[Pharmacokinetics of main activ ingredients of Guhong injection in cerebral ischemia rats].

To investigate the pharmacokinetic characteristics of active constituents of Guhong injection in rats with cerebral ischemia reperfusion injury. The middle cerebral artery occlusion (MCAO) model was established in our studies, and then all the rats received iv administration of Guhong injection (2.1 mL·kg⁻¹). The blood concentrations of aceglutamide and hydroxysafflor yellow A (HSYA) were determined by high performance liquid chromatography (HPLC) method at different time points. The concentration-time curves were drawn and pharmacokinetic data were obtained by DAS 3.2.6 software. The results showed that aceglutamide and HSYA showed good linear relationship within the ranges of 1.5-500 mg·L⁻¹ (R²=0.997 5) and 0.33-40 mg·L⁻¹ (R²=0.998 9) respectively. This quantitative method showed a high recovery rate, good precision and stability. The main pharmacokinetics parameters of t1/2α, t1/2ß, CL1, CL2, AUC0-t, AUC0-∞, Vd1, and Vd2 were (0.139±0.007) and (0.155±0.017) h, (0.803±0.046) and (2.233±0.410) h, (0.016±0) and (0.149±0.018) L·h⁻¹·kg⁻¹, (0.015±0.001) and (0.446±0.016) L·h⁻¹·kg⁻¹, (133.335±3.844) and (9.298±0.179) mg·h·L⁻¹, (143.851±3.595) and (14.464±1.451) mg·h·L⁻¹, (0.009±0.001) and (0.223±0.007) L·kg⁻¹, (0.006±0.001) and (0.212±0.032) L·kg⁻¹, respectively. The results showed that the established HPLC method was highly specific, and could be used for the simultaneous detection of aceglutamide and HSYA of Guhong injection in MCAO rats, which was conducive to pharmacokinetic studies. Pharmacokinetic data and parameters could provide reference for continuous administration and interval administration of the drug.

Machine learning integration of multi-modal analytical data for distinguishing abnormal botanical drugs and its application in Guhong injection.

BACKGROUND: Determination of batch-to-batch consistency of botanical drugs (BDs) has long been the bottleneck in quality evaluation primarily due to the chemical diversity inherent in BDs. This diversity presents an obstacle to achieving comprehensive standardization for BDs. Basically, a single detection mode likely leads to substandard analysis results as different classes of structures always possess distinct physicochemical properties. Whereas representing a workaround for multi-target standardization using multi-modal data, data processing for information from diverse sources is of great importance for the accuracy of classification. METHODS: In this research, multi-modal data of 78 batches of Guhong injections (GHIs) consisting of 52 normal and 26 abnormal samples were acquired by employing HPLC-UV, -ELSD, and quantitative 1H NMR (q1HNMR), of which data obtained was then individually used for Pearson correlation coefficient (PCC) calculation and partial least square-discriminant analysis (PLS-DA). Then, a mid-level data fusion method with data containing qualitative and quantitative information to establish a support vector machine (SVM) model for evaluating the batch-to-batch consistency of GHIs. RESULTS: The resulting outcomes showed that datasets from one detection mode (e.g., data from UV detectors only) are inadequate for accurately assessing the product's quality. The mid-level data fusion strategy for the quality evaluation enabled the classification of normal and abnormal batches of GHIs at 100% accuracy. CONCLUSIONS: A quality assessment strategy was successfully developed by leveraging a mid-level data fusion method for the batch-to-batch consistency evaluation of GHIs. This study highlights the promising utility of data from different detection modes for the quality evaluation of BDs. It also reminds manufacturers and researchers about the advantages of involving data fusion to handle multi-modal data. Especially when done jointly, this strategy can significantly increase the accuracy of product classification and serve as a capable tool for studies of other BDs.

Chemical composition, pharmacology and pharmacokinetic studies of GuHong injection in the treatment of ischemic stroke.

GuHong injection is composed of safflower and N-acetyl-L-glutamine. It is widely used in clinical for cerebrovascular diseases, such as ischemic stroke and related diseases. The objective of this review is to comprehensively summarize the most recent information related to GuHong in the treatment of stroke, including chemical composition, clinical studies, potential pharmacological mechanisms and pharmacokinetics. Additionally, it examines possible scientific gaps in current study and aims to provide a reliable reference for future GuHong studies. The systematic review reveals that the chemical composition of safflower in GuHong is more than 300 chemical components in five categories. GuHong injection is primarily used in clinical applications for acute ischemic stroke and related diseases. Pharmacological investigations have indicated that GuHong acts in the early and recovery stages of ischemic stroke by anti-inflammatory, anti-oxidative stress, anti-coagulation, neuroprotective and anti-apoptotic mechanisms simultaneously. Pharmacokinetic studies found that the main exposed substances in rat plasma after GuHong administration are hydroxysafflor yellow A and N-acetyl-L-glutamine, and N-acetyl-L-glutamine could exert its pharmacological effect across the blood-brain barrier. As a combination of Chinese herb and chemical drug, GuHong injection has great value in drug research and clinical treatment, especially for ischemic stroke disease. This article represents a comprehensive and systematic review of existing studies on GuHong injection, including chemical composition, pharmacological mechanism, and pharmacokinetics, which provides reference significance for the clinical treatment of ischemic stroke with GuHong, as well as provides guidance for further study.

