Study on the anti-mitochondrial apoptosis mechanism of Erigeron breviscapus injection based on UPLC-Q-TOF-MS metabolomics and molecular docking in rats with cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Erigeron breviscapus is a common medicine of eight ethnic minorities, including Miao, Naxi, and Yi. As early as the Ming Dynasty (AD 1368-1644), Lanmao's Materia Medica of Southern Yunnan (AD 1436) recorded that the medicine is used for the treatment of "Zuo tan you huan." In modern pharmacological research, Erigeron breviscapus injection is the most commonly used preparation in the treatment of ischemic stroke caused by acute cerebral infarction, but its mechanism of action in the treatment of ischemic stroke is not well understood. AIM OF THE STUDY: In this study, a metabonomics study based on ultraperformance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) was used in investigating the effect of a traditional Chinese medicine preparation Erigeron breviscapus injection on the rat model of focal cerebral ischemia-reperfusion and the affinity of its main components with the targets of mitochondrial apoptotic pathways. MATERIALS AND METHODS: This study used molecular docking technology to verify the effective binding ability of main effective components of Erigeron breviscapus injection to target proteins related to mitochondrial apoptosis pathway. This study developed a metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of focal cerebral ischemia-reperfusion model group and healthy control group was achieved. RESULTS: Erigeron breviscapus injection can significantly reduce the area of cerebral infarction, improve tissue morphological lesion in rats, and can increase the number of Nissl bodies. It may be a promoting factor by inhibiting hippocampal nerve cell apoptosis and Bax protein expression and by exerting effects against ischemia reperfusion after the induction of apoptosis. Thus, it plays a role in brain protection. Moreover, it can considerably promote the recovery of neurological deficiency signs in advance. Meanwhile, Erigeron breviscapus decreased malondialdehyde content and T-NOS activity. Its curative effect from strong to weak order: low dose > high dose > medium dose. The representative components of Erigeron breviscapus have good affinity with the active sites of mitochondrial apoptosis-related proteins. Metabolomics found that the potential biomarkers regulated by breviscapine are kynurequinolinic acid, succinylornithine, and leucine proline. It is speculated that it may participate in TRP-kynurequinolinic acid and succinylornithine-urea cycle-NO metabolic pathways. CONCLUSIONS: This paper revealed the potential biomarkers and metabolic pathways regulated by Erigeron breviscapus. It was speculated that the mechanism is related to its inhibition of mitochondrion-mediated apoptosis. Erigeron breviscapus could restore the metabolic profiles of the model animals to normal animal levels. The mechanism may be related to the potential biomarkers of quinolinic acid, succinylornithine, and leucine proline and the metabolic pathways involved. However, the exact mechanism by which Erigeron breviscapus inhibits mitochondrion-mediated apoptosis remains to be further explored.

Corrigendum: Induction of RIPK3/MLKL-mediated necroptosis by Erigeron breviscapus injection exhibits potent antitumor effect.

[This corrects the article DOI: 10.3389/fphar.2023.1219362.].

Induction of RIPK3/MLKL-mediated necroptosis by Erigeron breviscapus injection exhibits potent antitumor effect.

Colorectal cancer (CRC) is the second leading cause of tumor-related deaths worldwide. Resistance of tumor cells to drug-induced apoptosis highlights the need for safe and effective antitumor alternatives. Erigeron breviscapus (Dengzhanxixin in China) injection (EBI), extracted from the natural herb Erigeron breviscapus (Vant.) Hand.-Mazz (EHM), has been widely used in clinical practice for cardiovascular diseases. Recent studies have suggested that EBI's main active ingredients exhibit potential antitumor effects. This study aims to explore the anti-CRC effect of EBI and elucidate the underlying mechanism. The anti-CRC effect of EBI was evaluated in vitro using CCK-8, flow cytometry, and transwell analysis, and in vivo through a xenograft mice model. RNA sequencing was utilized to compare the differentially expressed genes, and the proposed mechanism was verified through in vitro and in vivo experiments. Our study demonstrates that EBI significantly inhibits the proliferation of three human CRC cell lines and effectively suppresses the migration and invasion of SW620 cells. Moreover, in the SW620 xenograft mice model, EBI markedly retards tumor growth and lung metastasis. RNA-seq analysis revealed that EBI might exert antitumor effects by inducing necroptosis of tumor cells. Additionally, EBI activates the RIPK3/MLKL signaling pathway, a classical pathway of necroptosis and greatly promotes the generation of intracellular ROS. Furthermore, the antitumor effect of EBI on SW620 is significantly alleviated after the pretreatment of GW806742X, the MLKL inhibitor. Our findings suggest that EBI is a safe and effective inducer of necroptosis for CRC treatment. Notably, necroptosis is a non-apoptotic programmed cell death pathway that can effectively circumvent resistance to apoptosis, which provides a novel approach for overcoming tumor drug resistance.

PK-PD Correlation of Erigeron Breviscapus Injection in the Treatment of Cerebral Ischemia-Reperfusion Injury Model Rats.

