Tetrahydrocurcumin ameliorates hepatic steatosis by restoring hepatocytes lipophagy through mTORC1-TFEB pathway in nonalcoholic steatohepatitis.

PURPOSE: To investigate the therapeutic effect and underlying mechanism of tetrahydrocurcumin (THC) on nonalcoholic steatohepatitis (NASH) induced by high-fat diet (HFD). METHODS: NASH rat model was established through long-term feeding HFD, and the steatosis cell model was stimulated via palmitate acid (PA). The therapeutic effect of THC was evaluated in terms of liver function, lipid metabolism, liver pathophysiology, inflammation and oxidative stress in vivo, and lipid accumulation in vitro. The alteration in lipophagy was identified by using western blot and immunofluorescence. mTORC1-TFEB signaling pathway was measured by qRT-PCR, western blot and protein-ligand docking. In addition, chloroquine and MHY1485 were further introduced to validate the effect of THC on lipophagy and mTORC1-TFEB signaling pathway, respectively. RESULTS: THC effectively improved hepatic steatosis, inflammation and oxidative stress in NASH rats, and reduced lipid accumulation in steatosis L02 cells and Hep G2 cells. THC promoted lipophagy with increasing LC3B-II as well as decreasing P62 expression via lysosomal biogenesis upregulation, which was greatly weakened after chloroquine intervention. mTORC1-TFEB is a critical pathway for regulating lysosome in autophagy, THC treatment induced TFEB nucleus translocation via inhibiting mTORC1 to upregulate lysosomal biogenesis. However, these effects were partly eliminated by mTORC1 activator MHY1485. CONCLUSION: THC restored lipophagy to reduce lipid accumulation by regulating mTORC1-TFEB pathway in NASH rats and steatosis hepatocytes. These findings suggested that THC represents a therapeutic candidate for NASH treatment.

Jian-Pi-Yi-Shen formula ameliorates renal fibrosis-induced anemia in rats with chronic kidney disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Jian-Pi-Yi-Shen (JPYS) formula is an effective herbal therapy against renal injury, and JPYS has been clinically applied to ameliorate chronic kidney disease (CKD) and CKD-associated anemia. Increasing evidence supports the link between renal fibrosis and anemia in CKD. JPYS possessed anti-fibrosis effects in experimental CKD. Nevertheless, research on the mechanisms of JPYS in ameliorating renal anemia (RA) through suppressing renal fibrosis remains to be clarified. AIM OF THE STUDY: Our study here was carried out to investigate the mechanisms of JPYS in protecting against RA. MATERIALS AND METHODS: An adenine-induced anemia model in rats with CKD at three different time points was established, and bio-samples taken from each group were analyzed. Biochemical analysis was employed to detect kidney function and hematological parameters. Masson staining was used to evaluate renal fibrosis of rats. Western blot and immunohistochemistry were utilized to evaluate the expressions of fibrotic markers, erythropoietin (EPO) and hypoxia inducible factor-2α (HIF-2α) in the kidneys of rats. Subsequently, transcriptomic analysis was conducted to disclose the possible mechanisms of JPYS in treating RA. Finally, the expression levels of key targets were analyzed and validated by using Western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: JPYS treatment improved kidney function, suppressed renal fibrosis and enhanced hematological parameters in CKD rats. Moreover, JPYS treatment restored the increased expression levels of fibrotic markers and the declined EPO with time dependence. In parallel, data indicated JPYS treatment stimulated the translocation of HIF-2α into nucleus in the renal interstitium and thus promoted the expression of EPO. Transcriptomic profiling disclosed that activations of both nuclear factor kappa B (NF-κB) and transforming growth factor-ß (TGF-ß)/Smad pathways were closely associated with RA. Ultimately, experimental validation results presented that the increased expressions of target proteins from the above-mentioned two pathways in the kidneys were decreased significantly after JPYS treatment. CONCLUSION: Our findings suggest that JPYS may improve RA by alleviating renal fibrosis, and the mechanisms of which involve in inhibiting the NF-κB and TGF-ß/Smad pathways.

