Canine distemper virus (CDV) was the most common cause of infectious ME in 10/16 dogs (62.5%). Of the total cases with a positive infectious disease result, 3/16 (18.7%) had normal CSF results and 13/16 (81.3%) had abnormal CSF results (p = 0.0078). MRI and CT abnormalities in the brain were detected in 74 and 39% of dogs with inflammatory CSF, respectively. MRI and CT abnormalities in the spinal cord were detected in 90 and 57% of dogs with inflammatory CSF results, respectively. Age was not significantly different between infectious ME and presumptive MUO groups (p = 0.15). Seizures were the most common clinical sign reported for both MUO (36.8% of cases) and infectious ME (31.2% of cases). In conclusion, presumptive MUO is significantly more common than infectious ME in this population of dogs. Furthermore, although normal CSF results were uncommon in dogs with infectious ME, this finding occurred in several patients (3/16), suggesting that infectious disease testing should be considered even in the face of normal CSF results. Finally, MRI was more sensitive than CT in the detection of abnormalities when dogs with ME had inflammatory CSF results but was not 100% sensitive, suggesting CSF analysis should be performed to rule out inflammation even when no abnormalities are detected on MRI or CT.This retrospective study evaluated canine patients with presumptively diagnosed meningoencephalomyelitis (ME) based on neurological clinical signs, cerebrospinal fluid (CSF) analysis, cross-sectional imaging, and infectious disease testing with a limited neurological-focused polymerase chain reaction (PCR) panel performed on blood and CSF. The first goal was to determine the proportion of dogs where the condition was caused by an infectious agent versus a probable immune-mediated etiology  in our geographic region. The secondary goals of this study were to examine and define associations between abnormal CSF test results and cross-sectional neuroimaging findings, in addition to defining the age and most common neurological clinical signs in each group of ME. A total of 168 dogs matched the inclusion criteria with magnetic resonance imaging (MRI) performed in 130 dogs and computed tomography (CT) performed in 38 dogs. Presumptive MUO was observed in 152/168 (90.5%) of dogs and infectious ME was identified in 16/168 (9.5%) of dogs ( The etiNeurologic clinical signs of ME depend on localization within CNS and severity . The mosDiagnosis usually is based on advanced imaging (CT or MRI), cerebrospinal fluid (CSF) analysis, and antibody and antigen tests to rule out an infectious disease. The correlation regarding inflammatory CSF results and MRI change findings was previously described in the study by Lamb et al. who reported that inflammatory CSF results were associated with normal MRI results in 24% of the cases. In neuroimaging, most cases of neoplastic lesions, which are generally unifocal, are differentiated from inflammatory diseases, which are usually multifocal , 6. The Differentiating infectious vs. non-infectious ME in the clinical patient is important . The proIn the present study, the medical records of 168 dogs with presumptive meningoencephalomyelitis of unknown origin (MUO) and infectious ME were retrospectively evaluated. Geolocation may influence the incidence of etiologies of ME . To this2.2.1.The present animal study was approved and reviewed by the Ethics Committee of the Pontifical Catholic University of Paraná . Written informed consent was obtained from the hospital owners for the participation of their animals in this study.2.2.Retrospective, transversal, and non-randomized.2.3.Data from a total of 168 dogs with CSF samples and neurological focused infectious disease PCR panel from 2015 to 2022 that were referred to a veterinary neurology service in a private veterinary hospital in Curitiba (Clinivet), Paraná, Brazil.Bartonella spp., Borrelia burgdorfi, Blastomyces dermatitidis, CDV, Coccidioides spp., Cryptococcus spp., Histoplasma capsulatum, Neospora spp., Toxoplasma gondii and West Nile Virus. All samples for the PCR panel were performed using blood and CSF samples and results were provided as positive or negative.All cases needed to fulfill the following criteria: detailed neurological exam; recorded neurological clinical signs; blood work including complete blood count, ALT, creatine, urea, albumin, glucose, ammonia; cerebrospinal fluid (CSF) analysis; cross sectional images; and neurological infectious disease panel (referred to going forward as PCR panel). The latter test is conducted by Idexx Laboratories  and includes the following: 2.4.All dogs were anesthetized using intravenous bolus of propofol (5 mg/kg) and intubated with a tracheal tube. The same person (FB) performed all CSF collections at the cisterna magna, using the technique described in the literature . The patCSF results were considered normal if there was <5 nucleated cells and no abnormal distribution of white cells, protein concentration < 30 mg/dL, 70% of serum glucose, and absence of microorganism in the sample.2.5.MRI testing  was performed with patients also anesthetized with intravenous propofol  at 5 mg/kg. These patients were intubated and placed in dorsal recumbency. Pulse sequences for the brain exams included, T2 sagittal plane, T1-weighted, T2-weighted, and FLAIR in transversal planes, Diffusion, ADC, and SWI in transverse planes, plus T1-weighted with fat suppression after administration of intravenous paramagnetic contrast medium  at a dose of 0.1 mmol/kg. For the spinal images pulse sequences included were sagittal plane T1-weighted, T2-weighted, HASTE and STIR, transverse plane T2-weighted, dorsal plane STIR and sagittal/transverse plane post-contrast T1-weighted with fat suppression after administration of intravenous paramagnetic contrast medium at the same dose that in brain exams. All MRI exams were performed in a human hospital.The CT scans  were performed with the patients under sedation using intravenous dexmedetomidine  10 mcg/kg and placed in dorsal recumbency, CT exams were performed without contrast and after intravenous contrast iohexol  at a dose of 1.5 mL/kg, injected intravenous. All CT exams were performed at Clinivet Veterinary Hospital.To avoid interobserver discrepancies, all MRI and CT images were prospectively evaluated by the same examiner (FB) who has more than 15 years of clinical experience in neurology, neuro-imaging studies, and neurosurgery.Dogs identified with solitary mass which was attached in the meninges with a dural tail which strongly suggested a diagnosis of meningioma were excluded. Dogs with neuroimaging that suggested vascular disease (ischemic) showing restriction on diffusion images and hyposignal on ADC images were also excluded. In addition, dogs were excluded with otitis media and communication with brainstem, due to a lack of PCR results in those cases.Cases with CT included 38 dogs and cases with MRI included 130 dogs.2.6.The indicators used for the diagnosis of dogs with MUO were neurological clinical signs, negative infectious disease panel results, and CSF analysis showing an increase in nucleated cells (>5 μL) with at least >50% lymphocytic/monocytic pleocytosis and/or protein (>30 mg/dL) .Neuroimaging with changes inclusive of the following was also part of this criteria: MRI cases that showed multifocal hyperintensity lesions on T2 or FLAIR, with or without contrast enhancement were also indicators  Figure . CT alsoIndicators for considering presumptive MUO as a diagnosis in a few dogs with normal CSF analysis and normal neuroimaging were as follows: neurological clinical signs, breed, and partial or full response to immunosuppressive therapy.2.7.The indicators used for ME of infectious origin were positive results in the infectious disease PCR panel in the blood sample and CSF.2.8.2.8.1.The following groups were compared by statistical analysis: Positive PCR vs. normal CSF results. Positive  included 38 dogs and cases with magnetic resonance imaging (MRI) included 130 dogs.All intracranial and spinal cord MRI cases were analyzed and compared in this manner: the total MRI performed in CSF normal cases, and the total MRI performed in CSF abnormal cases. MRI results  in patients with a normal CSF analysis; and MRI results  in patients with abnormal CSF analysis . MRI finAll intracranial and spinal cord CT cases were analyzed and compared in this manner: The total CT performed in CSF normal cases, and the total CT performed in CSF abnormal cases. CT results  in patients with a normal CSF analysis; CT results  in patients with an abnormal CSF analysis .2.9.p-values ≤0.05 were considered significant. The software MedCalc® Statistical Software version 20.027  was used for these calculations.Results were organized in contingency tables. Sensitivity and specificity were calculated . The following statistical tests were applied: Chi-square test (presumptive MUO cases versus cases diagnosed as infectious ME); and Fisher’s exact test (for PCR-positive results for infectious agents versus CSF results). Mann–Whitney test . The confidence interval (CI) was 95% and 3.p = 0.15). Among the 168 dogs in the present study, 30 breeds were included, and the two most affected breeds were mixed breed dogs (28%) and the Maltese (13%) . In the MUO group, the mean age was 5.5 years (4 months - 11 years), and in the ME infection group, the mean age was 4.5 years (2 months - 15 years). This difference was not significant (se (13%) .3.1.p < 0.0001). In the MUO group, 119/152 dogs had abnormal CSF results (78.3%), and 33/152 dogs had normal CSF results (21.7%). In the infectious ME group, the CSF analysis showed abnormal results in 13/16 (81.3%) versus 3/16 (18.7%), with normal CSF analysis. In addition, the presence of abnormal CSF results were significantly correlated with positive results in the infectious disease PCR panel (p = 0.0078), 95% CI of odds ratio: 0.025 to 0.728.Of the 168 CSF samples, a total of 152 dogs (90.5%) were diagnosed with presumptive MUO (probable immune-mediated etiology), and 16 dogs (9.5%) were diagnosed with infectious ME; this difference was significant (Toxoplasma gondii 2/16 (12.5%); 3) Neospora sp. Plus Toxoplasma gondii 1/16 (6.3%); 4) Borrelia burgdorferi 3/16 (18.7%).Four different infectious agents were diagnosed by the infectious disease PCR panel: 1) CDV 10/16 (62.5%); 2) Of the CDV cases, 7/10 (70%) showed abnormal CSF analysis. Interestingly, the predominant cell type observed in the CDV cases were > 50% lymphocytes and, in toxoplasmosis and neosporosis cases, more than 50% neutrophils were observed.Of the 168 dogs examined in this study, 132/168 (78.6%) had abnormal CSF analysis, and 36/168 (21.4%) were considered normal. Of the 132 total abnormal CSF results 92 dogs had increased nucleated cells >5 mm3 with or without accompanying elevated protein (>30 mg/dL) in their respective CSF samples and 71 dogs had elevated protein with or without accompanying increased nucleated cells. In those samples with abnormal CSF results, the mean number of nucleated cells was 108.25 . The mean concentration of protein was 67.05 mg/dL and (range 31 to 720 mg/dL). No samples with microorganisms were detected directly using CSF