Aconitum L. is a family of 1-year-to-perennial herbaceous plants within the Ranunculaceae that is both medicinal and poisonous. The plants are widely distributed in temperate zones of Northern Hemisphere, mainly in Asia, Europe, and North America. China is one of the countries with richest species resources of aconitum in the world, mainly distributed in Sichuan, Yunnan, Tibet and other provinces in Southwest China (1). There are more than 200 species of Aconitum in China, including Aconitum leucostomum, Aconitum kusnezoffii, Aconitum carmichaelii, Aconitum coreanum, and Aconitum pendulum (2). As a result of climate change and overgrazing, natural grasslands have degenerated, leading to a rapid increase in poisonous plants including aconitum, which has caused huge economic losses in animal husbandry in the grasslands. Animals can be poisoned after ingesting Aconitum, with clinical symptoms of salivation, vomiting, diarrhea, dyspnea, sensory paralysis, and eventually death from cardiopulmonary failure (3).

Studies have indicated that aconitum has pharmacological activities including analgesic (4), cardiac (5), anti-aging (6), anti-inflammatory (7), anti-tumor (8) and anti-viral activity (9). Aconitum contains more than 90 kinds of alkaloids, including aconitine, hypaconitine, and mesaconitine (10, 11). Aconitine, a diterpene alkaloid, is the principal toxic ingredient of Aconitum (Figure 1) (11). The reported LD₅₀ values of Aconitine in mice were 1.8 mg/kg orally and 0.308 mg/kg intraperitoneally (12). Zhou et al. reported Aconitum was highly toxic including cardio-toxicity and neurotoxicity, embryo toxicity and renal toxicity. Its toxicological mechanism has been shown in its effect on voltage-dependent sodium channels, release of neurotransmitters and changes in receptors, promotion of lipid peroxidation and cell apoptosis in heart, liver and other tissues (13). The toxicological research on aconitine has mainly focused on acute cardiotoxicity (14–16) and neurotoxicity (17–19). Peng et al. investigated the neurotoxic effects and underlying mechanisms of aconitine on cerebral cortex neuron cells prepared from neonatal SD (Sprague-Dawley) rats. They found aconitine may damage neuron cells through its inhibition of Na⁺-K⁺-ATP activity and neurotransmitters in the cells, which resulted in injuries to cell morphology and function (20). Subsequently, Gao et al. investigated the apoptotic effects of aconitine in H9c2 cardiac cells and concluded that aconitine might induce apoptosis through mitochondria-mediated signaling pathways (21).

However, there have been no systematic studies reported on the hematological and histopathological influence of the subacute administration of aconitine. Thus, our study was conducted to investigate the hematological indices and histopathological changes due to aconitine treatments in experimental animal models, to provide an important theoretical basis for the research on the toxic mechanism of aconitine. These results may contribute valuable information to toxicology.

Aconite is widely distributed, especially in the Xinjiang Autonomous Region, causing a large amount of economic loss every year. Aconitum alkaloids cause acute poisoning because of its high toxicity (22), however, subacute and chronic poisoning of animals are also common in the grasslands. The animals may display the typical symptoms of aconitine poisoning, such as nausea, vomiting, dizziness, palpitation, hypotension, arrhythmia, and respiratory spasm (3, 23). In our experiments, mice showed varying degrees of poisoning symptoms depending on the dosage. In particular, mice in the high-dose group showed diaphoresis, diarrhea, and obvious neurological symptoms. These results agree with the finding reported by Wada et al. (24), though the symptoms were slightly milder. No mice died during our entire experiment, which could be due to the different dosage of aconitine. The body weight of animals can generally reflect their health status (25). In our study, the treated animals had lower average weights than the control group. This indicates that aconitine can inhibit the growth and development of animals.

As the executor of the animal's physiological function, the organ coefficient can reflect the function and lesion status of the organ (26). A decrease in the organ coefficient indicates that the organ is atrophic and degenerative. An increase in the organ coefficient could reflect the organ's changes of congestion, edema, and hyperplasia (27). The organ coefficient of the liver, spleen, and brain all significantly increased, and the lungs and cerebellar organ coefficient were decreased, suggesting that aconitine can inhibit the growth and development of mice while causing damage to organs.