A comparative study of the protective effects of Guhong injection and its component on cerebral ischemia-reperfusion injury based on the oxidation index.

Guhong injection (GHI), a compound preparation of Chinese and Western medicine, is composed of safflower water extract and aceglutamide, and has a certain therapeutic effect on cerebral ischemia diseases. In this study, we investigated and compared the protective effects of GHI, Honghua injection (HHI), and aceglutamide (ACG) on cerebral ischemia-reperfusion injury in Sprague-Dawley (SD) rats randomly assigned to the following 5 groups: Sham, MCAO, MCAO + GHI, MCAO + HHI, and MCAO + ACG. The results revealed that GHI, HHI, and ACG improved neurological functions and reduced the infarct volume, the contents of HIF-1α, PKC, and EPO, and the expression of NOX-4 and HIF-1α mRNA. The protein expression of HIF-1α and iNOS treated with GHI, HHI, and ACG was decreased, while that of PHD2 was increased. Meanwhile, the BrdU+/NeuN+ cell counts of SGZ and SVZ areas in the brain tissues of the GHI, HHI, and ACG groups were greater than those of the MCAO rats. Thus, GHI, HHI, and ACG can confer protection against cerebral ischemia-reperfusion injury, possibly through antioxidation. Our research findings may provide evidence for the effectiveness of the combination of traditional Chinese and Western medicine.

Guhong injection mitigates myocardial ischemia/reperfusion injury by activating GST P to inhibit ASK1-JNK/p38 pathway.

BACKGROUND: Guhong injection (GHI), a novel compound preparation that is composed of a chemical drug, namely aceglutamide, and the aqueous extract of safflower (Carthamus tinctorius L.), exhibits extreme antioxidative, antiapoptotic, anti-inflammatory, and neuroprotective effects. Since oxidative stress, apoptosis, and inflammatory response are all the dominant mechanisms of myocardial ischemia/reperfusion (MI/R) injury, we probe into the protective mechanism of GHI on MI/R injury for the first time. METHODS: In this research, we first employed molecular docking to determine whether three active ingredients in GHI, acetylglutamine (NAG), hydroxysafflor yellow A (HSYA), and syringin, possessed the potential activity to modulate the protein, glutathione S-transferase P (GST P). We further identified the protective effect of GHI on myocardial tissue with TTC staining, HE staining, TUNEL staining, and ELISA, and on H9c2 with flow cytometry and ELISA. We next explored whether the cardioprotective effect of GHI on left anterior descending ligation-reperfusion in rats and hypoxia/reoxygenation (H/R) in H9c2 cells was related to activate GST P to inhibit ASK1-JNK/p38 pathway via approaches of qRT-PCR and Western blot. RESULTS: Results of molecular docking indicated that all three compounds spontaneously docked to GST P, among them the binding affinities of both HSYA and syringin to GST P were higher than NAG. In vivo, GHI reduced myocardial infarction size and mitigated myocardial pathological injury. In vitro, GHI enhanced cell viability and extenuated depolarization of mitochondrial membrane potential. In addition, the results of in vivo and in vitro studies demonstrated that the cardioprotection of GHI was associated with improving the mRNA and protein expression levels of GST P to modulate oxidative stress, and inhibiting the levels of mRNA expression and protein phosphorylation of ASK1, JNK, and p38. However, the suppressed effect of GHI on ASK1-JNK/p38 pathway was reversed by ethacrynic acid (EA, a GST inhibitor), indicating that the regulation of GHI on ASK1-JNK/p38 was related to the activity of GST P. Besides, the in vitro results of qRT-PCR and western-blot also certified that the inhibited JNK and p38 further reduced Bax expression and elevated Bcl-2 expression to reduce the expression of caspase-3 to exert anti-apoptosis effects. CONCLUSION: Taken together, the cardioprotection of GHI mainly incarnated in activating GST P to relieve oxidation properties, thereby inhibiting ASK1-JNK/p38 pathway to suppress apoptosis.

Targeted Energy Metabolomics Combined with Spatial Metabolomics Study on the Efficacy of Guhong Injection Against Cerebral Ischemia Reperfusion.