microscopy. Regarding the samples with abnormal CSF analysis, 119/132 (90.2%) had negative results for the infectious disease PCR panel and 13/132 (9.8%) had a positive result. For the samples with normal CSF analysis, 33/36 (91.7%) had negative PCR panel results and three normal CSF analysis samples 3/36 (8.3%) were positive for CDV per the PCR panel.3.2.A total of 130 MRI  and 38 CT scans  were performed. Of all 168 neuroimages, the spinal cord was examined in 32/168 cases (19.0%).3.2.1.The total number of brain MRI analyzed was 106 cases. The relative sensitivity value was 74% (95% CI: 63.1 to 83.1%) and specificity was 64% (95% CI: 42.5 to 82%), for the detection of changes in dogs with CSF results supportive of inflammation. A comparison of results: neuroimaging analysis versus results from CSF analysis is presented in Regarding myelopathies: The total number of spinal cord MRI analyzed included 24 cases. The relative sensitivity value was 90.5% (95% CI: 69.7 to 98.8%) and specificity was 66.9% (95% CI: 9.4 to 99.2%) for spinal cord MRI detected inflammatory changes in CSF results. A comparison of results found in the MRI neuroimaging analysis versus results from CSF analysis is presented in Of all cases where the brain was examined by MRI, the most common abnormal findings were multiple lesions with hyperintensity on T2 and FLAIR sequences with or without contrast enhancement in the meninges and/or in the lesions , 3. Of aOne representative brain MRI lesion is shown in 3.2.2.Brain CT results were available in 30 cases. The relative sensitivity value was 39.1% (95% CI: 19.7 to 61.4%) and specificity was 71.4% (95% CI: 29 to 96.3%) for brain CT, in the detection of changes in cases with inflammatory CSF results A comparison of results found in the brain CT neuroimaging analysis versus CSF analysis is shown on Regarding myelopathies: Spine CT imaging was analyzed in 8 dogs. The relative sensitivity value was 57.1% (95% CI: 18.4 to 90.1%) and specificity 25% (95% CI: 0.6 to 80.6%) for spine CT in the detection of changes in CSF inflammatory results. A comparison of results found in the spine CT neuroimaging analysis versus CSF analysis results is shown in Overall, in the CT cases, the most common abnormality was multiple hypoattenuating or hyperattenuating lesions with or without contrast enlargement in the meninges and/or inside the lesions.3.3.Of the 168 dogs, it was noted that the most common neurologic clinical signs were seizures. This included, in the presumptive MUO group, 56/152 (36.8%) of the cases and, in the ME infection group, 5/16 (31.3%) of the cases. This observation was followed by ataxia 37/152 (24.3%) in the MUO group and 3/16 (18.8%) in ME infection; complete neurological signs are presented in Myoclonus was detected in ten dogs and, of those, six had CSF abnormal findings, five were positive for CDV, and one was positive for toxoplasmosis and neosporosis.A total of 29/152 (17.3%) of dogs showed neurological clinical signs that suggested myelopathies, such as diffuse spine pain, neck pain, tetraparesis, and paraparesis.4.4.1.Presumptive MUO was significantly more common than infectious ME in Brazilian cases. Our findings corroborate a recent study in England that reported MUO 83.6%) as more common than infectious ME (16.4%) . In the % as moreOur study found, 4 agents were diagnosed by the PCR panel inclusive of CDV in 10 cases, toxoplasmosis in two cases, one case toxoplasmosis plus neosporosis, and borreliosis in three cases. This contrasts a recent study from England that reported more infectious ME caused by bacterial infections than CDV . CDV inf4.2.The investigation by Lamb et al. found abnormal MRI results in 76% of dogs with abnormal (inflammatory) results in the CSF analysis . ConversIn the present investigation, samples from 22.7% of dogs with an abnormal brain and spinal cord MRI that was suggestive of a diagnosis of ME had a normal result in the CSF analysis. This finding is consistent with another study that showed that dogs with abnormal MRI suggestive of MUO could have normal results in the CSF analysis . To thisThe present study found that MRI is almost two times more sensitive than CT for finding intracranial inflammation in dogs with abnormal (inflammatory) CSF results. This information is consistent with a previous study which reported that MRI was more sensitive than CT for detecting lesions associated with intracranial inflammation in dogs , 21. NotOf all the MRI performed in CDV cases, 50% showed some abnormality, as small areas with hyperintensity in T2 and FLAIR. This corroborates previous MRI findings of CDV cases that showed demyelination signs hypersintensity in T2 and FLAIR images .Regarding myelopathies, the present study showed that MRI is more sensitive than CT which is also consistent with previous studies that recommend MRI over CT for the detection of lesions in the spinal cord .4.3.The present investigation included a higher proportion of younger dogs with ME which is consistent with a previous investigation that reported dogs aged between 3 and 7 years are most commonly affected by all subtypes of MUO . In the Seizure is the most common neurological clinical sign observed in the presumptive MUO and infectious ME groups. Seizure activity was consistent with that reported in a previous study in dogs with MUO , 14. InfIn a previous report, 8% of dogs diagnosed with MUO presented with neurological signs suggestive of myelopathy . There aThe definition of median survival time (MST) was beyond the scope of this investigation. This information has been previously discussed in the literature , 8. Like4.4.The results of this paper may be influenced by the study location which was inclusive of one private practice veterinary hospital in Brazil. In addition, the case review and imaging interpretation also used one examiner (FB) which could add bias. In addition, histopathology was not performed in order to confirm each diagnosis. That is, some lesions identified as consistent with MUO, could be reflective of another disease such as neoplasia or infectious granuloma. In addition, the sensitivity and specificity of the infectious disease PCR panel are unknown. Moreover, this panel is limited to 10 agents and thus is not inclusive of all infectious agents that can induce ME in dogs. Lastly, cultures were not performed on CSF samples.5.The most common ME in this population of dogs was presumptive MUO, confirming our primary hypothesis. Testing with an infectious disease PCR panel is an important tool to exclude infectious ME, although, a larger PCR panel would be preferred. Normal CSF analysis does not preclude positive results in the PCR panel; however, a positive PCR panel usually accompanies an abnormal CSF analysis. MRI was more sensitive than CT to detect inflammatory changes in the brain and spinal cord, however, the use of CSF analysis is important to rule out inflammation even when MRI or CT are normal. An abnormal MRI or CT can be observed in the absence of abnormal CSF results. Additionally, the most common clinical signs in ME were seizures and age was not significantly different between groups with infectious ME and presumptive MUO cases.The original contributions presented in the study are included in the article/The animal study was approved by Reviewed by Ethics Committee of Pontifical Catholic University of Paraná . The study was conducted in accordance with the local legislation and institutional requirements.FB conducted neurological examinations, magnetic resonances, image analysis, manuscript writing, results, and discussion. CC has improved English syntax and grammar and analysis of results and discussion. AB helped with data collection, manuscript writing and referencing, and organization. JV assisted in the correction of the manuscript. FM-F assisted in writing the manuscript, syntax, and analysis of results and discussion. All authors contributed to the article and approved the submitted version.


Safety and immunogenicity of a single dose of MenACYW-TT were evaluated in 10-17 years old healthy adolescents vs a quadrivalent (MCV4-TT [Nimenrix®]) meningococcal vaccine licensed ex-US. Co-administration of MenACYW-TT with 9vHPV and Tdap-IPV vaccines was also studied.MenACYW-TT  conducted in Hungary, Italy, Spain, and Singapore, meningococcal vaccine-naïve adolescents or MenC-primed (when < 2 years of age) were randomized to receive either MenACYW-TT or MCV4-TT and co-administration with 9vHPV and Tdap-IPV vaccines. Serum bactericidal assays with human complement (hSBA) were used to measure anti-meningococcal antibodies against all 4 serogroups at baseline and 30 days post-vaccination. Antibodies against antigens contained in Tdap-IPV and 9vHPV were also measured. Safety data were collected within 30 days post-vaccination.(Table). When MenACYW-TT was administered alone or concomitantly with 9vHPV and Tdap-IPV vaccines, the immunogenicity results were comparable or higher in subjects receiving MenACYW-TT alone than in those receiving all vaccines concomitantly. The safety profiles were comparable between MenACYW-TT and MCV4-TT, administered alone or concomitantly with 9vHPV and Tdap-IPV vaccines, with no safety concerns observed during this study.A total of 463 subjects were enrolled in this study. Non-inferiority between MenACYW-TT and MCV4-TT, based on seroprotection rate (percentage of subjects achieving hSBA titers ≥ 1:8), was demonstrated for all 4 serogroups one month following meningococcal vaccination. Thirty days post vaccination, hSBA geometric mean titers (GMTs) were higher for MenACYW-TT vs MCV4-TT for serogroups C, Y and W, and comparable for serogroup A. The percentages of subjects with an hSBA vaccine seroresponse were higher for MenACYW-TT vs MCV4-TT for all 4 serogroups The trial met the primary objective. MenACYW-TT induced non-inferior immune responses to all serogroups based on seroprotection rates, and higher or comparable GMTs and seroresponse rates compared to MCV4-TT.Céline Zocchetti, MSc, Sanofi: Stocks/Bonds Olga Syrkina, MD, Sanofi: Stocks/Bonds Siham Bchir, MSc, Sanofi: Sanofi's Employee|Sanofi: Stocks/Bonds Isabelle Bertrand-Gerentes, MD, Sanofi: Stocks/Bonds