By measuring the cerebral infarction rate and neurological behavioral score of rats in a sham operation group, an MCAO model control group and an Erigeron breviscapus injection treatment group, we explored the therapeutic effects of Erigeron breviscapus injection on brain tissue and neuroethological injury in rats. Plasma samples were collected at 18 time points after intravenous injection of Erigeron breviscapus. The levels of scutellarin, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid and isochlorogenic acid B in rat plasma at the various time points were determined by an HPLC method, and drug concentration versus time plots were constructed to estimate the pharmacokinetic parameters. Finally, a PK-PD combined model was used to analyze the relationship between the blood concentration, time and therapeutic effects of the seven active components. The results of the pharmacodynamics studies showed that the cerebral infarction rate of rats in the Erigeron breviscapus injection group decreased significantly at 5 min, 10 min, 20 min, 6 h, 8 h, 18 h, 24 h, 32 h, 40 h and 48 h after cerebral ischemia. Abnormal neurological behavior scores were significantly reduced after 4 h of cerebral ischemia. The pharmacokinetics results showed that the seven chemical constituents in Erigeron breviscapus injection reached their highest detection value after 5 min of cerebral ischemia. The lowest detection values of scutellarin and isochlorogenic acid B appeared after 6 h of cerebral ischemia but could not be detected after 8 h. The lowest detection values of 5-caffeoylquinic acid and 4,5-dicaffeoylquinic acid were found in the third hour of cerebral ischemia but not after 4 h. The lowest detection values of 4-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and chlorogenic acid were found during the second hour of cerebral ischemia but not at the third hour. However, at 18 h, 24 h, 32 h and 40 h of cerebral ischemia, the cerebral infarction rates of rats in the Erigeron breviscapus injection group were significantly reduced, with decreased values of 6.22%, 11.71%, 6.92% and 4.96%, respectively, and the effects were stronger than those after 5-20 min of cerebral ischemia. The decreased values reached their highest value after 24 h of cerebral ischemia. Our results show that the effects of Erigeron breviscapus injection on reducing the cerebral infarct rate in MCAO model rats are characterized by a fast onset and long maintenance time. The 5-min blood concentration in cerebral ischemia was the highest test value, and after this time, the cerebral infarction rate of MCAO rats began to decrease. However, the peak value of the effects lagged behind that of the plasma concentration. The maximum effective time for Erigeron breviscapus injection appeared 24 h after cerebral ischemia, which provides a reference for the screening of specific drugs for ischemic stroke, optimal dosing regimens and rational clinical drug use. Graphical Abstract.

Optimization of the Best Proportion of Astragalus membranaceus Injection Combined with Erigeron breviscapus Injection against Cerebral Ischemia-reperfusion Injury in Rats by Baseline Geometric Proportion Increasing and Decreasing Design / 中国药房

OBJECTIVE： To screen the best proportion of Astragalus membranaceus injection combined with Erigeron breviscapus injection against cerebral ischemia-reperfusion injury in rats. METHODS： Male SD rats were randomly divided into sham operation group， model group and administration group [different A. membranaceus injection-E. breviscapus injection proportion groups， being A（0 ∶ 10）， B（2 ∶ 8）， C（4 ∶ 6）， D（6 ∶ 4）， E（8 ∶ 2）， F（10 ∶ 0）groups， set by baseline geometric proportion increasing and decreasing design]， with 8 rats in each group. Except for sham operation group， reperfusion injury model of middle cerebral artery occlusion were induced by modified suture method in rats. The each administration group was given relevant medicine intraperitoneally once immediately after inducing model， and then given again after 24 hours （medication interval between the two injections of 30 min）. Constant volume of normal saline was given to rats in sham operation group and model group. Forty-eight hours after reperfusion， Longa scoring method was used to evaluate neurological impairment of rats， and neurological impairment score was recorded. Serum content of MDA and activity of SOD were measured by colorimetry assay. TTC assay was used to detect cerebral infraction， and cerebral infarction rate was calculated. Kim’s formula was used to calculate the synergistic index （q） of rats in administration groups. RESULTS： Compared with sham operation group， neurological impairment score and serum content of MDA were increased significantly in model group， while activity of SOD was decreased significantly （P＜0.01）. The area of cerebral infarction increased significantly， and the rate of cerebral infarction increased significantly （P＜0.01）. Compared with model group， neurological impairment scores and serum contents of MDA were decreased significantly in group A， B， C， D and E； neurological impairment score of group C was significantly lower than those of group A and F； serum contents of MDA in group B， C， D and E were significantly lower than that of group F （P＜0.05 or P＜0.01）. Activities of SOD in group A， B， C， D and E were increased significantly， and group C was significantly higher than group F （P＜0.05 or P＜0.01）. The cerebral infarction area of rats in each administration group was reduced to varying degrees. The cerebral infarction rates of rats in group B， C， D and E were significantly reduced， and group C and D were significantly lower than group F， while group C was significantly lower than group A （P＜0.05 or P＜0.01）. The q values of group B， C， D and E were 0.90， 1.30， 1.00， 0.70 （neurological impairment score） and 0.79， 1.27， 0.98， 0.82 （cerebral infarction rate）. CONCLUSIONS： Different ratios of A. membranaceus injection and E. breviscapus injection have certain protective effects on cerebral ischemia-reperfusion injury model rats， can relieve their neurological deficits， alleviate their oxidative stress and reduce their cerebral infarction areas. The effect of the combination of the two drugs is better than that of single use， and the optimum ratio is 4 ∶ 6.