Optimizing the metabolic phenotype to improve cerebral function is critical for treatment of cerebral ischemia-reperfusion (I/R) injury. Guhong injection (GHI), which comprised safflower extract and aceglutamide, is widely prescribed in Chinese medicine for the treatment of cerebrovascular diseases. In this study, a combination of LC-QQQ-MS and MALDI-MSI were utilized to explore tissue-specific metabolic alterations in the brain of I/R, as well as to evaluate the therapeutic effect of GHI. Pharmacological evaluation demonstrated that GHI can significantly improve infarction rate, neurological deficit, cerebral blood flow, and neuronal damage in I/R rats. Based on LC-QQQ-MS, 23 energy metabolites were found to be significantly altered in the I/R group compared to the sham group (P < 0.05). After GHI treatment, 12 metabolites, including G6P, TPP, NAD, citrate, succinate, malate, ATP, GTP, GDP, ADP, NADP, and FMN showed a significant tendency of returning to baseline values (P < 0.05). Based on MALDI-MSI, 4 metabolites in glycolysis and TCA, 4 metabolites in nucleic acid metabolism, 4 amino acid metabolites, and 6 metabolites were discovered and compared between the different groups in the four special regions of cortex, hippocampus, hypothalamus, and striatum. Parts of these were found to have significant changes after I/R in the special brain region, and were regulated by GHI. The study provides comprehensive and detailed information for specific metabolic reprogramming of brain tissue in rats with I/R, and the therapeutic effect of GHI. Schema describing the discovery strategies of integrated LC-MS and MALDI-MSI to identify cerebral ischemia reperfusion metabolic reprogramming and GHI therapeutic effects.

Discovery of anti-stroke active substances in Guhong injection based on multi-phenotypic screening of zebrafish.

Ischemic stroke is a leading cause of death worldwide, and it remains an urgent task to develop novel and alternative therapeutic strategies for the disease. We previously reported the positive effects of Guhong injection (GHI), composed of safflower extract and aceglutamide, in promoting functional recovery in ischemic stroke mice. However, the active substances and pharmacological mechanism of GHI is still elusive. Aiming to identify the active anti-stroke components in GHI, here we conducted a multi-phenotypic screening in zebrafish models of phenylhydrazine-induced thrombosis and ponatinib-induced cerebral ischemia. Peripheral and cerebral blood flow was quantified endogenously in erythrocytes fluorescence-labeled thrombosis fish, and baicalein and rutin were identified as major anti-thrombotic substances in GHI. Moreover, using a high-throughput video-tracking system, the effects of locomotion promotion of GHI and its main compounds were analyzed in cerebral ischemia model. Chlorogenic acid and gallic acid showed significant effects in preventing locomotor dyfunctions. Finally, GHI treatment greatly decreased the expression levels of coagulation factors F7 and F2, NF-κB and its mediated proinflammatory cytokines in the fish models. Molecular docking suggested strong affinities between baicalein and F7, and between active substances (baicalein, chlorogenic acid, gallic acid, and rutin) and NF-κB p65. In summary, our findings established a novel drug discovery method based on multi-phenotypic screening of zebrafish, provided endogenous evidences of GHI in preventing thrombus formation and promoting behavioral recovery after cerebral ischemia, and identified baicalein, rutin, chlorogenic acid, and gallic acid as active compounds in the management of ischemic stroke.

Guhong injection promotes post-stroke functional recovery via attenuating cortical inflammation and apoptosis in subacute stage of ischemic stroke.

BACKGROUND: As a leading cause of death and disability, alternative therapies for stroke are still limited by its complicated pathophysiological manifestations. Guhong injection (GHI), consisting of safflower aqueous extract and aceglutamide, has been widely applied for the clinical treatment of cerebrovascular diseases, especially ischemic stroke and post-stroke recovery, in China. Recently, a series of studies have reported the positive effect of GHI against cerebral ischemia/reperfusion injury via targeting various molecular mechanisms. However, questions remain on whether treatment with GHI contributes to better functional recovery after stroke and if so, the potential mechanisms and active substances. PURPOSE: The aim of this work was to explore the potential therapeutic possibilities of GHI for the neurological and behavioral recovery after stroke and to investigate the underlying molecular mechanisms as well as active substances. METHODS: The neural and motor deficits as well as cortical lesions after GHI treatment were investigated in a mouse model of transient ischemic stroke. Based on the substance identification of GHI, network pharmacology combined with an experimental verification method was used to systematically decipher the biological processes and signaling pathways closely related to GHI intervention in response to post-stroke functional outcomes. Subsequently, ingenuity pathway analysis (IPA) analysis was performed to determine the anti-stroke active substances targeting to the hub targets involved in the significant molecular pathways regulated by GHI treatment. RESULTS: Therapeutically, administration of GHI observably ameliorated the post-stroke recovery of neural and locomotor function as well as reduced infarct volume and histopathological damage to the cerebral cortex in subacute stroke mice. According to 26 identified or tentatively characterized substances in GHI, the compound-target-pathway network was built. Bioinformatics analysis suggested that inflammatory and apoptotic pathways were tightly associated with the anti-stroke effect of GHI. Based on protein-protein interaction network analysis, the hub targets (such as NF-κB p65, TNF-α, IL-6, IL-1ß, Bax, Bcl-2, and Caspase-3) involved in inflammation and apoptosis were selected. On the one hand, immunofluorescence and ELISA results showed that GHI (10 ml/kg) treatment obviously reduced NF-κB p65 nuclear translocation as well as decreased the abnormally elevated concentrations of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) in damaged cortex tissues. On the other hand, GHI (10 ml/kg) treatment significantly downregulated the number of TUNEL-positive apoptotic cells in ischemic cortex and effectively restored the abnormal expression of Bax, Bcl-2, and Caspase-3. Based on the results of IPA, hydroxysafflor yellow A, baicalin, scutellarin, gallic acid, syringin, chlorogenic acid, kaempferol, kaempferol-3-O-ß-rutinoside, and rutin acted synergistically on core targets, which could be considered as the active substances of GHI. CONCLUSION: Overall, the current findings showed that the beneficial action of GHI on improving post-stroke functional recovery of subacute stroke mice partly via the modulation of cortical inflammation and apoptosis. These findings not only provide a reliable reference for the clinical application of GHI, but also shed light on a promising alternative therapeutic strategy for ischemic stroke patients.

