Mongolian medicine theory-based multidimensional evaluation of toxicity mitigation in Hezi-processed Caowu jointly mediated by powder dosage form and small dose.

Backgroud: Caowu (CW, Aconitum kusnezoffii Reichb.) is a well-known Mongolian medicine with the effects of dispelling cold and relieving pain. In China, it is widely used in the prevention and treatment of rheumatoid arthritis (RA). However, its cardiotoxicity and neurotoxicity seriously restrict its clinical application. Different from the use of CW as a decoction after being boiled in water in TCM, in Mongolian medicine, CW is processed using Hezi (HZ, dry ripe fruit of Terminalia chebula Retz. and T. chebula Retz. var. tomentella Kurt.) decoction or combined with HZ to prepare pills or powders, and administered at a small dosage, thereby ensuring medication safety. This way of medication is a useful experience of the minority. Thus, multi-dimensional research (quantitation of alkaloid + in vitro + in vivo + mechanism) is critical to elucidate the characteristics of the Mongolian ethnic group's detoxification experience. We investigated the content of alkaloids, the anti-inflammatory and analgesic effects, the cardiorenal-hepatic toxicities, and the oxidative stress levels in both powder and decoction of raw CW (SCW) and HZ-processed CW (HCW), with a focus on the toxicity mechanisms in H9c2 cells (specifically for the powder dosage). Methods: HPLC was used to quantify the content of main metabolites in SCW and HCW (in powder or decoction form). The pharmacological effects were evaluated using in vivo animal models (xylene-induced inflammation and formalin-induced pain). The toxicity of SCW and HCW was assessed via electrocardiographic analysis, histological analysis, and biochemical analysis. Subsequently, in vitro toxicity mechanism studies were conducted on H9c2 cells using techniques such as MTT, fluorescent probes, ELISA, and Western blotting. Additionally, the involvement of the p38/JNK signaling pathway in their cardiotoxicity was verified by treating cells with p38/JNK pathway inhibitors. Results: Using HPLC, we found that both the use of HZ as an excipient for processing CW and decocting CW with water affect the content of alkaloids in CW. In the xylene-induced ear edema model, SCW powder, SCW decoction, and HCW powder showed significant anti-inflammatory effects by regulating inflammation-related factors (IL-1β, IL-6, and IL-10); in contrast, HCW decoction did not show significant anti-inflammatory effects compared with the model group. In the formalin-induced pain model, both SCW and HCW exerted analgesic effects to varying degrees by regulating pain-related factors (5-HT and PGE2). In the toxicity study, SCW powder exhibited the strongest toxicity to the heart; in contrast, SCW decoction had the weakest toxicity, while HCW powder and HCW decoction fell in between. Furthermore, the types of arrhythmia induced by SCW powder were most complex. In addition, the cardiotoxicity of SCW was closely related to oxidative stress. Cell experiments showed that SCW induced H9c2 cell damage, which HCW partially mitigated by regulating the p38/JNK pathway. Conclusion: In conclusion, compared with SCW, HCW reduces toxicity while exerting pharmacological effects in powder dosage form. This attenuation is linked to a reduction in oxidative stress, inhibition of p38/JNK phosphorylation, and regulation of mitochondrial apoptosis-related protein expression. This finding advances our understanding of the coexistence of toxicity and efficacy in clinical application of CW.