Network pharmacology reveals that Berberine may function against Alzheimer’s disease via the AKT signaling pathway

ObjectiveTo investigate the mechanism underlying the effects of berberine (BBR) in the treatment of Alzheimer’s disease (AD).Methods3 × Tg AD mice were treated with BBR for 3 months, then the open field test (OFT), the novel object recognition test (NOR) and the Morris water maze (MWM) test were performed to assess behavioral performance. Hematoxylin–eosin (HE) staining, Nissl staining were used to examine histopathological changes. The pharmacological and molecular properties of BBR were obtained from the TCMSP database. BBR-associated AD targets were identified using the PharmMapper (PM), the comparative toxicogenomics database (CTD), DisGeNet and the human gene database (GeneCards). Core networks and BBR targets for the treatment of AD were identified using PPI network and functional enrichment analyses. AutoDock software was used to model the interaction between BBR and potential targets. Finally, RT-qPCR, western blotting were used to validate the expression of core targets.ResultsBehavioral experiments, HE staining and Nissl staining have shown that BBR can improve memory task performance and neuronal damage in the hippocampus of AD mice. 117 BBR-associated targets for the treatment of AD were identified, and 43 genes were used for downstream functional enrichment analysis in combination with the results of protein–protein interaction (PPI) network analysis. 2,230 biological processes (BP) terms, 67 cell components (CC) terms, 243 molecular function (MF) terms and 118 KEGG terms were identified. ALB, EGFR, CASP3 and five targets in the PI3K-AKT signaling pathway including AKT1, HSP90AA1, SRC, HRAS, IGF1 were selected by PPI network analysis, validated by molecular docking analysis and RT-q PCR as core targets for further analysis. Akt1 mRNA expression levels were significantly decreased in AD mice and significantly increased after BBR treatment (p < 0.05). Besides, AKT and ERK phosphorylation decreased in the model group, and BBR significantly increased their phosphorylation levels.ConclusionAKT1, HSP90AA1, SRC, HRAS, IGF1 and ALB, EGFR, CASP3 were core targets of BBR in the treatment of AD. BBR may exert a neuroprotective effect by modulating the ERK and AKT signaling pathways

The Role of the Rho/ROCK Pathway in Ang II and TGF-β1-Induced Atrial Remodeling.

To study the role of the Rho/ROCK pathway in Ang II and TGF-β1-induced atrial remodeling.A canine atrial fibrillation (AF) model was established by rapid atrial pacing (RAP) of the left atrium. The roles of TGF-β1, the RhoA/ROCK signaling pathway and connective tissue growth factor (CTGF) in atrial remodeling were studied via both in vitro and in vivo experiments. Each of the dogs that received RAP developed persistent AF within 4 weeks. The mRNA expression levels of TGF-β1 (1.32±0.38), Collagen-I(1.33±0.91), CTGF(5.83±3.71), RhoA(1.23±0.57) and ROCK-1 (1.02±0.27) in the left atrium were significantly increased following 4 weeks of RAP. Angiotensin II (Ang II) induced the proliferation of atrial fibroblasts and up-regulated the expression of both CTGF and ROCK-1 in a dose-dependent manner. Simvastatin and Y27632 reversed Ang II-induced CFs proliferation, as well as ROCK-1(0.89±0.05 and 1.27±0.03, respectively) and CTGF (0.87±0.04 and 0.91±0.02, respectively) expression. The expression mRNA of ROCK-1(1.74±0.13) and CTGF (2.28±0.11) can upregulated by TGF-β1, and down-regulated by Simvastatin (1.22±0.03 vs 2.27±0.11), Y27632 (1.01±0.04 vs 1.64±0.03), Los (1.04±0.11 vs 1.26±0.05), respectively. Losartan and Simvastatin attenuated the effects of TGF-β1, inhibited RhoA activity as opposed to RhoA protein expression. Y27632 had no effect on either the expression or the activity of RhoA.The increased expression of profibrotic factors (CTGF, ROCK1 and Smad2/3) played an important role in our RAP-induced AF model. Increased atrial profibrotic factors involve the activation of either the TGF-β1/RhoA/ROCK-1 or the TGF-β1/Smad2/3 signaling pathway

The Role of the Rho/ROCK Pathway in Ang II and TGF-β1-Induced Atrial Remodeling - Fig 2

<p>The upper panel depicts mRNA expression levels of TGF-β1 (A), CTGF (B), collagen-I (C), RhoA (D) and ROCK-1 (E) in the canine left atrium following 4 weeks of RAP. The middle panel depicts the protein expression levels of TGF-β1 (F), CTGF (G), collagen I (H) and ROCK-1 (I) in the canine left atrium following 4 weeks of RAP. The lower panel depicts the immunohistochemical results pertaining to collagen-I (J), CTGF (K) and ROCK-1 (L) in the hearts (×400). C, control group; P, pacing group. *P<0.05, **P<0.01 vs group C. (n = 6 for Group C, n = 9 for Group P).</p

Representative HE staining (up panel) and Masson’s trichrome staining (down panel) of the atrial myocardium.

<p>(A) Structurally normal atrial myocardium in the control group and severe fibrosis in the pacing group (×100). (B) Atrial myocardium with HE staining under ×400 light microscopy. (C) A small amount of collagen is visible within the atrial myocardium of the control group, and an increased amount of collagen is visible within the atrial myocardium following pacing (×100). The red areas represent myocytes, and the blue areas represent collagen. (D) Atrial myocardium with Masson’s trichrome staining under ×400 light microscopy. C, control group; P, pacing group. (n = 6 for Group C, n = 9 for Group P).</p

The effect of TGF-β1 on the Smad signal pathway.

<p>(A) TGF-β1 upregulated the expression of Smad2/3. (B) Sim and Losinhibited the protein expression of Smad2/3 in the atrial CFs treated with TGF-β1. (C) The ratio of GTP-RhoA/total RhoA following different treatments. *P<0.05, **P<0.01 vs TGF-β1 group, #<i>P</i><0.05, ##<i>P</i><0.01 vs Y27632+TGF-β1 group. (n = 5 for each group).</p

The changes in echocardiographic indices before and after rapid atrial pacing (mm, x±s).

<p>The changes in echocardiographic indices before and after rapid atrial pacing (mm, x±s).</p

The changes in ERPA, ERPAV, CSNRT and AVN-Wen (x±s).

<p>The changes in ERPA, ERPAV, CSNRT and AVN-Wen (x±s).</p

The effect of the Ang II pathway on atrial CF proliferation (upper panel) and the effect of Ang II on the mRNA expression of the AF factors in the atrial CFs (lower panel).

<p>(A) The effects of different concentrations of Ang II on the proliferation of the atrial CFs. (B, C, D) The effects of different concentration of Los (B), Sim (C) and Y27632 (D) on Ang II induced cell proliferation. Ang II upregulated the expression of ROCK-1 (E), CTGF (F) and TGF-β1 (G). Sim, Los and Y27632 downregulated the Ang II induced expression of TGF-β1 (J), CTGF (I) and ROCK-1 (H). *P<0.05, **P<0.01 vs Group C, #<i>P</i><0.05, ##<i>P</i><0.01 vs Group Ang II.(n = 5 for each group).</p

Comparison of hydroxyproline expression between the two groups.

<p>LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. *P<0.05, **P<0.01. (n = 6 for Group C, n = 9 for Group P).</p