[Meta-analysis on indirect comparison of Erigeron breviscapus injection and Breviscapus injection in treatment of acute ischemic stroke].

Erigeron breviscapus injection(DZXI) and Breviscapus injection(DZSI) are two popular injections in treatment of acute ischemic stroke in China. Both of them are manufactured from a same herbal medicine, E. breviscapus, but DZXI is an herbal extract(mixture) preparation and DZSI is a pure compound injection. This article was aimed to systemically evaluate and compare their efficacy and safety in treatment of acute ischemic stroke. The randomized controlled trials(RCTs) were collected for comparing DZXI and DZSI with Salvia miltiorrhiza injection(FDI) as the medium, and they were compared with indirect Meta-analysis(ITC). Thirty-nine RCTs with 4 180 patients were included. Meta-analysis results showed that both DZXI and DZSI had better efficacy than FDI in acute ischemic stroke. In the indirect comparison, DZSI had a higher total efficacy than DZXI, with significantly statistical differences[OR=0.634, 95%CI = (0.432,0.928), P<0.000 01], but there was no significant difference in improvement of neurological deficit [MD=-1.19, 95%CI=(－3.57,1.19), P=0.953]. On the safety aspect, adverse reaction rate of DZXI was 1.14%, mainly including head swelling, fever and chills while DZSI had no significant adverse reactions. The limited evidences in this study showed that Breviscapine injection had higher total efficiency and safety than E. breviscapine injection, but due to the low quality of the included RCTs, these two medicines should be comprehensively compared in further high-quality clinical trials.

Meta-analysis on indirect comparison of Erigeron breviscapus injection and Breviscapus injection in treatment of acute ischemic stroke / 中国中药杂志

Erigeron breviscapus injection(DZXI) and Breviscapus injection(DZSI) are two popular injections in treatment of acute ischemic stroke in China. Both of them are manufactured from a same herbal medicine, E. breviscapus, but DZXI is an herbal extract(mixture) preparation and DZSI is a pure compound injection. This article was aimed to systemically evaluate and compare their efficacy and safety in treatment of acute ischemic stroke. The randomized controlled trials(RCTs) were collected for comparing DZXI and DZSI with Salvia miltiorrhiza injection(FDI) as the medium, and they were compared with indirect Meta-analysis(ITC). Thirty-nine RCTs with 4 180 patients were included. Meta-analysis results showed that both DZXI and DZSI had better efficacy than FDI in acute ischemic stroke. In the indirect comparison, DZSI had a higher total efficacy than DZXI, with significantly statistical differences[OR=0.634, 95%CI = (0.432,0.928), P<0.000 01], but there was no significant difference in improvement of neurological deficit [MD=-1.19, 95%CI=(－3.57,1.19), P=0.953]. On the safety aspect, adverse reaction rate of DZXI was 1.14%, mainly including head swelling, fever and chills while DZSI had no significant adverse reactions. The limited evidences in this study showed that Breviscapine injection had higher total efficiency and safety than E. breviscapine injection, but due to the low quality of the included RCTs, these two medicines should be comprehensively compared in further high-quality clinical trials.

Study of protective effect of erigeron injection on renal ischemia-reperfusion injury / 中国生化药物杂志

Objective To investigate the protective effect of breviscapine on ischemia-reperfusion renal injury, which provides scientific theoretical basis for clinical prevention and treatment of renal ischemia-reperfusion injury. Method 36 SPF male healthy SD rats were randomly divided into 3 groups, 12 rats in each group. Group A was ischemia-reperfusion group, group B was erigeron breviscapus injection preconditioning group, and group C was sham operation group. Rats in group A and C were injected with normal saline, while rats in group B were given 12 ml/kg erigeron breviscapus injection by intraperitoneal injection., After 14 days, renal function, renal antioxidant indexes, renal cell apoptosis indexes and expression of Bcl-2, Bax in renal tissue of three groups were compared. Results The indexes of renal function showed that blood urea nitrogen (BUN) and Serum creatinine (SCr) in group B were significantly lower than group A (P<0.05), but significantly higher than group C (P<0.05). Renal antioxidant indexes showed that superoxide dismutase (SOD) and malondialdehyde (MDA) in group B were significantly higher than group A (P<0.05), but significantly lower than group C(P<0.05). The index of renal cell apoptosis showed that the apoptosis index AI in group B was significantly lower than group A (P<0.01), but significantly higher than group C (P<0.01). The results of immunohistochemical staining showed that the expression of Bcl-2 in group B were significantly higher than that of group A and group C (P<0.05), and the expression of Bax in group B was significantly lower than that in group A (P<0.05) while significantly higher than group C (P<0.01). The ratio of Bcl-2/Bax in group B was significantly higher than group A and group C (P<0.05). Conclusion Erigeron Breviscapus Injection may have a protective role on renal ischemia reperfusion injury though antioxidant and anti-apoptosis.