Cerebrospinal fluid (CSF) is crucial for maintaining neuronal homeostasis, providing nutrition, and removing metabolic waste from the brain. However, the relationship between neuronal activity and CSF solute transport remains poorly understood. To investigate the effect of regional neuronal activity on CSF solute transport, Sprague–Dawley rats  under anesthesia received an intracisternal injection of a fluorescent tracer  and were subjected to unilateral electrical stimulation of a forelimb. Two groups (n = 10 each) underwent two different types of stimulation protocols for 90 min, one including intermittent 7.5-s resting periods and the other without rest. The control group was not stimulated. Compared to the control, the stimulation without resting periods led to increased transport across most of the cortical areas, including the ventricles. The group that received intermittent stimulation showed an elevated level of solute uptake in limited areas, i.e., near/within the ventricles and on the ventral brain surface. Interhemispheric differences in CSF solute transport were also found in the cortical regions that overlap with the forelimb sensorimotor area. These findings suggest that neuronal activity may trigger local and brain-wide increases in CSF solute transport, contributing to waste clearance. Thus, efficient clearance of waste from the brain is essential to maintain its normal homeostasis. Suboptimal waste clearance and aberrant brain lymphatic function have been observed in several pathological conditions, including stroke3, traumatic brain injury4, normal pressure hydrocephalus5, as well as physiological factors such as hypertension6 and aging8. Although the direct relationship between waste clearance and its buildup in the brain is still under intense investigation, progressive accumulation of metabolic byproducts and their derivatives, such as beta-amyloid and tau-protein plaques, is a hallmark sign in Alzheimer's disease9. Similarly, inefficient waste clearance from the brain is found to be associated with the accumulation of alpha-synuclein proteins in Parkinson's disease10 and dementia with Lewy bodies12.The brain is a highly metabolic organ that produces a significant amount of waste byproducts from neuronal activity13. Although the detailed cytoarchitecture and biophysical mechanism involved in the lymphatic function of the central nervous system (CNS) is not fully understood, it is known that the CNS lymphatic system is different in both anatomy and function from those found in other organs16. Studies have suggested that waste produced by neuronal cells, in the form of solutes, is transported to the interstitial fluid (ISF) and subsequently exchanged with the cerebrospinal fluid (CSF) via various mechanisms, including aquaporin-4 (AQP4) water channels18, osmotic pressure gradient19, active ion transport20, and passive diffusion21, before its ultimate exit from the CNS to the peripheral lymphatic system. Thus, CSF solute transport in the brain plays an important role in removing waste products from the brain23.The lymphatic system, other than its important function in mediating immune responses and fluid balance, plays a critical role in removing waste from organs24 whereby CSF solutes move along the perivascular space (PVS) which runs parallel to cerebral blood vessels, facilitating CSF/ISF solute exchange. The flow of CSF within the PVS has been shown to be driven by the contraction of smooth muscle cells surrounding the cerebral arterioles28. Meanwhile, neuronal activity is accompanied by localized vascular responses , such as changes in local cerebral blood flow (CBF)/cerebral blood volume (CBV), which are mediated by vessel wall dilation/contraction29. Recent studies have demonstrated the potential of vagus nerve stimulation to modulate CSF flow in rats31. Moreover, the cerebral vasodilation triggered by whisker stimulation in sensory regions of mice has been accompanied by increased CSF influx velocity within the activated hemisphere32. Additionally, photic stimulation of the visual cortex in humans yielded large-scale CSF flow that is aligned with visually evoked hemodynamic responses33. Despite these recent studies of CSF dynamics that are associated with neuronal activity, evaluation of the location and the degree of CSF solute transport responding to region-specific brain stimulation warrants further investigation.The presence and directionality of CSF movement around the cerebral vasculature have recently been identified by Mestre et al.31. Furthermore, the unilateral sensorimotor stimulation scheme allows for interhemispheric comparison, unlike the bilateral photic stimulation employed in the study by Williams et al.33 We evaluated the effects from two stimulation conditions that differ in the number of stimulations and their timing (with or without an introduction of resting periods), which are known to contribute to different cerebral hemodynamic responses34. Herein, we refer to the sustained stimulation without the introduction of a time gap as the sequential condition ('SEQ'), and the stimulation that accompanied a 7.5 s resting period between the batch of stimulation as the intermittent condition ('INT'). The SEQ condition would lead to an increase in blood flow until reaching a static level35, while the 7.5 s resting between stimuli in the INT condition would yield periodic hemodynamic changes36, thereby inducing dynamic vascular responses. To assess CSF solute transport impacted by the stimulation, we adopted a well-established technique involving the intracisternal injection of a fluorescent CSF tracer37. The tracer injection was conducted prior to initiating the stimulation, after which we evaluated the distribution of the tracer on the dorsal brain surface and within coronal sections that encompassed the stimulated brain regions.In the present study, we investigated the impact of region-specific neuronal activity within the sensorimotor cortex elicited by unilateral electrical stimulation of a forelimb on CSF solute transport in anesthetized Sprague–Dawley (SD) rats. The selection of forelimb stimulation mitigates potential confounding cardiovascular and respiratory effects on CSF solute transport associated with cranial nerve stimulationhttps://arriveguidelines.org/). Eight week-old male SD rats  were socially housed (two per cage) and maintained on a 12 h/12 h light/dark cycle  with ad libitum access to food and water.All animal procedures were conducted in accordance with the guidelines of the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women's Hospital (protocol 2020N000013). The reporting in this study is in accordance with the ARRIVE guidelines (i.p.) injection of a ketamine-xylazine mixture (80:10 mg/kg). The choice of ketamine-based anesthesia was based on prior research demonstrating a minimal effect of ketamine on sensory evoked potentials38. The depth of anesthesia was determined by the absence of pedal reflex to a toe pinch, and additional doses of anesthesia were administered as needed. After removing the fur over the scalp using clippers and depilation lotion, the animals were placed on a stereotaxic frame  after the application of ophthalmic ointment to the eyes, with the head tilted down ~ 30° ventrally for surgical access to the cisterna magna. The CSF tracer was administered via intracisternal injection using a previously established surgical protocol39. Briefly, a midline incision was made between the ears along the neck. Subcutaneous tissues and posterior vertebral muscles were retracted to expose the atlantooccipital membrane, and a 30-gauge needle filled with sterile artificial CSF  was inserted into the cisterna magna space at a depth of ~ 1–2 mm. Cyanoacrylate glue  was applied around the injection site to seal the puncture while securing the needle in place. Texas-Red OA  in aCSF (0.5 wt% concentration) was loaded in a polyethylene tube  and delivered into the CSF space at a rate of 2 µL/min for 10 min using a syringe pump . Following tracer administration, the open end of the tube was sealed to prevent backflow of the tracer solution, and the animal's head was returned to its natural prone position. The body temperature of the animals was maintained throughout the procedure using a water-circulating pad .The rats were anesthetized using intraperitoneal  at a frequency of 2 Hz, with 10 s intervals between initiation of each pulse train, for a duration of 90 min. The SEQ condition delivered electrical pulses with the same pulse durations and frequency (in 90 min), but without introducing the intervals in-between. Two electrical stimulation conditions were used to assess the effects of non-stimulated resting period  on the CSF solute transport. The choice of a relatively low stimulation frequency (2 Hz) was intended to mitigate the risk of neuronal adaptation during sustained stimulation (for 90 min) during the SEQ condition, while producing robust evoked responses43.Overall experimental procedure is illustrated in Fig. 44, we monitored heart rate and oxygen saturation (SpO2) every 5 min during the stimulation period using a pulse oximeter . The respiration rates were counted manually.The electrical stimulation was delivered through a pair of cuff electrodes, with the positive wire above the manus and the negative wire wrapped beneath the cubitus, using a stimulator . The stimulation intensity for each rat was individually adjusted between 7 and 14 mA until paw muscle twitching was observed. The rats in the control group underwent the same surgical and stimulation preparations but without the application of electrical stimulation. To evaluate whether the CSF solute transport was affected by heart rate or anesthetic depth45. Both bright-field and fluorescence images were taken under a fixed exposure time (0.1 s and 1 s), respectively.Animals were sacrificed immediately after the electrical stimulation through transcardial perfusion of 150 mL normal saline followed by 150 mL of 4% paraformaldehyde solution  at a flow rate of 25 mL/min. The skulls were detached then immersion-fixed for an additional 24 h in PFA prior to brain harvesting. After harvesting the brain, the dorsal surface of the brain was imaged using an inverted fluorescent microscope  equipped with an ultrawide field CMOS sensor  at an image size of 4912 × 2760 pixels. Bright-field and fluorescence images were acquired using a broadband light source and a fluorescent light configuration (EM: 560 nm/EX: 620 nm), respectively. Then, a block encompassing ± 2 mm  from bregma was then cut and embedded in low gelling-temperature agarose  and sectioned using a vibratome  in the rostral-caudal direction from + 1 to + 0.2 mm (from bregma) in 200 µm-thick slices, yielding five slices that contain the brain regions associated with forelimb stimulation46. The dorsal surface fluorescent images from each animal were aligned together based on the corresponding bright-field images, guided by the rostral edge of the neocortex and the interhemispheric fissure as references. After alignment, the images were smoothed with a 2D Gaussian filter (standard deviation of 2) and down-sampled (492 × 276 pixels) to reduce the contribution of cortical edges associated with cerebral blood vessels to allow for group-averaging of the fluorescence images.The transport of CSF solutes was quantified from both surface and sectional fluorescent brain images after extracting red fluorescence using ImageJ software47  was calculated from fluorescent images across the five sectioned brain slices per animal. Region-specific solute transport was measured by separately calculating MPI from 14 regions-of-interest (ROIs) manually segmented from a co-registered rat brain atlas in relation to the corresponding sectional images2) across experimental conditions over the 90 min stimulation period were evaluated using multi-way ANOVA. One-way ANOVA was used to assess the difference in animal weights across groups and to compare the fluorescent intensity values (MPI) obtained from the brain block as well as from the individual ROIs across experimental conditions. Differences in solute uptake among groups were examined using a Fisher's least significant difference (LSD) post-hoc test. A one-tailed paired t test was used for pixel-wise