Piperlonguminine attenuates renal fibrosis by inhibiting TRPC6.

ETHNOPHARMACOLOGICAL RELEVANCE: Liuwei Dihuang (LWDH) is a classic prescription that has been used to the treatment of "Kidney-Yin" deficiency syndrome for more than 1000 years in China. Recent studies have confirmed that LWDH can prevent the progression of renal fibrosis. Numerous studies have demonstrated the critical role that TRPC6 plays in the development of renal fibrosis. Due to the complex composition of LWDH and its remarkable therapeutic effect on renal fibrosis, it is possible to discover new active ingredients targeting TRPC6 for the treatment of renal fibrosis. AIM OF STUDY: This study aimed to identify selective TRPC6 inhibitors from LWDH and evaluate their therapeutical effects on renal fibrosis. MATERIALS AND METHODS: Computer-aided drug design was used to screen the biologically active ingredients of LWDH, and their affinities to human TRPC6 protein were detected by microcalorimetry. TRPC6, TRPC3, and TRPC7 over-expressed HEK293 cells were constructed, and the selective activities of the compounds on TRPC6 were determined by measuring [Ca2+]i in these cells. To establish an in vitro model of renal fibrosis, human renal proximal tubular epithelial HK-2 cells were stimulated with TGF-ß1. The therapeutic effects of LWDH compounds on renal fibrosis were then tested by detecting the related proteins. TRPC6 was knocked-down in HK-2 cells to investigate the effects of LWDH active ingredients on TRPC6. Finally, a unilateral ureteral obstruction model of renal fibrosis was established to test the therapeutic effect. RESULTS: From hundreds of LWDH ingredients, 64 active components with oral bioavailability ≥30% and drug-likeness index ≥0.18 were acquired. A total of 10 active components were obtained by molecular docking with TRPC6 protein. Among them, 4 components had an affinity with TRPC6. Piperlonguminine (PLG) had the most potent affinity with TRPC6 and blocking effect on TRPC6-mediated Ca2+ entry. A 100 µM of PLG showed no detectable inhibition on TRPC1, TRPC3, TRPC4, TRPC5, or TRPC7-mediated Ca2+ influx into cells. In vitro results indicated that PLG concentration-dependently inhibited the abnormally high expression of α-smooth muscle actin (α-SMA), collagen I, vimentin, and TRPC6 in TGF-ß1-induced HK-2 cells. Consistently, PLG also could not further inhibit TGF-ß1-induced expressions of these protein biomarkers in TRPC6 knocked-down HK-2 cells. In vivo, PLG dose-dependently reduced urinary protein, serum creatinine, and blood urea nitrogen levels in renal fibrosis mice and markedly alleviated fibrosis and the expressions of α-SMA, collagen I, vimentin, and TRPC6 in kidney tissues. CONCLUSION: Our results showed that PLG had anti-renal fibrosis effects by selectively inhibiting TRPC6. PLG might be a promising therapeutic agent for the treatment of renal fibrosis.

[End point determination by HPLC chromatographic fingerprint in processing prepared Rehmannia].

To establish HPLC chromatographic fingerprints to control the quality of Chinese herbal medicine. In this study, fingerprints were established based on HPLC-DAD chromatographs. And with these fingerprints, content variations of three important active components catalpol, 5-hydroxymethylfurfural and acteoside in Rehmannia rhizome were analyzed during processing, as well as changes of the fingerprints. Fingerprints comparing with the standard prepared Rehmannia fingerprints which came from the mean of prepared ones randomly chosen for standard was done to seek optimal processing time. The results indicated that catalpol decreased quickly as braising prolonged and almost vanished in the end. While the active component of 5-HMF increased linearly throughout the process of braising. And the content of acteoside did not show obvious change. Similarity to standard prepared Rehmannia reached summit after braising for 26 hours. So 26 hours could be considered to be the optimum time for braising prepared Rehmannia. Chromatographic fingerprint is convenient for revealing changes of constituents and for accurately controlling quality during processing prepared Rahmannia.

[Study on difference of biological characteristics and resistance to powdery mildew of different Astragalus populations].

OBJECTIVE: To study difference among populations which belong to Astragalus membranaceus and A. membranaceus var. mongholicus on morphology, habit, characteristics of physiology and resistance to powdery mildew, and classify them in order to provide theoretical basis for breeding and improving varieties. METHOD: Morphology, habits, isozyme and soluble protein electrophoretograms were compared among the populations. They were categorized by cluster analysis based on those electrophoretograms. Different ability of resistance to powdery mildew was also studied through comparing disease indices among six populations. RESULT: The results showed A. membranaceus var. mongholicus was distinctly different from A. membranaceus. There was a special type in colonies of A. membranaceus, which showed hairy upper epidermis of leaflets and later florescence. CONCLUSION: Astragalus for medicine could be categorized in three types A. membranaceus var. mongholicus, A. membranaceus early florescence type and A. membranaceus late florescence type. Among them A. membranaceus var. mongholicus is most resistant to powdery mildew, while A. membranaceus is easily infected, and the early florescence type is even more easily infected.