Efficacy of Guhong injection versus Butylphthalide and Sodium Chloride Injection for mild ischemic stroke: A multicenter controlled study.

BACKGROUND: Most studies on Guhong injection have involved a single center with a small sample size, and the level of clinical evidence is low. AIM: To assess the safety and efficacy of Guhong injection for mild ischemic stroke (IS). METHODS: A total of 399 IS patients treated at six hospitals from August 2018 to August 2019 were retrospectively analyzed. The patients were given Guhong injection (experimental group) or Butylphthalide and Sodium Chloride Injection (control group). Changes in National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS) scores were observed before treatment and at 1, 2, and 3 wk after treatment in each group. The efficacy and safety of Guhong injection for IS were assessed. Other medications taken by the patients were confounding factors for efficacy assessment. These factors were controlled by propensity score matching, and the results were further analyzed based on the matching. RESULTS: The marked response rates at three follow-up visits were 64.64%, 74.7%, and 66.7% in the experimental group, and 48.26%, 45.4%, and 22.2% in the control group. The marked response rates increased significantly in the experimental group compared with the control group (P < 0.05). The overall response rate at the first visit (days 7 ± 2) did not differ significantly between the two groups, but differed significantly at the second (days 14 ± 2) and third visits (days 21 ± 3) (P < 0.05). The proportion of patients without any symptoms in the experimental group was significant different at the first visit (P < 0.05), but not significantly different at the second visit. The two groups showed no significant difference in the baseline distribution of mRS scores. At the first and second visits, the change in mRS scores was -2 and -1 in the experimental and control groups, respectively, which were significantly different (P < 0.05). After propensity score matching, the overall response rate and marked response rate were 97.29% and 100% in the experimental group (P > 0.05) and 64.0% and 47.7% in the control group (P < 0.05) at the first visit, respectively. The decreased NIHSS scores in the two groups were significant different (P < 0.05). The overall response rate and marked response rate differed significantly between the two groups at the second visit (P < 0.05). There was no significant difference in the incidence of adverse events between the two groups. No severe adverse events occurred in either group. CONCLUSION: Guhong injection is safe and more effective than Butylphthalide and Sodium Chloride Injection for treatment of IS.

Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

Guhong injection (GHI) is a drug for ischemic stroke created by combining safflower, a traditional Chinese medicine, and aceglutamide, a Western medicine. In this study, we investigated the curative effect of GHI on cerebral ischemia-reperfusion (I/R) injury via the PKC/HIF-1α pathway in rats. Adult male Sprague Dawley rats were randomly divided into seven groups: sham-operated, middle cerebral artery occlusion (MCAO), GHI, nimodipine injection (NMDP), MCAO + LY317615 (PKC inhibitor), GHI + LY317615, and NMDP + LY317615. After establishing an MCAO rat model, we performed neurological deficit testing, 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin (HE) staining, enzyme-linked immunosorbent assay, Western blotting, and q-PCR to detect the brain damage in rats. Compared with the MCAO group, the GHI and GHI + LY317615 group showed neurological damage amelioration as well as decreases in serum hypoxia-inducible factor-1α (HIF-1α), protein kinase C (PKC), and erythropoietin levels; brain HIF-1α and inducible nitric oxide synthase protein expression; and brain HIF-1α and NOX-4 mRNA expression. These effects were similar to those in the positive control groups NMDP and NMDP + LY317615. Thus, our results confirmed GHI can ameliorate cerebral I/R injury in MCAO rats possibly via the PKC/HIF-1α pathway.