comparison of fluorescence intensity value obtained from dorsal surface images within each group. The significance level was set at p < 0.05 except for the pixel-by-pixel analysis of the fluorescence from the dorsal brain surface (p < 0.01).Statistical analysis was conducted using Matlab software . The differences in vital signs . The vital signs (respiratory/heart rates and SpO2) measured during the stimulation session are shown in Table p > 0.7).There was no difference in body weight among the three groups  = 0.07, p < 0.001), suggesting both SEQ and INT conditions yielded a higher total CSF solute uptake compared to the control. A post-hoc LSD analysis showed that the SEQ condition resulted in significantly higher MPI (30.7 ± 5.4) than the INT by 26.3%  and the control condition by 61.5% . Additionally, the INT condition exhibited a significantly higher MPI than the control condition by 27.9% (p < 0.05).The aligned fluorescence images of brain slices were averaged within each condition to represent the group-level distribution of CSF solute transport from the brain block Fig. a. One-wap < 0.05, detailed statistical results are presented in Supplementary Table post-hoc analysis showed that the SEQ condition showed the highest fluorescence value in both medial areas compared to the INT and the control conditions . The comparison of solute transport between the INT and control groups revealed higher MPI values in the INT from the MV area , but no difference was found in the MD .The individual MPI values obtained from each ROI are reported in Supplementary Table post-hoc test following one-way ANOVA, p < 0.05; detailed statistical results are reported in Supplementary Table post-hoc, all p < 0.05), while the marginal difference was observed in ipsilateral VS, TC, and OT regions .In addition to these medial brain regions, all bi-hemispheric ROIs Fig. d exhibitp < 0.05). On the other hand, the SEQ showed lower fluorescent intensity in the contralateral M1, S1, and OT compared to the opposite hemisphere . The control condition showed no significant interhemispheric differences.When the MPI values from these ROIs were compared between the hemispheres (CL vs IL) within each group . Pixel-wise comparison between hemispheres, conducted via one-tailed paired t test, revealed areas that showed distinctive hemispheric differences . In the present study, we examined the effect of region-specific brain activity elicited by electrical forelimb stimulation on the transport of an intracisternally injected CSF tracer, Texas Red OA, in rats.Despite extensive research, the exact routes of waste clearance from the brain are still unknown. However, increasing evidence suggests the importance of solute transport through the CSF in the clearance of waste produced by neuronal activity  were applied. Group-averaged fluorescence images of the coronal brain sections showed that neuronal activity increased CSF solute transport, and the overall OA uptake was higher in both the SEQ and INT stimulated groups compared to the unstimulated control condition Fig. a. The reion Fig. b. As to 57, were not different across experimental conditions, suggesting that their contributions to the enhanced CSF solute transport are not likely. We note that several physiological parameters known to impact CSF flow, such as intracranial pressure (ICP)58 and CO2 level59, were not directly monitored in our study as the procedure would involve invasive means to implement sensors. A study conducted by Illif et al. showed that ICP during intracisternal CSF tracer infusion in mice (1 µL/min for 10 min) resulted in a transient, mild elevation of (~ 2.5 mmHg) of ICP that rapidly recovered back to the pre-infusion level upon cessation25. A slightly higher infusion rate of 2 µL/min in our study, which was aimed to introduce enough tracer into a rat brain having much greater volume (by approximately four times), is unlikely to induce significant changes in ICP. It is supported by a study where a 60-min intracisternal infusion of a fluorescent tracer at a rate of 1.6 µL/min in rats did not perturb ICP60. The measured respiration rate and SpO2, which are closely linked to vascular CO2 level61, remained within the normal physiological range  under the SEQ condition compared to the control condition, while animals subjected to INT stimulation showed higher OA uptake in the MV Fig. c. Based 66). We conjecture that transport of intracisternally injected OA, following established major routes of CSF  was expedited in the SEQ condition compared to INT, resulting in the observed widespread solute uptake across the brain areas.Furthermore, ROI-based analysis of MPI demonstrated that the SEQ stimulation led to increased transport of CSF solutes across all identified ROIs when compared to the control condition. This suggests that localized neuronal activity can cause changes in CSF solute transport at a macroscopic scale beyond the primary site of the neuronal activity. Animals under the INT condition, however, showed limited locations of ROIs with a significant increase in OA uptake compared to the control condition , medial ventral (MV) region, as well as the temporal cortex (TC)/olfactory tubercles (OT) contralateral (CL) to the limb stimulation). Although not statistically significant, the INT condition still showed a degree of elevation of OA uptake in most of the ROIs. As to the mechanism behind this observation, we hypothesize that fewer neuronal events (with the introduction of wait time of 7.5 s) during the INT condition compared to SEQ resulted in a lower degree of neurovascular response, which caused CSF solutes not being able to reach all ROIs under given observation time (90 min). We observed that the regions that showed a marginal elevation of solute uptake from the INT condition (compared to control) aligned with the known pathway of intracisternally injected tracers either along the ventricular compartment or along the ventral surface of the brain that connected to PVS adjacent to the middle cerebral arteries  from activated astrocytes, resulting in a subsequent reduction of neuronal activity68. We also note that contribution from potentially persistent vasodilation69 or enlargement of cell volume70 might have played a role in impeding CSF movement within the PVS or interstitial solute transport during sustained neuronal stimulation during SEQ, subsequently reducing the solute transport efficiency71. Although our study primarily focused on the macroscopic distribution of CSF solute, in-vivo imaging techniques such as multi-photon microscopy24 may provide more detailed information about the tracer's movement and distribution in the PVS.Interhemispheric differences in CSF solute transport following unilateral limb stimulation were found from the bilateral ROIs Fig. d. Despit72.Fluorescence images of the dorsal brain surface also revealed a difference in OA uptake between hemispheres Fig. , support33, revealing the enhanced and region-specific solute transport associated with neuronal activity in anesthetized animals. It should be pointed out that previous studies on rodents have demonstrated that the state of brain activity, whether in an awakened or anesthetized state, significantly influences CSF solute transport73. Functional neuroimaging studies have further revealed spatial differences in neuronal response depending on the state of brain activity, with a higher spatial specificity during wakefulness in contrast to the widespread distribution observed under anesthesia74. Recent literature focusing on whisker stimulation also accentuated the presence of elevated CSF flow within the stimulated brain region under anesthetized conditions but not in awake mice32. Therefore, the possible effects of anesthetic agents on neuronal evoked potentials75 and CSF dynamics require further investigation in awake and natural sleep states.The findings of this study are consistent with previous observations on the interplay between neuronal activity and CSF solute transport77. A prior study that engaged an intermittent sensory stimulation with a higher-frequency (5 Hz)32 showed an increase in CSF solute transport only in the stimulated hemisphere, where our results demonstrated that low-frequency stimulation influences CSF solute transport in both the stimulated and opposite hemispheres. This variation might be attributed to the unilateral 2 Hz stimulation evoking bilateral neuronal activation42. We also note that stimulation of a specific nerve may lead to activation of a corresponding subregion of the brain78, which may influence the CSF flow in that region. Therefore, further exploration using diverse stimulation parameters and targets would be necessary to fully understand the relationship between local neuronal activity and CSF solute transport.Further direction for the current research may involve a more comprehensive investigation into diverse stimulation paradigms. The present study has demonstrated a significant difference in CSF solute transport depending on the hemodynamic responses induced by stimulation protocols, with and without the inclusion of intermittent resting intervals. However, it is important to note that various stimulation parameters such as intensity, rate, and duty cycle can exert a nonlinear impact on hemodynamic responsesW of 45 kDa) was based on its size similarity to the neuroactive Aβ oligomers, which are widely associated with Alzheimer's disease79. Although there was no direct evidence in the present study suggesting that neural activity improves waste clearance, it is possible that the wait time after the injection of the CSF tracer might have allowed the tracers to start exiting the brain80. While we have not examined the degree of tracer clearance to the cervical lymph nodes (primary sites of solute drainage from the brain), it is plausible to conjecture that neuronal activity may promote the solute clearance whereby prior research has established a functional relationship between a degree of CSF solute influx to brain parenchyma and its lymphatic drainage81. Thus, the effects of neuronal activity in the transport and clearance of different sizes of solutes in both CSF and interstitial space, including the assessment of solute drainage to cervical lymph nodes, will cast interesting subjects of future investigation.The choice of an OA as a CSF tracer (MWe presented early evidence of the neuronal activity elicited by electrical forelimb stimulation which increased regional CSF solute transport in the sensorimotor areas, with the inclusion of other brain areas that are in CSF transport pathways. Interhemispheric differences in CSF solute transport were observed in the stimulated somatosensory area, whereby the sustained stimulation without resting periods which reduced CSF transport efficiency may be associated with synaptic fatigue. The present work provides new insights to the link between neuronal activity and CSF solute transport. Further investigations are necessary to fully comprehend the scope of the relationship between neuronal activity and waste transport/clearance for the potential discovery of novel strategies for neurodegenerative disorders related to aberrant brain waste clearance.Supplementary Information.


