprazole alone may not be sufficient to produce significant short-term neuroprotection in ICH mice. Second, the duration and frequency of lansoprazole administration may have been too limited (only three doses over 3 days). Increasing the treatment duration and frequency (e.g., daily administration for 7 days) may be necessary for lansoprazole to improve short-term neurological outcomes. We next evaluated the effect of lansoprazole on long-term neurological function by assessing learning and memory in ICH mice. As hypothesized, lansoprazole improved cognitive performance, suggesting that its treatment contributes to the restoration of long-term neurological function following ICH.

There are several limitations in this study. First, only the translocation of intestinal LPS was investigated. Previous reports have shown that stroke increases intestinal permeability and impairs intestinal barrier function, promoting the translocation and dissemination of commensal bacteria. These bacteria, once disseminated to inappropriate organs, may contribute to the worsening of ICH outcomes⁵. Therefore, future studies should aim to clarify the role of bacterial translocation and dissemination in this context. Second, based on previous studies^26,27,28, the current investigation assessed the effects of lansoprazole on neurological function using only a single dosage. To better understand potential dose-dependent effects, future research should include multiple dosage groups, ideally three, to evaluate the efficacy across a range of doses. Third, this study did not assess whether combination therapy, such as lansoprazole with neuroprotective agents or anti-inflammatory agents, could improve short-term neurological function in ICH mice. Exploring such combinations may reveal more effective strategies for early-stage intervention following ICH. Fourth, the gastrointestinal protective effect of lansoprazole may play a role in nutrient absorption, which could influence the evaluation of neurological function. This study can not exclude the possibility of this potential off-target mechanism.

In conclusion, this study demonstrated that lansoprazole not only ameliorated ICH-induced pathological injury in the intestine but also restored intestinal barrier dysfunction. As a result, it inhibited the translocation and dissemination of LPS from the host intestine. Subsequently, lansoprazole reduced systemic inflammation, BBB impairment, and neuroinflammation. While lansoprazole did not improve short-term neurological function, it significantly enhanced long-term neurological outcomes in ICH mice. Therefore, it indicated that PPI use did not lead to an increased risk of dementia in ICH. Conversely, PPI administration in the early stage of ICH may enhance patients' learning and memory, which is worth investigating and confirming in future clinical trials.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA kit	Beyotime Biotechnology	P0009
Collagenase	Sigma-Aldrich	128M4107v
Evans blue dye	Sigma-Aldrich	MKCG7507
FITC-dextran	Sigma-Aldrich	46944
GraphPad Prism 7.0	GraphPad statistical Software, Boston, MA, USA, www.graphpad.com	Prism 7.0
Lansoprazole	Shandong Yuxin pharmaceutical Co., Ltd.	524093053
LPS ELISA kit	Cusabio biotech Co., Ltd.	M25036407
Mice	Jinan Pengyue Experimental Animal Breeding Co., Ltd.	SCXK20220006
Microinfusion pump	World Precision Instruments	Micro 3
Microsyringe	Hamilton Company	SYR 10 μL
Olympus microscope	Olympus Co., Ltd.	IX-70
Rotarod	Panlab Co., Ltd.	LE8205
Stereotaxic frame	Kopf Instruments	Model 902-A
TNF-α ELISA kit	Beyotime Biotechnology	PI301
Transmission electron microscope	JEOL Ltd.	JEM-100SX
Video Tracking Software	Panlab Co., Ltd.	Smart 3.0