Protective Effect of Naoxintong Capsule () Combined with Guhong Injection () on Rat Brain Microvascular Endothelial Cells during Cerebral Ischemia-Reperfusion Injury.

OBJECTIVE: To investigate the synergistic effect of Naoxintong Capsule (NXTC, ) and Guhong Injection (GHI, ) on cerebral ischemia-reperfusion (I/R) injury. METHODS: Forty-eight Sprague-Dawley rats were divided into 6 groups: control group, oxygen and glucose deprivation (OGD) group, nimodipine group (9.375 mg/kg), NXTC group (0.5 g/kg), GHI group (5 mL/kg) and NXTC+GHI group (0.5 g/kg NXTC+5 mL/kg GHI), after the onset of reperfusion and once per day for the following 7 days. Blood was collected 1 h after final administration, and the sera were collected. Cultured primary rat brain microvascular endothelial cells (rBMECs) were subjected to OGD to establish a cell injury model. Untreated rBMECs were used as blank control. The cell counting kit-8 assay was used to assess cell viability using the sera. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were assessed using an enzyme-linked immunosorbent assay. Apoptosis was evaluated after Hoechst33342 staining using fluorescence microscopy and flow cytometry. JC-1 staining was performed to assess changes in mitochondrial membrane potential. RESULTS: Statistical analysis indicated that more than 95% of the cells were rBMECs. Compared with the OGD group, the cellular morphology of the all drug delivery groups improved. In particular, the combined drug group had the most significant effect. Compared with the OGD group, all drug intervention groups induced a decrease in the apoptotic rate of rBMECs, increased the SOD levels, and decreased the MDA levels (all P<0.01). Compared with the mono-therapy groups, the NXTC+GHI group exhibited a significant improvement in the number of apoptotic rBMECs (P<0.01). All drug intervention groups showed different degrees of increase in membrane potential, and the NXTC+GHI group was higher than the NXTC or GHI group (P<0.01). CONCLUSION: The combinationa application of NXTC and GHI on cerebral I/R injury clearly resulted in protective benefits.

Guhong Injection Protects Against Apoptosis in Cerebral Ischemia by Maintaining Cerebral Microvasculature and Mitochondrial Integrity Through the PI3K/AKT Pathway.

Guhong injection (GHI) can be used for the treatment of ischemic stroke. We investigated the antiapoptotic activity of GHI, its ability to repair the cerebral microvessels and mitochondria, and the PI3K/AKT signaling pathway of GHI against cerebral ischemia. Western blot and immunohistochemical analyses were used to determine the expression of cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), cytochrome c (Cyt-c), basic fibroblast growth factor (BFGF), vascular endothelial growth factor (VEGF), transforming growth factor-ß1 (TGF-ß1), and proteins in the PI3K/AKT signaling pathway. Transmission electron microscopy and scanning electron microscopy were used to evaluate the structures of the cerebral microvasculature and cells. Hoechst 33342 staining was used to evaluate the nuclear morphology. FITC-AV/PI double staining was used to measure the antiapoptotic effects. The fluorescent dye JC-1 was used to measure mitochondrial membrane potential. The enzyme-linked immunosorbent assay (ELISA) was used to detect the activities of matrix metalloproteinase-9 (MMP-9). Biochemical assay kits were used to detect the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA). Compared with the middle cerebral artery occlusion (MCAO) group, there was decreased infarct volume and significantly improved neurological deficits in the GHI group. In addition, the expression of Bcl-2 was significantly upregulated, while the expression of Cyt-c, Bax, and cleaved caspase-3 was notably downregulated. GHI administration attenuated the pathological change and morphology of the cerebral microvasculature, and immunohistochemical staining indicated that the expressions of BFGF, VEGF, and TGF-ß1 were significantly increased. The cell morphology, cell viability, cell nuclei characteristics, and mitochondrial morphology normalized following GHI treatment, which decreased the release of Cyt-c and the mitochondrial membrane potential. The levels of LDH, MMP-9, and MDA decreased, while SOD increased. Moreover, GHI administration inhibited the activation of the PI3K/AKT signaling pathway in rat brain microvascular endothelial cells (rBMECs) following oxygen/glucose deprivation (OGD) injury. Therefore, our results show that GHI administration resulted in antiapoptosis of cerebral cells and repair of cerebral microvessels and mitochondria via the PI3K/AKT signaling pathway.

Efficacy and safety of Guhong injection for treating coronary microvascular disease: study protocol for a randomized controlled trial.