Diabetes is a serious and quickly expanding global health problem. Cardiovascular disease is the leading cause of mortality in type 2 diabetes mellitus (T2DM) patients. Coronary slow flow (CSF) is characterised by delayed distal perfusion during coronary angiography with normal coronary arteries..This study aimed to investigate the correlation between CSF and inflammatory markers regarding glycemic status in T2DMThis cross-sectional study included 120 patients who were divided equally into 4 groups according to their glycemic control and presence or absence of coronary slow flow: Group I included patients with T2DM with good glycemic control without CSF; Group II included patients with T2DM with good glycemic control and CSF; Group III included patients with T2DM with poor glycemic control without CSF; and Group IV included patients with T2DM with poor glycemic control and CSF. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), C-reactive protein (CRP), platelets, hematocrit, and haemoglobin were also evaluated as risk factors for coronary slow flow.r = 0.53) with CSF in poorly controlled T2DM. NLR cutoff > 2.1 could predict CSF in poorly controlled T2DM with a modest sensitivity and specificity. A 1.9 increase in HbA1c increases the likelihood of coronary slow flow. Dylipidemia increases the likelihood of coronary slow flow by 0.18 times. Other predictors for coronary slow flow include NLR, PLR, CRP, platelets, hematocrit, and hemoglobin. The effect of the predictors is still statistically significant after being adjusted for glycemic status, age, and sex (p < 0.001).This study showed that body mass index (BMI), hematocrit level, NLR, and CRP demonstrated a moderate but significant correlation (Poor glycemic control increases the incidence of CSF. This supports the hypothesis that CSF is related to endothelial dysfunction as poor glycemic control causes endothelial dysfunction due to inflammation.ZU-IRB#9419–3-4–2022 Registered 3 April 2022, email. IRB_123@medicine.zu.edu.eg. In 2019, the International Diabetes Federation (IDF) predicted that Egypt ranks ninth among the world’s countries, with around 8,850,400 cases and a frequency of 15.2% in adults. Egypt is expected to be the seventh-largest country in the world by 2045 [Coronary heart disease (CHD), heart failure, arrhythmias, and sudden cardiac death are examples of macrovascular complications. Cerebrovascular disease and peripheral artery disease are both types of cardiovascular disease in T2DM. Complications contribute significantly to morbidity and death in both types of diabetes. Cardiovascular disease is the leading cause of mortality in T2DM patients .Glycemic control is evaluated by haemoglobin A1c (HbA1c), continuous glucose monitoring (CGM) with either time in range (TIR) and/or glucose management indicator (GMI), and blood glucose monitoring (BGM) .One of the major causes of atherosclerosis is diabetes. Inflammation has been clearly established to have a significant role in the initiation, development, and progression of atherosclerosis. CSF was a marker of endothelial activation and inflammation. HbA1c is also a significant inflammatory marker .Inflammation has a role in many cardiovascular diseases, and inflammatory pathways have been recognised in the setting of CSF. Other inflammatory indicators, such as red cell distribution width and serum uric acid levels, have also been linked to the presence of CSF. Inflammatory parameter abnormalities may be a sign of endothelial dysfunction, both of which lead to CSF ..Therefore, the current study aimed to investigate the correlation between CSF and inflammatory markers regarding glycemic status in T2DMWe enrolled 120 diabetic patients of type 2 with chronic coronary syndrome (CCS) at our Cardiology Department, Faculty of Medicine, Zagazig University, Egypt.All T2DM patients who were referred for coronary angiography and had coronary slow flow were included in the study. The presence of typical chest discomfort or positive or conflicting results from noninvasive screening measures for myocardial ischemia served as the justification for coronary angiography. If non-invasive assessment indicates high risk occurrences, coronary angiography is recommended. The CSF is basically defined as a delay in the progression of the contrast that is injected into the coronary arteries during coronary angiography .Patients with valvular heart disease, atrial fibrillation, cardiomyopathies, congenital heart disease and hematological disorders were excluded.n = 30); Group II: patients with T2DM with good glycemic control and CSF (n = 30); Group III: patients with T2DM with poor glycemic control without CSF (n = 30); Group IV: patients with T2DM with poor glycemic control and CSF (n = 30). NLR was measured in each group to see how it related to glycemic control parameters and predicted the occurrence of CSF. Other predictors of the occurrence of CSF were also investigated.This study included all patients with T2DM on conventional glycemic control (CGC); Admission blood glucose, 180–200 mg/dl) by conventional therapy using insulin and Dipeptidyl-peptidase 4 (DPP4) inhibitors who referred for coronary angiography who also had CSF. The patients were divided into four groups based on their glycemic control and the presence or absence of CSF. Group I: patients with T2DM with good glycemic control without CSF  are all considered.According to the ESH/ESC Guidelines for the Management of Arterial Hypertension, hypertension is defined as blood pressure levels of 140 mmHg SBP and/or 90 mmHg DBP .Diabetes mellitus was diagnosed on basis listed by American Diabetes Association (2010) as: Fasting blood sugar ≥ 126 mg/dl or 2 h postprandial blood sugar ≥ 200 mg/dl or HBA1C ≥ 6.5 or Symptoms of diabetes plus casual plasma glucose concentration ≥ 200 mg/dl. Casual is defined as any time of day without regard to time since last meal. The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss .Smoking was defined as active smoking in the last 6 months .Dyslipidemia was considered according to recommendations of Third Report of the National Cholesterol Education Program (NCEP), when any of the following was present: serum cholesterol ≥ 200 mg/dl, LDL ≥ 100 mg/dl, HDL < 40 mg/dl for high-risk patients [Family history of premature CAD, defined by the presence of at least a first degree relative with a cardiovascular event or premature SCD at a young age (< 65 years for women and < 55 years for men) .2, resulting from mass in kilograms and height in meters [Blood pressure, BMI, and electrocardiogram (ECG) were measured to all patients. Body Mass Index (BMI), defined as the body weight divided by the square of the body height in meters, and is universally expressed in units of kg/mn meters .The diagnostic criteria for CSF are based on the recommendations of Beltrame . At coroAll patients underwent TTE using the commercially available system  on admission for the coronary angiography procedure. Standard images were obtained using a 3.5–5 MHz transducer at a depth of 16 cm in the parasternal and apical views. The TTE study was performed according to the recommendations of the American Society of Echocardiography and European Association of Echocardiography, and measurements were indexed to body surface area when indicated. Assessment of resting regional wall motion abnormalities, wall motion score index (WMSI), ejection fraction and LV end-diastolic and end-systolic volumes were calculated using the biplane Simpson’s method and resting wall motion abnormalities , fasting plasma glucose, HbA1c, and hsCRP were studied. The NLR of the patients is calculated by dividing the absolute neutrophil number by the absolute lymphocyte number. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), C-reactive protein (CRP), platelets, hematocrit, and haemoglobin were also evaluated as risk factors for coronary slow flow.. Continuous data were represented as mean and SD, and categorical data were represented as event and percentage. One-way ANOVA, independent t-test, chi-square test, or exact Fisher test, and Pearson correlation were used. ROC curve analysis and the Kappa test were used to detect the ideal cut-off values of the predictors with the greatest sensitivity and specificity. When the probability of error is less than 5% (p < 0.05), the result is significant.Version 26 of the Statistical Program for Social Science (SPSS) was used to analyse the dataThis study revealed that there was no statistically significant difference between groups in terms of age, gender, or CAD family history.This investigation revealed that there was no statistically significant difference in haemoglobin levels across groups.p3, p5, p6 < 0.05) , and c-reactive protein (CRP) were significantly lower in patients without CSF  compared to those with CSF  (5) Table .Table 1LCSF correlates positively with NLR in patients with inadequate glucose management Table .Table 2Cr = 0.548) (Table p < 0.0001) (Table The current study looked at the relationship between coronary slow flow and NLR in relation to glycemic status. Coronary slow flow shows a moderately significant correlation with NLR (8) Table . When co1) Table , 3 and 4Inflammation plays a role as an important pathogenic factor for various cardiovascular diseases, along with coronary heart disease. So, the neutrophil-to-lymphocyte ratio (NLR) has been reported to affect various inflammatory diseases, including some cardiovascular diseases. NLR is also an important factor in cardiovascular diseases in terms of morbidity and mortality .In patients with T2DM and CSF, our study showed that NLR had a moderately significant correlation with glycemic control parameters. The study performed by Sefil et al.  reportedIn a prospective study on a non-diabetic 38,074-strong cohort, an average of 6-year follow-up NLR was associated with the incidence and prevalence of T2DM. This finding suggests that the NLR is a predictor of diabetes development .Our study found no significant relationship between NLR and glycemic control parameters in patients with T2DM who did not have CSF. This agreement with Mendes et al.  showed tOur study showed that NLR was higher in patients with CSF than in patients without CSF, especially in patients with poor glycemic control; this agrees with Cetin et al. ; OylumluDespite the studies mentioned above, the association between CSF and inflammation remains unclear due to the limited number of patients in these studies and conflicting data. In a study established by Altun et al. , they foOur study showed that poor glycemic control increases the incidence of CSF. A one-degree increase in FBS increases the likelihood of CSF by 2.4. The increase in HbA1c increases the incidence of CSF by 1.9. This can ensure the hypothesis that coronary slow flow is caused by endothelial dysfunction, as chronic hyperglycemia is well known to accelerate the development of endothelial dysfunction and the pathological process of atherosclerosis, resulting in more diffuse coronary artery lesions and worse clinical outcomes .n = 120), as it was a single-center investigation. Patients with type 2 diabetes were the only ones included. All diabetic individuals should be included in future trials. Our research did not include those on chronic medication. It's possible that the long-term use of medicine contributes to the onset of coronary sluggish flow. Therefore, additional long-term studies are suggested to examine the connection between medications and CSF occurrence, preferably including the medication of patients.The current study was restricted by its modest sample size .Poor glycemic control increases the incidence of CSF. This support the hypothesis that CSF is related to endothelial dysfunction as poor glycemic control cause endothelial dysfunction.Other factors  significantly increase in patients with CSF and can be used as predictors of CSF.Further large caliber, multicenter studies are needed to consolidate our findings. Further long-term studies which include patient medication are recommended to evaluate the relation between drugs and CSF occurrence. NLR may be useful as an easily measurable, non-invasive and cost-effective parameter for follow up of glycemic control in patients with CSF and also for prediction of CSF. Good glycemic control is mandatory as poor glycemic control leads to various cardiovascular events.