Physiological and biochemical indices of blood are sensitive indicators of the health of animals, reflecting the metabolism, organ function and nutritional level (28, 29). In this study, both sexes of rats were used in this study, and there was no apparent difference between males and females in the measured parameters. The results of the Hematological indexes showed that the RBC, HGB and HCT in the experimental group were significantly decreased with a time and dose-effect relationship. WBC and Gran in the low-dose group increased, but the middle-dose group and the high-dose group increased first and then decreased. The results indicated that low doses of aconitine improve the body's immune function while high doses are immunosuppressive.

The liver is one of the major target organs of exogenous poisoning. Exogenous toxicants can damage liver cells, and the degree of cellular response can range from mild changes to the cytoplasm to cell death. Measuring the activity of ALT and AST is helpful for determining the extent of liver cell damage (30, 31). Kidneys are the major excretion route of exogenous toxicants; the content of CRE and BUN in serum can be used to evaluate the glomerular filtration, which is of great significance for judging the degree of renal damage (31, 32). We found that with an increase in the time and dose, the activity of ALT and AST increased; the content of CRE and BUN also showed an upward trend with obvious time and dose-effect relationship. This showed that subacute aconitine poisoning can cause various degrees of liver and kidneys functional damage in mice. In addition, the TP and ALB in the mice in each experimental group were significantly decreased. This is commonly seen in malnutrition, protein synthesis and synthesis disorders, functional liver diseases, nephrotic syndrome and other diseases. We demonstrated that aconitine can cause systemic poisoning involving multiple organs and systems in mice.

There are many animal diseases, with complex etiology and pathogenesis. The pathological examination of tissues contributes not only to disease diagnosis but also demonstrating that the poisoning may cause damage (33). Zhu et al. treated Mdr1a⁻/⁻ mice with 0.1 mg/kg aconitine then observed Edema, enlarged tissue space, and karyopyknosis in the hippocampus were observed in the brain, Meanwhile, myocardium fragmentation, deformation, and necrosis of the myocardium were observed in the heart (34). Subsequently, to evaluate the antidotal effect of processed borax against acute and subacute toxicity, cardiac toxicity and neuro-muscular toxicity caused by raw aconite, Sarkar et al. found that the observed changes in aconitine poisoned animals were moderate cell and fluid effusion and severe hemorrhage in lungs (35). Compared with these early studies, similar pathological changes were found in brain, heart and lungs tissues in our experiment. Moreover, there were significantly more megakaryocytes in the spleen of the middle-dose group than the high-dose group. This result was also consistent with the WBCs and Gran alteration in this study. We speculate that middle-dose aconitine may promote immune function in mice, whereas high-doses have significant immunosuppressive effects. These histopathological changes may be the reason for the alteration in the organ coefficient in this study. While combined with the results of hematology, it demonstrates that aconitine could cause chronic hemorrhagic anemia in mice. With the biochemistry detection and previous research results (34–36), we speculate that the target organ of aconitine may be heart, cerebellum, liver, kidney and spleen, which further leads to dysfunction of the systemic organs of mice, causing irreversible toxic damage.

Our study for the first time explored the effects of subacute aconitine poisoning on hematological and histopathological influences in mice. The results demonstrated that aconitine has marked effects on growth and development in mice, causing systemic poisoning involving multiple organs and systems in a significant time and dose-effect relationship. These results can provide an important theoretical basis for further investigating the mechanism of aconitine toxicity.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Ethical Principles [Animal (Scientific Procedures) Act 2012] in Animal Research adopted by the China College of Animal Experimentation and were approved by the College of Veterinary Medicine-Northwest A&F University.

HL: funding acquisition and writing-original draft. LM, ZG, KW, YZha, ST, YZhu, and BZ: visualization. LM, ZG, KW, YZha, ST, YZhu, HL, and BZ: methodology and writing-review and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the grants from the National Natural Science Foundation of China (No. 32072929) and the Science and Technology Special Fund Aid to Qinghai Province (No. 2020-QY-210).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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