BACKGROUND: Coronary microvascular disease (CMVD) can be described as one of the cardiovascular diseases with normal coronary angiography but evidence of myocardial ischemia or microcirculatory lesions, often presenting as angina pectoris attacks. Coronary artery microtubular dysfunction is one of the pathogenic features of coronary heart disease, but its occurrence and development and the current CMVD-intervention therapy needs further research. Chinese traditional medicine (TCM) has advantages for the treatment of cardiovascular diseases. Hence, this article describes an ongoing randomized controlled clinical trial based on the theory of TCM for the purpose of evaluating the efficacy and safety of Guhong injection versus placebo in patients with CMVD. METHODS/DESIGN: This is a multicenter, randomized, parallel-arm, open-label, double-blind, placebo-controlled clinical trial. A total of 260 eligible patients will be allocated and randomly assigned, in a ratio of 1:1, to either the experimental group or the control group. The treatment course is 10 consecutive days, and with an 8-week follow-up. The primary outcome is therapeutic efficacy. Secondary outcomes include the quantitative score of TCM syndromes (a series of TCM symptoms and signs of coronary heart disease), the average frequency of anginal attacks, electrocardiogram (ECG) changes, inflammatory response, endothelial function indicators and myocardial metabolites. DISCUSSION: This trial is strictly designed in accordance with principles and regulations issued by the China Food and Drug Administration (CFDA). The results should provide high-quality evidence on the efficacy and safety of Guhong injection in the treatment of CMVD. TRIAL REGISTRATION: Chinese Clinical Trials Registry, ID: ChiCTR1900022902. Registered on 27 April 2019.

Guhong Injection promotes fracture healing by activating Wnt/beta-catenin signaling pathway in vivo and in vitro.

Guhong Injection (GHI), composed of aceglutamide and safflower aqueous extract, has been applied to the clinical treatment of orthopedic diseases, but the relevant mechanism by which GHI exerts effects on bone remodeling has not been reported. In the present study, we investigated the effects of various concentrations of GHI (2.5, 5 and 10 ml/kg) in accelerating rat tibia healing progress by observing haematoxylin and eosin (HE) stained sections, detecting the activity of bone metabolism biochemical markers such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), osteocalcin (OC) and C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-1) in rat serum, as well as measuring the expressions of collagen I (COL-1) and collagen II (COL-2) in rat tibia. Also, we investigated the effects of different concentrations of GHI (30, 60 and 90 µl/ml) on the proliferation and differentiation of osteoblasts (OBs) through proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and type I collagen (COL-1). At the same time, the expression of important factors of Wnt/beta-catenin signaling pathway including Wnt-3a, beta-catenin, disheveled-1 (Dvl-1), glycogen synthase kinases-3beta (GSK-3beta), lymphoid enhancing factor-1 (LEF-1) and axis inhibition protein-2 (Axin-2) after GHI intervention was detected by quantitative real-time PCR (q-PCR), immunohistochemistry and Western blotting. In vivo, rats of tibia fracture model treated with intraperitoneal injection (ip) of GHI had more mature fibroblasts, as well as shorter period formation of new bone. The levels of BMP-2, TGF-beta and OC in rat serum were significantly up-regulated, while the level of CTX was down-regulated. After 4 weeks of drug treatment, the level of COL-1 in the rat tibia increased, but there was no significant change in the level of COL-2. In vitro, after drug intervention, the number of OBs increased significantly, the activities of PCNA, ALP and COL-1 were enhanced. Treatment with GHI increased the mRNA and protein expression of Wnt-3a, beta-catenin, Dvl-1 and LEF-1, and decreased the expression of mRNA of Axin-2 and GSK-3beta. All results demonstrate that GHI accelerates the proliferation of OBs and shortens the recovery time of bone structure, and the Wnt/beta-catenin signaling pathway is involved in the regulation process.

Pharmacokinetics of Active Components From Guhong Injection in Normal and Pathological Rat Models of Cerebral Ischemia: A Comparative Study.

Background and Objectives: Guhong Injection (GHI) is usually administered for the treatment of stroke in clinics. Aceglutamide and hydroxyl safflower yellow A (HSYA) are its key ingredients for brain protective effect. To investigate the pharmacokinetics of aceglutamide and HSYA under pathological and normal conditions, the pharmacokinetic parameters and characteristics of middle cerebral artery occlusion (MCAO) and normal rats given the same dosage of GHI were studied compared. Methods: 12 SD rats were divided into two groups, namely, MCAO and normal groups. Both groups were treated with GHI in the same dosage. Plasma samples were collected from the jaw vein at different time points and subsequently tested by high-performance liquid chromatography (HPLC). Results: After administration of GHI, both aceglutamide and HSYA were immediately detected in the plasma. Ninety percent of aceglutamide and HSYA was eliminated within 3 h. For aceglutamide, statistically significant differences in the parameters including AUC(0-t), AUC(0-∞), AUMC(0-t), AUMC(0-∞), Cmax (P < 0.01), and Vz (P < 0.05). Meanwhile, compared with the MCAO group, in the normal group, the values of AUC(0-t), AUMC(0-t), VRT(0-t), and Cmax (P < 0.01) for HSYA were significantly higher, whereas the value of MRT(0-t) was significantly lower in the normal group. Conclusions: The in vivo trials based on the different models showed that, the pharmacokinetic behaviors and parameters of aceglutamide and HSYA in GHI were completely different. These results suggest that the pathological damage of ischemia-reperfusion has a significant impact on the pharmacokinetic traits of aceglutamide and HSYA.