Liver fibrosis is a progressive histological manifestation that happens in almost all chronic liver diseases. An unabated liver fibrosis may eventually develop into liver cirrhosis or hepatocellular carcinoma. Yet, the strategy for reversal of liver fibrosis is still limited. Herein, a biomimetic nano‐regulator (P‐ZIF8‐cirDNAzyme) is developed to affect both collagen synthesis and degradation in liver to remodel collagen microenvironment. It is found that Zn (II) interference can efficiently inhibit collagen synthesis in activated hepatic stellate cells (aHSC) by inactivating proline 4 hydroxylase and affecting many fibrosis‐related signaling pathways. Meanwhile, Zn (II)‐dependent circular DNAzymes (cirDNAzymes) are used to efficiently silence tissue inhibitors of metalloproteinase‐1, accelerating the degradation of collagen. They act in concert to recover the balance between collagen deposition and degradation. Additionally, ZIF‐8‐cirDNAzyme is coated by platelet membrane (PM) for precisely targeting aHSC via PM's inflammatory tropism and CD62p–CD44 interaction. In carbon tetrachloride‐induced fibrotic mice, P‐ZIF‐8‐cirDNAzyme shows a potent anti‐fibrotic effect, greatly reducing the expression of collagen by 73.12% and restoring liver function nearly to normal. This work proposes a prospective platform enabling ion interference and gene silencing, collectively acting in aHSC for reversal of liver fibrosis. The biomimetic nano‐regulator precisely targets hepatic stellate cell and produces Zn (II) interference and gene silencing. Zn (II) interference effectively inhibits collagen deposition simultaneously activating circular DNAzyme to silence tissue inhibitor of metalloproteinase 1 mRNA, which accelerates the degradation of accumulated collagen. This mutual promotion strategy recovers the balance between collagen deposition and degradation and remodels the collagen microenvironment. Although several antifibrotic agents, including Resveratrol (for reduction of oxidative stress), obeticholic acid (for downregulation of collagen), and simtuzumab (for inhibition of collagen‐crosslinking), have been tested in clinical trials; limited efficacy and serious side effects remain major barriers limiting their application. Therefore, the development of efficient and specific strategy for reversal of liver fibrosis is highly desired.Liver fibrosis is a progressive histological manifestation of chronic liver disease associated with alcohol abuse, viral infection, cholestasis, and autoimmune diseases.1 If n Specifically, aHSC contain Proline 4 hydroxylase (P4H) enzyme with high activity. P4H can promote the formation of stable collagen by catalyzing the hydroxylation of collagen proline residues, which is a major mechanism for inducing large amount of collagen deposition. In addition, the tissue inhibitor of metalloproteinase 1 (TIMP‐1) in aHSC is overexpressed, which can bind and inhibit matrix metalloproteinase (MMP) in liver. Since MMP is an essential enzyme in degradation of collagen, the highly expressed TIMP‐1 is considered as another central mechanism for inducing liver fibrosis. Therefore, simultaneously regulating P4H and TIMP‐1 in aHSC may be a potential strategy to disturb the imbalance between collagen deposition and degradation for reversal of liver fibrosis.Liver fibrosis is characterized by persistent activation of hepatic stellate cells (aHSC), leading to the imbalance between collagen deposition and degradation.6 Spec ion interference could be an attractive strategy for inhibiting P4H enzyme activity. It has been reported that Zn (II) can efficiently inhibit P4H activity by replacing the Fe (II) core in the enzyme. Interestingly, lots of clinical investigations have shown that serum zinc deficiency is common in patients with chronic liver disease. It has also been demonstrated that Zn (II) has great potential in liver protection. However, the use of “Zn (II) interference” in aHSC for inhibiting P4H enzyme activity has not been reported.Since P4H enzyme is a member of Fe (II)‐dependent dioxygenase superfamily,10 ion However, the gene silencing efficacy remains limited due to the relatively low stability of siRNA. DNAzyme is a single‐stranded DNA molecule with excellent RNA cleavage activity in the presence of metal ion. Compared with siRNA, DNAzyme possesses attractive biostability and does not perform through the formation of RNA‐induced silencing complex. But precisely delivering DNAzyme to aHSC and providing enough metal cofactor for implementing the RNA‐cleaving activity remain a challenge.Except for inhibiting P4H enzyme activity, to recover balance between collagen deposition and degradation, strategies for promoting the degradation of deposited collagen are needed. Gene therapy has been applied as an efficient strategy for downregulation of collagen level in liver by using siRNA to interfere TIMP‐1 mRNA expression.14 How becoming a smart self‐driven nanoplatform and allowing for simultaneous ion interference and gene silencing to regulate aHSC. Meanwhile, it is critical to deliver nano‐formulation into the effector cells (aHSC) to exert maximum therapeutic effect. PM exhibits natural aggregation and adhesion to inflammatory sites. More importantly, its surface protein CD62p can specifically bind to the CD44 receptor which is highly expressed by aHSC. Meanwhile, PM has an excellent ability to escape elimination by mononuclear macrophage system and avoid inducing apparent immune response, showing great potential for targeted delivery of nano‐formulation to aHSC.Nano‐scaled zeolitic imidazolate framework‐8 (ZIF‐8) nanoparticles have been developed as a nanocarrier for DNAzyme delivery. Benefiting from its pH‐responsive structural collapse, ZIF‐8 can escape from lysosome and concurrently release Zn (II) and DNAzyme into cytoplasm,17 becSchemeHerein, we designed P‐ZIF‐8‐cirDNAzyme, an aHSC‐targeted biomimetic nano‐regulator composed of platelet membrane, ZIF‐8, and TIMP‐1 circular DNAzyme (cirDNAzyme), which acts on the dual pathway of inhibiting collagen synthesis and promoting collagen degradation to remodel the collagen microenvironment. As shown in 22.1 so we constructed a cirDNAzyme to achieve high mRNA‐silencing efficiency. CirDNAzyme was synthesized through the hybridization of complementary TIMP‐1 DNAzyme sequences, and T4 DNA ligase was used to achieve a highly efficient nick closing. The successful preparations of cirDNAzyme were verified by agarose gel electrophoresis, and we confirmed that cirDNAzyme can resist degradation by exonuclease I  , we first needed to design a TIMP‐1 DNAzyme. The “8‐17” deoxyribozyme was selected and converted into a therapeutic DNAzyme that specifically recognizes and cleaves TIMP‐1 mRNA. It has been reported that cyclization of the DNA structure could further improve the stability of oligonucleotide drugs in vivo,20 so −1), likely due to the strong electrostatic interaction between the metal nodes of the framework and cirDNAzyme's backbone phosphates, as well as the porous structure confinement of the nanoparticles. Additionally, we tested that the loading of Zn(II) in P‐ZIF‐8‐DNAzyme was about 19.54 wt% via inductively coupled plasma mass spectrometry (ICP‐MS). This provided a foundation for regulation of intracellular zinc ion concentration.To characterize the successful preparation of nanoparticles, transmission electron microscopy (TEM) was first performed. It showed that P‐ZIF‐8‐cirDNAzyme displayed a uniform spherical structure and with a core–shell morphology Figure . Besidesarticles.21 Add−1 correspond to the stretching vibrations of C—H in imidazole rings of ZIF‐8 and the peak at 3440 cm−1 correspond to the stretching vibration of O—H in organic materials (here may be mainly cirDNAzyme). And only the preparation containing PM showed a peak at 1653 cm−1, indicating that the flexural vibration of O—H in PM is presented. All the evidence above supported the successful fabrication of P‐ZIF‐8‐cirDNAzyme.Afterward, X‐ray diffraction (XRD) analysis revealed the P‐ZIF‐8‐cirDNAzyme retained a high crystallinity Figure , and FouTo confirm the successful translocation of PM onto the surface of P‐ZIF‐8‐cirDNAzyme, which can protect nanoparticles from immune clearance, we analyzed the protein composition of P‐ZIF‐8‐cirDNAzyme by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. It was found that the protein profiles were very similar between P‐ZIF‐8‐cirDNAzyme and PM Figure . Further2.2 Combined with the unstable feature of PM under acidic conditions, it is expected that the P‐ZIF‐8‐cirDNAzyme can release Zn (II) and cirDNAzymes in low pH environment. To investigate the drug release behavior of the nanoplatform, P‐ZIF‐8‐cirDNAzyme was incubated at different pH solution. TEM showed that the morphology of P‐ZIF‐8‐cirDNAzyme collapsed in acidic pH solution, indicating the acidic sensitivity of ZIF‐8  on P4H enzyme, we measured the activity of P4H under different Zn (II) concentration with an activity detection kit. It was found that Zn (II) inhibited P4H enzymatic reaction in a dose‐dependent manner Figure . To furtFurther analysis was then performed to determine whether P‐ZIF‐8‐cirDNAzyme can release adequate Zn (II) serving as cirDNAzyme's cofactor for biocatalysis. First, we verified the function of cirDNAzyme to silence TIMP‐1 substrate. As expected, the substrate‐cleaving efficiency displayed tight Zn (II) concentration‐dependence by polyacrylamide gel electrophoresis (PAGE) analysis Figure . Next, wTo explore the stability of P‐ZIF8‐cirDNAzyme, we dispersed PM‐coating and non‐PM‐coating nanoparticles in phosphate‐buffered saline (PBS) and 10% fetal bovine serum (FBS) over the course of 1 week. There were no obvious changes in the size and polydispersity index (PDI) of P‐ZIF‐8‐cirDNAzyme during 1 week. Besides, the encapsulation efficiency of cirDNAzyme in P‐ZIF‐8‐DNAzyme is still nearly 95% in seventh day. As a comparison, the size and PDI of ZIF‐8‐cirDNAzyme gradually increased, and the encapsulation efficiency was getting lower during the week, indicating that nanoparticle stabilization can be facilitated by cell membrane coating Figure . It demo2.3Figure. We also measured CD44 expression in different cells. A higher expression level of CD44 protein was found on HSCT6 cells than that on AML‐12 cells and Quiescent stellate cells , macrophages (Raw 264.7) and mouse hepatocyte (AML‐12). To conduct the study, we labeled cirDNAzyme with Cy5 and investigated the uptake differences of nanoparticles among HSCT6, Raw 264.7, and AML‐12. The flow cytometry analysis Figure3A showeAfter efficient cell internalization, to achieve cytoplasmic gene silencing, the escape of cirDNAzymes from lysosomes is essential. To visualize the endosomal escape process, cirDNAzymes were labeled with Cy5 and the endosomes were stained with lysotracker green. As shown in Figure 256 cells  level, we used Zinquin AM, a Zn (II) probe, to detect the alteration of intracellular Zn (II) concentration after incubated with P‐ZIF‐8‐cirDNAzyme. We found that the blue fluorescent intensity obviously increased