RESUMEN
Background: Morus alba L. (Sangzhi) alkaloid (SZ-A) is a new antidiabetic drug approved by the China National Medical Products Administration in 2020. Diabetic nephropathy (DN) is a common diabetic complication and an important cause of morbidity and mortality in patients with diabetes. The effects of SZ-A on DN remain unknown. Purpose: This study evaluated the effects of SZ-A on DN in Zucker diabetic fatty (ZDF) rats and explored the underlying mechanisms based on nitrosative stress, inflammation, and fibrosis. Methods: Diabetic ZDF rats were orally administered 100 and 200 mg/kg of SZ-A once daily for 9 weeks. The glucose metabolism and kidney function were assayed. The pathological injury and fibrosis of the kidneys were separately evaluated using hematoxylin and eosin staining and Masson's staining. The oxidative and nitrosative stress and inflammation were assayed by determining the levels of related indices in the blood and kidneys and quantifying the related gene and protein expression. The expression of transforming growth factor ß1 (TGFß1) gene and protein were assayed by quantitative real-time PCR and immunohistochemistry, respectively. The renal transcriptomics was analyzed using RNA sequencing. Results: Repeated treatment with SZ-A significantly improved glucose metabolism, dose-dependently decreased the levels of blood urea nitrogen, urinary albumin, and ß2-microglobulin, and evidently relieved the renal injury in diabetic ZDF rats. As for the mechanisms, SZ-A remarkably ameliorated systemic nitrosative stress through lowering the levels of blood inducible nitric oxide synthase and nitric oxide, and significantly relieved systemic and renal inflammation by reducing the levels of blood interleukin-1ß and monocyte chemoattractant protein-1 (MCP-1) and decreasing the levels of renal C-reactive protein content and expression of tumor necrosis factor-α in the kidneys. SZ-A also improved renal fibrosis by lowering the expression of TGFß1 in the kidneys. Additionally, SZ-A significantly lowered the expression of stimulator of chondrogenesis 1 in the kidneys. Conclusion: Repeated treatments with SZ-A significantly ameliorates DN by regulating systemic nitrosative stress, renal inflammation, and renal fibrosis partially through inhibition of the cytokine-NO and TGF-ß1 signaling in ZDF rats, providing evidence for the additional application of SZ-A in clinical use for the treatment of DN.
RESUMEN
Background: Morus alba L. (Sangzhi) alkaloids (SZ-A), extracted from the Chinese herb Morus alba L. (mulberry twig), have been shown to ameliorate hyperglycemia in type 2 diabetes and have been approved for diabetes treatment in the clinic. However, their versatile pharmacologic effects and regulatory mechanisms are not yet completely understood. Purpose: This study explored the protective effects of SZ-A on islet ß cells and the underlying mechanism. Methods: Type 2 diabetic KKAy mice were orally administered SZ-A (100 or 200 mg/kg, once daily) for 11 weeks, and oral glucose tolerance, insulin tolerance, intraperitoneal glucose tolerance and hyperglycemia clamp tests were carried out to evaluate the potency of SZ-A in vivo. The morphology and ß-cell dedifferentiation marker of KKAy mouse islets were detected via immunofluorescence. The effect of SZ-A on glucose-stimulated insulin secretion was investigated in both the islet ß-cell line MIN6 and mouse primary islets. Potential regulatory signals and pathways in insulin secretion were explored, and cell proliferation assays and apoptosis TUNEL staining were performed on SZ-A-treated MIN6 cells. Results: SZ-A alleviated hyperglycemia and glucose intolerance in type 2 diabetic KKAy mice and improved the function and morphology of diabetic islets. In both MIN6 cells and primary islets, SZ-A promoted insulin secretion. At a normal glucose level, SZ-A decreased AMPKα phosphorylation, and at high glucose, SZ-A augmented the cytosolic calcium concentration. Additionally, SZ-A downregulated the ß-cell dedifferentiation marker ALDH1A3 and upregulated ß-cell identifying genes, such as Ins1, Ins2, Nkx2.2 and Pax4 in KKAy mice islets. At the same time, SZ-A attenuated glucolipotoxicity-induced apoptosis in MIN6 cells, and inhibited Erk1/2 phosphorylation and caspase 3 activity. The major active fractions of SZ-A, namely DNJ, FAG and DAB, participated in the above regulatory effects. Conclusion: Our findings suggest that SZ-A promotes insulin secretion in islet ß cells and ameliorates ß-cell dysfunction and mass reduction under diabetic conditions both in vivo and in vitro, providing additional supportive evidence for the clinical application of SZ-A.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Morus alba L. (Sangzhi) alkaloids (SZ-A) tablets have been approved by the China National Medical Products Administration for T2DM treatment. Our previous study (Liu et al., 2021) revealed that SZ-A protected against diabetes and inflammation in KKAy mice. However, the mechanism and components in SZ-A exerting anti-inflammatory effects are unclear. AIM OF THE STUDY: Investigate the effects and molecular mechanisms of SZ-A on inflammation, and identify anti-inflammatory active components in SZ-A. MATERIALS AND METHODS: The major ingredients in SZ-A were analyzed by HPLC and sulfuric acid - anthrone spectrophotometry. The inhibitory activities of SZ-A on lipopolysaccharide (LPS)-stimulated inflammation were determined in bone marrow-derived macrophage (BMDM) and RAW264.7 cells. The cytokine levels of IL-6 and TNF-α in cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression levels of IL-6 and TNF-α were detected by qRT-PCR. The levels of protein phosphorylation of p38 MAPK, ERK, and JNK were analyzed by Western blot. RESULTS: The main components in SZ-A were found to be 1-deoxynojirimycin (DNJ), 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), fagomine (FAG), polysaccharide (APS), and arginine (ARG). SZ-A reduced the levels of IL-6 and TNF-α secreted by LPS-induced RAW264.7 and BMDM cells. Simultaneously, the mRNA expression levels of IL-6 and TNF-α were all significantly suppressed by SZ-A in a concentration-dependent manner. Furthermore, SZ-A inhibited the phosphorylation of p38 MAPK, ERK, and JNK in BMDM and the activation of ERK and JNK signaling in RAW264.7 cells. We also observed that DNJ, DAB, FAG, and ARG markedly downregulated IL-6 and TNF-α cytokine levels, while APS did not have an obvious effect. CONCLUSIONS: SZ-A attenuates inflammation at least partly by blocking the activation of p38 MAPK, ERK, and JNK signaling pathways. DNJ, FAG, DAB, and ARG are the main constituents in SZ-A that exert anti-inflammatory effects.
