The safety evaluation of Shenze Shugan capsule and mechanism of apoptosis induced by five potentially nephrotoxic components.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenze Shugan capsule is a prescription of traditional Chinese medicine for nonalcoholic steatohepatitis treatment. It includes Rhei Radix et Rhizoma (RR), Cassiae Semen (CS) and Alismatis Rhizoma(AR), which widely contains rhein, emodin, aurantio-obtusin, alisol A and alisol B 23-monoacetate. AIM OF THE STUDY: In this study, we aimed to explore the safety of the medicine, and further elucidate the mechanism of apoptosis induction in HK-2 cells by five components, including rhein, emodin, aurantio-obtusin, alisol A and alisol B 23-monoacetate. MATERIALS AND METHODS: We investigated the nephrotoxicity of Shenze Shugan capsule, including RR, CS, AR and mixed herbs given for two months in rats. Superoxide dismutase (SOD) in kidney tissues, urea nitrogen (BUN) and creatinine (CRE) in serum were detected, and renal pathology analysis was performed. In cell experiments, the apoptotic rate and cell cycle distribution of HK-2 cells were tested by flow cytometry. The levels of mitochondrial membrane potential (ΔΨm) and related protein expression in mitochondrial pathway were measured as well. RESULTS: We confirmed that two months of administering high doses(60 times the dose for clinical use in adults) of RR, CS or mixed herbs upregulated the levels of CRE and RUN, inhibited SOD activity, and increased the degree of tubular degeneration and glomerular dilatation, but Shenze Shugan capsule has no significant differences in renal structure or renal function. In addition, we found that five components all concentration-dependently inhibited HK-2 cells proliferation and induced apoptosis, especially aurantio-obtusin as the novel nephrotoxic component. Rhein and emodin significantly induced S/M accumulation, but aurantio-obtusin, alisol A and alisol B 23-monoacetate significantly induced G1/M accumulation in HK-2 cells. Similarly, they could induce Caspase3 activation, loss of mitochondrial membrane potential (ΔΨm), and down-regulation of Bcl-2 and up-regulation of Bax. CONCLUSIONS: Through a two-month subchronic toxicity study in rats, our preliminary determination is that this formulation is safe and reliable for long-term use. Interestingly, the potentially toxic herbs such as RR, CS, AR can reduce toxicity by drug compatibility. When further exploring the mechanism of action of toxic herbs, we found that mitochondrial pathway is involved in the apoptosis of HK -2 cells induced by rhein, emodin, aurantio-obtusin, alisol A and alisol B 23-monoacetate. Our findings provide new ideas for safety studies of Shenze Shugan capsule.

Transcriptome data-based identification of candidate genes involved in metabolism and accumulation of soluble sugars during fruit development in 'Huangguan' plum.

Fruit sweetness being an important factor of organoleptic quality directly affects the consumers' preferences for fresh fruit consumption, and is influenced by the composition and quantity of sugars. In this study, four soluble sugars (sucrose, fructose, glucose, and sorbitol) were identified and quantified in plum fruits cv. 'Huangguan' at four different maturity stages (fruitlet, green, veraison, and mature stage). The results revealed that sucrose and glucose are major soluble sugar components at the fruitlet and mature stages, respectively. RNA-Seq analysis was carried out and 6,778 differentially expressed genes (DEGs) were identified, including 121 genes involved in sugar metabolism. Furthermore, a total of 39 transcripts of 8 gene families encoding key enzymes related to the metabolism and accumulation of soluble sugars were separately identified. ERD6L (gene 103322904) was involved in keeping a balance of glucose between the inside and outside of vacuole. SS (gene 103333990) and SDH (gene 103335104) regulated the accumulation of fructose at the green stage. SDH (gene 103335104) controlled the degradation of sorbitol at the green stage. SS (gene 103333990) and PFK (gene 103333391) regulated the degradation of sucrose at the early stages of fruit development. Moreover, NINV (gene 103331108) regulated the accumulation of total sugar in plum. Genes 103321334 and 103335689 were important bZIP transcription factors that regulate the accumulation of glucose and fructose in fruits. Twelve DEGs were selected and validated to observe the relative accuracy of transcriptome sequencing data using qRT-PCR. Gene expression patterns were consistent between qRT-PCR and RNA-Seq data, indicating the reliability of RNA-Seq data. PRACTICAL APPLICATIONS: The results of this study provided new insights into comprehensive understanding of the genetic control of sugar metabolism and accumulation in plum fruits.