Guhong injection protects against focal cerebral ischemia-reperfusion injury via anti-inflammatory effects in rats.

Guhong injection (GHI), composed of aceglutamide and safflower aqueous extract, has been used clinically for the treatment of cerebrovascular diseases such as cerebral embolism, hemorrhage and mental deterioration. In this paper, we reported the results of the first study on the anti-inflammatory effects of GHI in murine focal cerebral ischemia-reperfusion (I/R) injury. Adult male SD rats were randomly divided into six groups: Sham group, I/R group, GHI-L group (2.5 mL/kg), GHI-M group (5 mL/kg), GHI-H group (10 mL/kg) and Nimodipine group. I/R injury was induced by middle cerebral artery occlusion (MCAO) for 1.5 h followed by reperfusion for 24 h. Compared with I/R group, rats treated with GHI showed dose dependent reductions in neurological defect scores and cerebral infarct volume. GHI obviously down-regulated nitric oxide (NO), inducible NO synthase (iNOS), myeloperoxidase (MPO), interleukin-1ß (IL-1ß), TNF-α (tumor necrosis factor-α) and C reactive protein (CRP) levels in serum. Moreover, histological examination by H&E staining showed that clear cell outline, less vacuolated spaces and largely surviving neurons were observed in GHI-treated rats. The immunohistochemical staining revealed that GHI administration significantly diminished the positive expressions of intercellular cell adhesion molecule-1 (ICAM-1) and nuclear factor-κB p65 (NF-κB p65) in brain tissues. Western blot analysis for ICAM, NF-κB p65 and iNOS further solidified the above findings. All these results demonstrate that GHI exerts a strong and ameliorative effect on cerebral I/R injury in rats possibly through the inhibition of inflammation.

Protective Effect of Naoxintong Capsule () Combined with Guhong Injection () on Rat Brain Microvascular Endothelial Cells during Cerebral Ischemia-Reperfusion Injury / 中国结合医学杂志

OBJECTIVE@#To investigate the synergistic effect of Naoxintong Capsule (NXTC, ) and Guhong Injection (GHI, ) on cerebral ischemia-reperfusion (I/R) injury.@*METHODS@#Forty-eight Sprague-Dawley rats were divided into 6 groups: control group, oxygen and glucose deprivation (OGD) group, nimodipine group (9.375 mg/kg), NXTC group (0.5 g/kg), GHI group (5 mL/kg) and NXTC+GHI group (0.5 g/kg NXTC+5 mL/kg GHI), after the onset of reperfusion and once per day for the following 7 days. Blood was collected 1 h after final administration, and the sera were collected. Cultured primary rat brain microvascular endothelial cells (rBMECs) were subjected to OGD to establish a cell injury model. Untreated rBMECs were used as blank control. The cell counting kit-8 assay was used to assess cell viability using the sera. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were assessed using an enzyme-linked immunosorbent assay. Apoptosis was evaluated after Hoechst33342 staining using fluorescence microscopy and flow cytometry. JC-1 staining was performed to assess changes in mitochondrial membrane potential.@*RESULTS@#Statistical analysis indicated that more than 95% of the cells were rBMECs. Compared with the OGD group, the cellular morphology of the all drug delivery groups improved. In particular, the combined drug group had the most significant effect. Compared with the OGD group, all drug intervention groups induced a decrease in the apoptotic rate of rBMECs, increased the SOD levels, and decreased the MDA levels (all P<0.01). Compared with the mono-therapy groups, the NXTC+GHI group exhibited a significant improvement in the number of apoptotic rBMECs (P<0.01). All drug intervention groups showed different degrees of increase in membrane potential, and the NXTC+GHI group was higher than the NXTC or GHI group (P<0.01).@*CONCLUSION@#The combinationa application of NXTC and GHI on cerebral I/R injury clearly resulted in protective benefits.