after P‐ZIF‐8‐cirDNAzyme treatment Figure . Afterwas Figure , which i1 Figure . We alsoα‐smooth muscle actin (α‐SMA) protein in P‐ZIF‐8‐cirDNAzyme were also reduced  assay showed, compared with the other four groups , P‐ZIF‐8‐cirDNAzyme treatment could downregulate TIMP‐1 mRNA to 31.3% Figure , which rd Figure . These r2.4R2 > 0.95) was in the same treatment groups, but no significant correlations between control and P‐ZIF‐8‐cirDNAzyme  were downregulated, while MMPs that promote collagen breakdown and several genes that promote apoptosis and inhibit proliferation were upregulated. Additionally, as illustrated by the gene ontology (GO) enrichment analysis , which broadly participate in the fibrosis process, such as activation and proliferation of hepatic stellate cells, the polarization of Kupffer cells, and ECM production. In short, these results above implied that P‐ZIF‐8‐cirDNAzyme not only inhibited P4H activity and silenced TIMP‐1 mRNA as demonstrated in our experiments but also could participate in multiple anti‐fibrotic pathways more broadly.The result of the volcano map of differentially expressed genes (DEGs) further revealed that, of the 1551 DEGs, 843 (54%) were downregulated and 708 (46%) were upregulated in P‐ZIF‐8‐cirDNAzyme treated group Figure4A. Subss Figure , the DEGs Figure  showed t2.5Inspired by the excellent performance in vitro, the therapeutic effect of P‐ZIF‐8‐cirDNAzyme in vivo was further explored. First, we tested P‐ZIF‐8‐cirDNAzyme's biocompatibility in mice. P‐ZIF‐8‐cirDNAzyme consisted of excellent hemocompatibility under repeated intravenous dosing within 6 weeks ‐induced fibrotic mice and naked Cy5‐cirDNAzyme was used as a control group. As shown in Figure Notably, there are a large number of parenchymal cells in the liver, such as hepatocytes, which are mainly responsible for the metabolism of nanoparticles. Thus, we still needed to verify whether the nanoparticles could precisely target aHSC in the liver. Immunofluorescence images showed that the aHSC marker  colocalized with the fluorescence signal of the nanoparticles  in P‐ZIF‐8‐cirDNAzyme group  were injected via the tail vein into the carbon tetrachloride , interleukin 6 (IL‐6), and transforming growth factor β (TGF‐β) were highly downregulated with P‐ZIF‐8‐cirDNAzyme treatment  interference could inhibit the activity of P4H, thereby preventing the hydroxylation of collagen proline residues. Therefore, we tested the level of hydroxyproline in the liver to determine whether Zn (II) interference is effective in vivo  contained nanoparticles  treatment greatly decreased the level of hydroxyproline, indicating that Zn (II) interference indeed could inhibit the hydroxylation of collagen proline residues in vivo. Notably, P‐ZIF‐8‐cirDNAzyme treated had the lowest level of hydroxyproline, which was highly possibly attributed to cirDNAzyme's function of degradation of existing collagen. Accordingly, ICP‐MS also proved the increase of Zn (II) content within aHSC after P‐ZIF‐8‐cirDNAzyme treatment . Overall, this work represents a fascinating therapeutic platform enabling ion interference and gene silencing, collectively acting in aHSC for the reversal of liver fibrosis, which provides a new idea and paradigm for delaying the progression of liver disease.In conclusion, we developed a biomimetic nano‐regulator (P‐ZIF‐8‐cirDNAzyme) for precisely and efficiently regulating aHSC to remodel collagen microenvironment. We demonstrated that the nanoparticles with platelet membrane coating could escape the clearance of the mononuclear scavenger system and move toward the inflammatory fibrotic liver, significantly enhancing the accumulation of the nanoparticles in the liver. Furthermore, the platelet membrane surface protein CD62p can specifically bind to the CD44 receptor overexpressed on aHSC. This strategy indicated platelet membrane may act as an attractive material for precisely targeting aHSC. The accumulation of Zinc ions in aHSC significantly inhibited collagen synthesis via ion interference. Meanwhile, the circular DNAzyme exhibited high stability and downregulated TIMP‐1 mRNA expression in aHSC, greatly promoting the degradation of collagen. Consequently, P‐ZIF‐8‐cirDNAzyme recovered the balance between collagen deposition and degradation, and restored liver function nearly to normal. Besides, it showed a broad influence on multiple fibrosis–related signaling pathways 2·6H2O), 2,4‐dimethylimidazole 2 and enzyme collagenase were purchased from Sigma‐Aldrich . T4 DNA ligase and DNase I  were purchased from Novoprotein Scientific (China). Zinquin AM  was purchased from MedChemExpress (MCE) company. TPEN  was purchased from Aladdin. P4H activity assay kit  was purchased from Qiyuan Biotechnology . Lyso‐Tracker Red and Hoechst 33 342 (100 ×) were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. Bicinchoninic acid protein assay kit and D‐PBS  were purchased from Beijing Solarbio Science and Technology Co., Ltd. . Anti‐CD41 , anti‐CD47 , anti‐CD62p , anti‐GAPDH antibody , and goat anti‐rabbit IgG secondary  were purchased from Proteintech (China). Anti‐TIMP‐1 antibody  was purchased from Beijing Biosscn Biotechnology Co., Ltd. Anti‐Collagen I monoclonal antibody  and anti‐α‐SMA monoclonal antibody  were purchased from Abcam . Goat anti‐mouse IgG secondary antibody conjugated with Cy3  were purchased from Proteintech (China). DMEM/F12, HEPES, AML‐12 cell specific culture‐medium  and RPMI‐1640 were purchased from Procell Life Science and Technology Co., Ltd. Cell Counting Kit‐8  was purchased from Beyotime Biotech. Inc. . Lipofectamine 2000 Transfection Reagent  was purchased from Thermo Fisher. Thrombin , ADP , and TXB2  ELISA kits were purchased from Shanghai Jianglai industrial Limited By Share Ltd. Cy5‐DNAzyme and DNA substrate were purchased from Tsingke Biotech Co., Ltd. GelRed Nucleic acid dye  was purchased from Tsingke Biotech Co., Ltd. DEPC water was purchased from Sangon Biotech, Shang hai, China. TRIzon Reagent , 6× DNA Loading Buffer, and 1 kb DNA Marker were purchased from JiangSu CoWin Biotech (CWBIO). HiScript II 1st Strand cDNA Synthesis Kit  was purchased from Nanjing Vazyme Biotech Co., Ltd. qPCR assay kit  was purchased from TransGen Biotech in Beijing. Hydroxyproline assay kit and ELISA kit  were purchased from Shanghai Enzyme‐linked Biotechnology.Zinc nitrate hexahydrate (Zn(NOθ). Size distribution and zeta potential were measured by a Zeta sizer . The UV–vis spectrum was obtained using a UV–vis spectrometer . TGA profiles were acquired using a gravimetric analyzer . The cell viability was measured using a microplate reader . The fluorescence images were obtained from a laser scanning confocal microscopy . ICP‐MS results were acquired from an inductively coupled plasma mass spectrometer . In vivo biodistribution was detected using a small animal imaging system . Blood biochemistry analysis was conducted by an Auto biochemical analyzer . Blood routine was examined by Auto Hematology Analyzer .TEM images were obtained by a TEM . XRD patterns were gained from X‐ray power diffractometer  in the range of 5–50° 2·6H2O (30 mg mL−1) and 40 µL of cirDNAzyme (100 µm) were dissolved in deionized water (0.5 mL). 1 mL of 2‐methylimidazole (33 mg mL−1) was dissolved in methanol. Under stirring, the aqueous zinc nitrate and cirDNAzyme were added into the solution of 2‐methylimidazole and were incubated for 5 min. The product was collected by centrifugation and washed three times with a mixture of H2O and ethanol. ZIF‐8‐cirDNAzyme nanoparticles were obtained by vacuum drying. ZIF‐8 nanoparticles were synthesized by the similar method. P‐ZIF‐8‐cirDNAzyme was prepared by adding platelet membrane vesicles aqueous solution to the above products and the mixture was sonicated for 10 min. The obtained P‐ZIF‐8‐cirDNAzyme nanoparticles were dried in a vacuum.0.5 mL Zn (NO−1) was sufficiently dissolved into different pH  for different time points, respectively. Subsequently, the mixture was centrifuged at 12 000 rpm for 20 min, the supernatant was collected for detecting Zn (II) concentrations by ICP‐MS. Meanwhile, sediment was utilized to examine the change of UV absorbance from 200 to 400 nm by a UV–vis spectrometer .P‐ZIF‐8‐cirDNAzyme nanoparticle (100 µg mLP‐ZIF‐8‐cirDNAzymes (cirDNAzyme was labeled with Cy5) were incubated in pH 7.4, 6.8, and 5.5 at 37 °C, 150 rpm, and lucifuge environment for different time points. Subsequently, the mixture was centrifuged at 12 000 rpm for 20 min, the supernatant was collected and fluorescence intensity was measured by microplate reader . The DNA concentration was calculated by fluorescence intensity according to a standard curve of free Cy5‐cirDNAzyme.m) was incubated with an equal amount of TIMP‐1 substrate (500 nm) (Table m). After incubation, the cleavage efficiency was verified by polyacrylamide gel electrophoresis. P‐ZIF‐8‐cirDNAzyme (100 µg mL−1) and TIMP‐1 substrate (500 nm) were incubated at different pH  for 4 h. The cleavage efficiency was verified by polyacrylamide gel electrophoresis.The experiment of cirDNAzyme cleavage in vitro was carried out: cirDNAzyme  were incubated with Exo‐I at 37 °C for 2 h in PBS buffer and then the stability was analyzed by agarose gel electrophoresis. The experiment of P‐ZIF‐8‐cirDNAzyme stability in vitro was carried out: DNAzyme and P‐ZIF‐8‐cirDNAzyme with equivalent amount of DNAzyme were incubated with DNase I at 37 °C for 0, 10, 30, and 60 min, and then the stability was analyzed by PAGE.2+), 2‐oxoglutrate (2‐OG), and its catalytic substrate peptide complex system 1. To investigate the effect of Zn (II) on the catalytic activity of P4H, system 2 was constructed in this study, and the difference from the initial results of the system is that Fe2+ was replaced by Zn (II), and all structures were energy minimized under the Amber 99SB force field. The molecular simulation experiment was commissioned by Phad Calculation The P4H protein used in this study was encoded by mouse prolyl 4 hydroxylase subunit alpha‐1 gene (UniProt number Q60715). The 3D structure of P4H protein was downloaded from Alpha Fold 2 database under the number AF‐Q60715‐F1. Due to the high homology of the P4H protein, the crystal structure of the viral collagen prolyl hydroxylase (PDB number 5C5T) was used as the template to construct the cofactor metal iron ion (Fe2+ and Zn (II) was calculated. The binding free energy ΔGbind of protein P4H and polypeptide substrate was calculated using the MM/GBSA. The specific method is described as follows. The binding energy was calculated according to the following formula using the program gmx‐MMPBSA according to the kinetic simulation trajectory fileTo investigate the effect of Zn (II) on the ability of P4H to catalyze prolyl‐containing peptides, the binding capacity of P4H to the catalytic substrate peptides in the presence of cofactors FeGGAS is the kinetic energy difference between receptor and ligand before and after binding in vacuum, which is further subdivided into Eint, Evdw, and Eele, where Eint represents the energy change of bond, bond angle, and dihedral angle, Evdw is the van der Waals energy change before and after binding, and ΔEele is the electrostatic interaction change. ΔGsolv is a solvent effect term, which is divided into polar term ΔEGB and non‐polar term ΔESURF. The calculation of ΔEGB was relatively complex and time‐consuming, so APBS program was used to calculate this part. ∆ESURF was calculated by solution accessible surface area, where γ = 0.0227 kJ (mol nm2)−1, β = 3.85 kJ mol−1. TΔS is the entropy change, which was the most time‐consuming and difficult to calculate. At present, the calculation accuracy of this term is low, and this energy had little influence on the contribution of binding free energy. Therefore, this term was regarded as 0 when calculating the binding free energy.