Discovery Of Potential Nephrotoxic Components In Zuojin Pills And Its Mechanisms

Zuojin Pill was originally derived from the"Danxi Heart Method"written by the famous medical expert Zhu Danxi,"Zuojin Pill,cure liver fire,a Huiling Pill",and its original prescription is a combination of Coptis and Evodia according to a 6:1 compatibility.It has the effects of clearing the liver and purging fire,reducing the adverse effects and relieving vomiting,and is mainly used to treat the symptoms of liver fire and stomach attack.Afterwards,famous medical experts of various generations continued to improve the ratio of Coptis and Evodia,and then explored Zhulian Pills（a collection of medical prescriptions）,Yulian Pills（Introduction to Medicine）,and Anti-Zuojin Pills.Zuojin Pills are widely used in the treatment of various gastrointestinal diseases such as gastric ulcers,gastric mucosal damage and Helicobacter pylori infection,with a wide range of clinical applications.Relevant pharmacological studies in recent years have shown that Zuojin Pill has an important role in treating cancer,lowering blood pressure and inhibiting bacteria.However,as the clinical use of Zuojin Pills becomes more and more widespread,the contraindications and potential toxic effects of Zuojin Pills are still unclear.In 1978,the Singapore government recognized Coptis,the single drug of Zuojin Pill,as a toxic traditional Chinese medicine and strictly forbids its clinical use.Clinical studies have shown that excessive or improper use of Coptis can induce and aggravate neonatal jaundice,which has triggered the international community to think about the safety of Chinese medicine and seriously hindered Chinese medicine Internationalization process.In addition,the single-medicine evodia in Zuojin Wan was first recorded as a toxic traditional Chinese medicine in the"Shen Nong’s Materia Medica",and it was recorded as a small toxic traditional Chinese medicine in the pharmacopoeia in 2021.The clinically mainly processed evodia is a finished product.Studies have shown that evodia base（EVO）has potential liver and kidney and gastrointestinal toxicity.After continuous administration of evodia,there will be potential liver and kidney toxicity.At present,clinical drug poisoning events caused by improper use of evodia have also occurred.With the widespread development and use of traditional Chinese medicines around the world,the safety of traditional Chinese medicines has attracted increasing attention.However,domestic and foreign research mainly focuses on pharmacology,and its toxicity studies are less,its toxicity mechanism is not clear,and there are potential risks during clinical use.In order to explore whether there are potential nephrotoxic components in Zuojin Pills and provide experimental basis for clinical rational use,this study uses high-content experimental technology to screen Zuojin Pills and related components for renal cell damage in vitro,and to determine the possibility of related components The mechanism of kidney injury is discussed.1.The toxicity of the aqueous extracts of the monomers in Zuojin Pills and the main monomers on renal cellsUsing HEK-293T cells and HK-2 cells as carriers,the effects of the water extract of Coptis Rhizoma,the water extract of Evodia and the water extract of Evodia on the morphology,LDH release rate and activity of the two kidney cells were studied.The results showed that the cytotoxicity of each water extract was in the order of raw evodia water extract>berberine water extract>evodia water extract.Further study through the high-content toxicity screening technology,from the number of nuclei,nucleus density,mitochondrial membrane potential and cell membrane permeability,the results showed that the cytotoxicity of each water extract is in the order of Rhizoma Coptidis water extract>raw Evodia edulis water extract>Water extract of Evodia.In order to explore the renal cytotoxic components of each single drug,the main alkaloids were selected for further study Eight monomers of lactone and synephrine were investigated.The results showed that berberine and berberine in Coptis chinensis have potential renal cytotoxicity at high concentrations.Evodia in Evodia can significantly reduce the number of nuclei,affect cell membrane permeability,and reduce mitochondrial membranes when it is greater than 0.2μmmol·L^-1.The electric potential induces cell death.2.Evodiamine induces autophagy and apoptosis by affecting calcium ion homeostasisBased on the above research,it is preliminarily confirmed that the main nephrotoxic component in Zuojin Pill is evodialine（EVO）.The study investigated the changes in the cell morphology and cell viability of HK-2 cells treated with evodia（C/0.1/0.2/0.4/0.8/1.6/3.2μmol·L-1）for 48 hours,and the results showed that the evodialine 3.2μmol·L-1 caused significant changes in cell morphology and LDH release rate,but the results of CCK-8 did not show significant toxicity due to its redox properties.In order to further verify the renal cytotoxicity of EVO,transmission electron microscopy was used to investigate the microstructure of HK-2 cells.The results showed that the mitochondria of the cells swelled significantly after treatment with EVO（0.1μmol·L-1）,and the formation of autophagosomes was observed..Confocal laser and flow cytometry were used to detect cell apoptosis,autophagy and intracellular calcium ion content.The results showed that EVO can promote the occurrence of cell apoptosis and autophagy.In order to study the mechanism of EVO’s toxicity to renal cells,transcriptomics technology was used to analyze the cell death induced by EVO.The analysis results show that EVO can significantly induce the activation of PI3K/AKT/m TOR signaling pathway in HK-2 cells.In order to further confirm the PI3K/AKT/m TOR signaling pathway induced by EVO,the experiment used Western-Blot technology to investigate the The expression of PI3K/AKT/m TOR signaling pathway,apoptosis and autophagy-related protein.The results showed that EVO can mediate calcium Ion overload regulates PI3K/AKT/m TOR signaling pathway to induce autophagy.3.In vivo studies to investigate the renal toxicity of EVOIn the study,Kunming mice were used as a model.After 14 days of continuous intragastric administration,the rats’body weight and organ ratio were recorded,and the mice’s blood was collected for biochemical index detection.The pathological sections were used to further confirm the renal toxicity of EVO.The results of the study showed that after EVO was administered for 14consecutive days,the body weight of the mice was significantly different,and the biochemical indicators related to kidney injury were significantly increased.The pathological results showed that EVO can cause kidney inflammation,glomerular and renal capsule damage,and mild to mild edema of renal tubular epithelial cells in mice.In order to clarify the mechanism of EVO’s renal toxicity,metabolomics was used to analyze the differential metabolites in mouse plasma.The results show that EVO can significantly reduce the body’s aldosterone reabsorption and steroid hormone synthesis process.Conclusion:In this study,the nephrotoxic components contained in Zuojin Pills were explored at the cellular level and animal level,and the potential nephrotoxic component EVO was screened for the first time.Further,through the combination of transcriptomics and Western-blot technology,we have deeply explored the toxic mechanism of EVO,and may regulate PI3K/AKT/m TOR-related signaling pathways by inducing intracellular calcium ion overload,leading to the combined effect of autophagy and apoptosis.In addition,EVO may participate in the kidney damage effect of EVO by affecting the sodium ion reabsorption process affected by aldosterone.The above results indicate that improper use of EVO in the clinic may lead to cumulative kidney injury,and provide early warning of toxicity for the safe use of Evodia in clinic.