Mechanism of Guhong Injection Against Cerebral Ischemia Based on Transcriptome Analysis / 中国实验方剂学杂志

Objective::Based on the protective effect of Guhong injection (GH) on cerebral ischemia, mechanism of GH against cerebral ischemia was identified using RNA-seq transcriptome and bioinformation analysis. Method::The model of middle cerebral artery occlusion (MCAO) was established through thread embolization. Sham group, model group, low-dose GH group (0.625 mL·kg-1·d-1), high-dose GH group (2.5 mL·kg-1·d-1), positive group (Ginaton, 8 mL·kg-1·d-1) were set up. Ludmila Belayev 12-point scoring method was applied to assess the protective effect of GH against MCAO-induced cerebral ischemia. And the differentially expressed genes after treatment with GH were identified by RNA-Seq technology. Enrichment analysis, cluster analysis and association analysis on disease targets of cerebral ischemia were carried out through such databases as DAVID, String and The Human Phenotype Ontology. Finally, the regulatory network was constructed by Cytoscape3.4.0. Result::Compared with the sham group, the neurological impairment was obvious in the model group (P<0.01), and the neurological impairment was alleviated in the GH group compared with the model group (P<0.05). RNA-Seq technology analysis showed that GH regulated genes involving such biological processes as cell apoptosis, inflammation, oxidative stress, toll-like signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway. Twenty disease targets and 64 MAPK signaling pathway genes were associated with differentially expressed genes after GH treatment, in which 23 genes were involved in apoptosis and inflammation. Conclusion::GH protected against cerebral ischemia in many ways, among which MAPK signaling pathway is an important way to exert its effect in inhibiting apoptosis and inflammation.

Mechanism of Guhong Injection against Hypoxia/Reoxygenation Injury of H9c2 Cardiomyocytes Based on PI3K/AKT Signaling Pathway / 中国药学杂志

OBJECTIVE: To explore the protectiveness of Guhong injection (GHI) on H9c2 cardiomyocytes injured by hypoxia/reoxygenation (H/R) based on PI3K/AKT signaling pathway. METHODS: H9c2 cardiomyocytes were cultured and the optimal GHI doses were screened by CCK-8. The cells were randomly subjected into 8 groups: control group, model group, low, medium and high GHI dose groups (30, 60 and 90 μL•mL-1 respectively), positive drug group (verapamil injection 7.5 μL•mL-1), GHI+LY294002 (PI3K inhibitor) group and LY294002 group. Cells were established H/R model with hypoxia for 4 h and reoxygenation for 16 h excepted the control group. The CK-MB, LDH, MDA content and SOD activities in each group were detected by Elisa, and the expression levels of p-PI3K, PI3K, p-AKT, AKT and GSK-3β in each group were detected by Western blot. RESULTS: Compared each drug-treated group with the model group, LDH and CK-MB contents were decreased (P<0.05), MDA content was decreased (P<0.01), and SOD activity was increased (P<0.01). Meanwhile, the expression levels of p-PI3K/PI3K, p-AKT/AKT, and GSK-3β protein were elevated (P<0.01). Compared with the model group, LDH, CK-MB, MDA and SOD activity in LY294002 group were no significant difference, while the expression levels of p-PI3K/PI3K, p-AKT/AKT and GSK-3β protein decreased (P<0.01). CONCLUSION: GHI could represent anti-oxidative and anti-apoptotic effect which reduced the damage of injured H9c2 myocardial cells caused by H/R. These effects might be related to the PI3K/AKT signaling pathway.

Pharmacokinetics of main activ ingredients of Guhong injection in cerebral ischemia rats / 中国中药杂志

To investigate the pharmacokinetic characteristics of active constituents of Guhong injection in rats with cerebral ischemia reperfusion injury. The middle cerebral artery occlusion (MCAO) model was established in our studies, and then all the rats received iv administration of Guhong injection (2.1 mL·kg⁻¹). The blood concentrations of aceglutamide and hydroxysafflor yellow A (HSYA) were determined by high performance liquid chromatography (HPLC) method at different time points. The concentration-time curves were drawn and pharmacokinetic data were obtained by DAS 3.2.6 software. The results showed that aceglutamide and HSYA showed good linear relationship within the ranges of 1.5-500 mg·L⁻¹ (R²=0.997 5) and 0.33-40 mg·L⁻¹ (R²=0.998 9) respectively. This quantitative method showed a high recovery rate, good precision and stability. The main pharmacokinetics parameters of t1/2α, t1/2β, CL₁, CL₂, AUC0-t, AUC0-∞, Vd1, and Vd2 were (0.139±0.007) and (0.155±0.017) h, (0.803±0.046) and (2.233±0.410) h, (0.016±0) and (0.149±0.018) L·h⁻¹·kg⁻¹, (0.015±0.001) and (0.446±0.016) L·h⁻¹·kg⁻¹, (133.335±3.844) and (9.298±0.179) mg·h·L⁻¹, (143.851±3.595) and (14.464±1.451) mg·h·L⁻¹, (0.009±0.001) and (0.223±0.007) L·kg⁻¹, (0.006±0.001) and (0.212±0.032) L·kg⁻¹, respectively. The results showed that the established HPLC method was highly specific, and could be used for the simultaneous detection of aceglutamide and HSYA of Guhong injection in MCAO rats, which was conducive to pharmacokinetic studies. Pharmacokinetic data and parameters could provide reference for continuous administration and interval administration of the drug.