Transcriptomics and UHPLC-QQQ-MS analyses reveal the dysregulation of branched chain amino acids metabolism in renal fibrotic rats.

The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB, the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.

A novel enzyme-based functional correlation algorithm for multi-omics reveals the potential mechanisms of traditional Chinese medicines: Taking Jian-Pi-Yi-Shen formula as an example.

The integrated analysis of host metabolome and intestinal microbiome is an opportunity to explore the complex therapeutic mechanisms of traditional Chinese medicines. Currently, researchers mainly employ various statistical correlation analytical methods to investigate metabolome-microbiome correlations. However, these conventional correlation techniques often focus on statistical correlations and their biological meanings are always ignored, especially the functional relevance between them. Here, we developed a novel enzyme-based functional correlation (EBFC) algorithm to further improve the interpretability and the identified scope of microbe-metabolite correlations based on the conventional Spearman's analysis. The proposed EBFC algorithm is successfully utilized to reveal the therapeutic mechanisms of Jian-Pi-Yi-Shen (JPYS) formula on the treatment of adenine-induced chronic kidney disease (CKD) rats. JPYS, a TCM formula for treating CKD, has beneficial clinical effects. We tentatively revealed the potential mechanism of JPYS for treating CKD rats from the perspective of the serum metabolome, gut microbiome, and their interactions. Specifically, 11 metabolites and 19 bacterial genera in the CKD rats were significantly regulated to approaching normal status after JPYS treatment, suggesting that JPYS could ameliorate the pathological symptoms of CKD rats by reshaping the disturbed metabolome and gut microbiota. Further correlation analysis between the significantly perturbed metabolites, microbiota, and the related enzymes provided more strong evidence for the study of host metabolism-microbiota interactions and the therapeutic mechanism of JPYS on CKD rats. In conclusion, these findings will help us to deeply understand the pathogenesis of CKD and provide new insights into the therapeutic mechanism of JPYS.

Jian-Pi-Yi-Shen formula restores iron metabolism from dysregulation in anemic rats with adenine-induced nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Jian-Pi-Yi-Shen (JPYS) is a herbal decoction being used to relieve the symptoms of chronic kidney disease (CKD) and its complications, including anemia, for over twenty years. Nonetheless, it is unclear how JPYS influences renal anemia and iron metabolism. AIM OF THE STUDY: An analysis of network pharmacology, chemical profiling, and in vivo experiments was conducted to identify the impact of JPYS on JAK2-STAT3 pathway and iron utilization in renal anemia and CKD. MATERIALS AND METHODS: The chemical properties of JPYS and its exposed ingredients were detected in vivo. And based on the aforesaid chemical compounds, the potential targets and signaling pathways of JPYS for renal anemia treatment were predicted by network pharmacology. Afterward, an adenine-feeding animal model of CKD-related anemia was developed to verify the mechanism by which JPYS modulates iron recycling to treat renal anemia. Renal injury was estimated by serum creatinine (Scr), blood urea nitrogen (BUN), histopathological examinations and fibrosis degree. Western blot, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry approaches were utilized to assess the levels of JAK2, STAT3 and iron metabolism-related factors. RESULTS: There were 164 active ingredients identified in JPYS, including prototypes and metabolites in vivo were identified in JPYS, and 21 core targets were found through network pharmacology based on topological characteristics. Combined with the core targets and pathway enrichment analysis, the majority of the candidate targets were associated with the JAK2-STAT3 signaling pathways. Experimental results indicated that JPYS treatment significantly decreased the expression of BUN and Scr, restored renal pathological damage, down-regulated fibrosis degree, and improved hematological parameters such as red blood cell, hemoglobin and hematocrit in CKD rats. Furthermore, JPYS significantly restored iron metabolism from dysregulation by increasing the levels of iron and ferritin in the serum, inhibiting the production of hepcidin in liver and serum, and regulating transferrin receptor 1 in bone marrow. Meanwhile, the expression of JAK2 and STAT3 was suppressed by JPYS treatment. CONCLUSIONS: Based on these results, JPYS reduces hepcidin levels by inhibiting the activation of JAK2-STAT3 signaling, thereby protecting against iron deficiency anemia.