Δ2 and all experiments were conducted on cells in the exponential growth phase.HSCT6 cells were purchased from the Kunming Cell Bank of Chinese Academy of Sciences . Raw 264.7 cells were purchased from Cell Bank of Chinese Academy of Science . HSCT6 cells were maintained DMEM/F‐12 supplemented with 10% FBS (Gibco). Raw 264.7 cells were maintained in DMEM (high glucose medium) with 15% FBS . All cells were cultured at 37 °C in a humidified incubator containing 5% CO5 per dish and cultured for 12 h. After that, cells were washed with PBS for three times and incubated with Cy5‐cirDNAzyme, ZIF‐8‐Cy5‐cirDNAzyme, and P‐ZIF‐8‐Cy5‐cirDNAzyme (10 µg mL−1) for 4 h. For anti‐CD62p/anti‐CD44 antibody block group, first anti‐CD62p antibody was incubated with P‐ZIF‐8‐cirDNAzyme or anti‐CD44 antibody was incubated with HSCT6 cells for 2 h. Then the pre‐treated P‐ZIF‐8‐cirDNAzymes were incubated with HSCT6 cells for 4 h. After incubation, cells were washed with PBS three times and stained with 1 mL Hoechst 33342 for 10 min. After staining, images were acquired using a Leica laser scanning confocal microscope . Last, the cells were collected by trypsin digestion. The fluorescence intensity of collected cells was detected by a flow cytometer .HSCT6, Raw 264.7, and AML‐12 cells were seeded at 35 mm confocal dish at 1 × 105 per dish and cultured for 12 h. After that, cells were washed with PBS for three times and incubated with P‐ZIF‐8‐Cy5‐cirDNAzyme (10 µg mL−1) for 0.5, 1, 2, and 4 h. After incubation, cells were washed with PBS for three times and incubated with Lyso‐Tracker Red (75 nm) for 15 min. Then cells were washed with PBS for three times and stained with 1 mL Hoechst 33342 for 10 min. After staining, images were acquired and the person correlation coefficients were analyzed by a Leica laser scanning confocal microscope .Cells were seeded at a 35 mm confocal dish at 1 × 10−1), cells were seeded at 35 mm confocal dish at 1 × 105 per dish and cultured for 12 h. After that, cells were washed with PBS for three times and incubated with P‐ZIF‐8‐cirDNAzyme (10 µg mL−1) for 4 h. For TPEN group, HSCT6 cells were first incubated with 10 µg mL−1 P‐ZIF‐8‐cirDNAzyme for 4 h and then treated with 1 µm TPEN for 0.5 h. After incubation, cells were washed with PBS for three times. Subsequently, the Zinquin  were incubated with cells for indicating intracellular free Zn (II) accumulation. Last, a Leica laser scanning confocal microscope was utilized for characterizing fluorescence intensity.To dectect the intracellular free Zn (II) accumulation by P‐ZIF‐8‐cirDNAzyme  for 4 h. After incubation, cells were washed with PBS for three times and collected by trypsin digestion. The collected cells were resolved by nitric acid and hydrogen peroxide . The mixture was added with deionized water to 10 mL. The final solutions were used to measure the concentration of Zn (II) by ICP‐MS.Cells were seeded at a 35 mm confocal dish at 1 × 105 per well and cultured for 12 h, and then were incubated with 10 µg mL−1 of different nanoparticles for 24 h. After incubation, precipitate of cells was completely collected by trypsin digestion and centrifugation . Then mRNA was isolated with TRIzol Reagent kit. 2 µg of RNA sample was utilized to obtain cDNA. 2 µL of cDNA sample and specific primers  and washed twice with PBS. Then RIPA and PMSF were used to Crack cells. The protein content was measured by Bicinchoninic Acid Protein quantification kit. Western blot analysis was conducted using standard method and the GAPDH was used as internal references.Cells were seeded at 6‐well plates at 1 × 105 per well and cultured for 12 h, and then were incubated with 10 µg mL−1 of different nanoparticles for 24 h. After incubation, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then after permeabilized and closured, they were incubated with collagen antibody at 4 °C overnight. Afterward, they were stained with fluorescent secondary antibody. After staining, images were acquired using a fluorescence microscope.Cells were seeded at 6‐well plates at 1 × 104 per well and cultured in 100 µL fresh complete medium for 12 h. Another complete medium containing P‐ZIF‐8‐cirDNAzyme  were added, respectively. After incubating for 24 h, 10 µL CCK8 was added into the medium and kept for another 1 h. The optical density (OD) was measured at 450 nm by a microplate reader . Cell viability of different nanoparticles was calculated by the following formula: cell viability (%) = (ODsample − ODblank/ODcontrol − ODblank) × 100%.The in vitro cytotoxicity assay of P‐ZIF‐8‐cirDNAzyme was conducted with CCK8 assay following the literature procedure. HSCT6 cells were seeded in 96‐well plates at 1 × 105 per well and cultured for 12 h, and then were incubated with 10 µg mL−1 P‐ZIF‐8‐cirDNAzyme for 24 h. Untreated HSCT6 cells were used as control group. Each group had three parallel replicates. After P‐ZIF‐8‐cirDNAzyme incubation, cells were washed, digested, and centrifugally collected for transcriptome analysis. RNA was extracted with Trizol Reagent kit. RNA sequencing and bioinformatic data collection were performed by Majorbio .HSCT6 cells were seeded at 6‐well plates at 1 × 104, diluted in mineral oil (1:3), was given by intraperitoneal (i.p.) injection two times a week for 6 weeks (4 µLg−1 body weight); mice in the nonfibrotic control group received oil alone.C57BL/6J mice  were purchased from Hunan SJA Laboratory Animal Company, and fed at the condition of 25 ± 2 °C and 55% humidity with 12 h light/dark cycle. The license number is SCXK (xiang) 2019‐0004. All the animal experiments were performed in accordance with the guidelines of the Regional Ethics Committee for Animal Experiments and the Care Regulations approved by the Institutional Animal Care and Use Committee of Zhengzhou University. The animal laboratory's accreditation number is 110 322 211 102 955 054. Mice were first randomly divided into groups containing at least five mice per group. Then, CCl4 induced liver fibrosis mice were divided into five groups. The different formulations (P‐ZIF‐8‐cirDNAzyme 10 µg mL−1) were intravenously injected once a day for 3 days. The blood of mice was collected after injection 24 h. The samples were utilized to examine those parameters of blood biochemistry and blood routine by Auto biochemical analyzer  and Auto Hematology Analyzer , respectively.The CClm nylon strainer was used to filter out undigested tissue. The cells were washed three times with Hank's solution without calcium and magnesium ions. The first and third times were centrifuged at 1700 r min−1 at 4 °C for 6 min, and the supernatant was discarded. After a second centrifugation at 400 r min−1 for 5 min at 4 °C, the supernatant was aspirated. The precipitate was resuspended with 1 mL Percoll and then added to the top layer of 8 mL 60% Percoll gradient centrifugation solution for centrifugation at 4 °C and 9600 r min−1 for 21 min. After centrifugation, the top layer of the liquid was the yellow‐white hepatocyte debris layer, and the lower layer of white material was the aHSC layer. This layer of material was carefully sucked through a straw, resuspended to 50 mL, and washed twice with RPMI‐1640 supplemented with antibiotics at 4 °C, 1700 r min−1, centrifuged for 6 min, and the precipitate was the aHSC.Activated hepatic stellate cells were obtained by in situ liver perfusion and density gradient centrifugation. The specific operation is as follows. After the limbs were fixed, 75% alcohol was sprayed to disinfect the chest and abdomen. The abdomen was cut through a wedge to fully expose the subhepatic inferior vena cava and portal vein. The suprahepatic inferior vena cava was clamped, the portal vein was punctured with a 24 G vein trocar, and the fixed cannula was ligated. Hank's solution without calcium and magnesium ions was infused, and the infrahepatic inferior vena cava was cut as the outflow tract. The hearts were punctured and the mice were sacrificed, then 1 mL of collagenase was injected into a 24 G venous cannula. The portal ligament and falciform ligament were cut, and the liver was removed intact and placed in Hank's solution without calcium and magnesium ions at 4 °C. The liver was ground into small pieces (diameter < 1 mm), then 9 mL collagenase was added and poured into a small beaker and sealed. The digestion was carried out in a water bath at 37 °C for 30 min with shaking. Then RPMI‐1640 medium containing serum at 4 °C was added and diluted for 2 min with stirring. The 100 µ−1 body weight) were injected into fibrotic mice at different times via the tail vein. Mice were anesthetized and their hairs were removed through the shaving device. The mice were imaged 4, 8, 12, 24, and 48 h later using an in vivo imaging system . Corresponding ex vivo imaging assays were also performed using excised organs.For in vivo imaging studies, free Cy5‐cirDNAzyme, ZIF‐8‐Cy5‐cirDNAzyme, or P‐ZIF‐8‐Cy5‐cirDNAzyme (1 mg kg−1) were injected into fibrotic mice via the tail vein. Twenty‐four hours after the injection, mice were sacrificed and liver tissue was harvested and frozen‐sectioned. Sections were then stained with a rabbit anti‐mouse α‐SMA antibody  at 4 °C overnight to label HSCs. Sections were next incubated with DyLight 488‐labeled goat antirabbit secondary antibody 37 °C for 45 min, followed by staining of nuclei with 4,6‐diamidino‐2‐phenylindole . Finally, frozen sections were imaged with a MIDI Slide scanner .For localization studies, ZIF‐8‐Cy5‐cirDNAzyme and P‐ZIF‐8‐Cy5‐cirDNAzyme . Histological analysis was as follows: Paraffin‐embedded liver tissues were sectioned at 5 µm, then deparaffinized and hydrated. Thereafter, sections were stained with a H&E staining Kit  or with 0.1% w/v Sirius Red  in a saturated aqueous solution of picric acid for 1 h. Slides were then rinsed twice in 0.01 t‐tests for two‐group comparisons and one‐way analysis of variance (ANOVA) with post hoc Tukey's test for multiple‐group comparisons . Statistical analyses were performed using IBM SPSS Statistics 26 software.All studies were evaluated in at least three independent experiments for each condition to ensure reproducibility. Data were expressed as mean ± SD. Significant differences between different groups were determined using two‐tailed unpaired Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.