Karacoline, identified by network pharmacology, reduces degradation of the extracellular matrix in intervertebral disc degeneration via the NF-κB signaling pathway / 药物分析学报

Karacoline is a compound found in the plant Aconitum kusnezoffii Reichb. Although Aconitum kusnezoffii Reichb is widely used for the treatment of pain, very few studies have been carried out on the use of karacoline due to its potential toxicity. In this study, we selected key matrix metalloproteinases (MMPs), collagen II, and aggrecan as targets due to their association with intervertebral disc degeneration (IDD). Using these targets, we then used network pharmacology to predict a series of molecules that might exert therapeutic effects on IDD. Of these molecules, karacoline was predicted to have the best effect. Tumor necrosis factor (TNF)-αis known to promote the degeneration of the extracellular matrix in IDD. We therefore applied different concentrations of karacoline (0, 1.25, or 12.88μM) along with 100 ng/mL TNF-αto rat nucleus pulposus cells and found that karacoline reduced the expression of MMP-14 in IDD by inhibiting the nuclear factor (NF)-κB pathway, while collagen II and aggrecan expression was increased. This suggested that extracellular matrix degradation was inhibited by karacoline (P<0.05). Our data therefore reveal a new clinical application of karacoline and provide support for the use of network pharmacology in predicting novel drugs.

Effect of the sonic hedgehog inhibitor GDC-0449 on an in vitro isogenic cellular model simulating odontogenic keratocysts / 国际口腔科学杂志·英文版

Odontogenic keratocysts (OKCs) are common cystic lesions of odontogenic epithelial origin that can occur sporadically or in association with naevoid basal cell carcinoma syndrome (NBCCS). OKCs are locally aggressive, cause marked destruction of the jaw bones and have a propensity to recur. PTCH1 mutations (at ∼80%) are frequently detected in the epithelia of both NBCCS-related and sporadic OKCs, suggesting that PTCH1 inactivation might constitutively activate sonic hedgehog (SHH) signalling and play a major role in disease pathogenesis. Thus, small molecule inhibitors of SHH signalling might represent a new treatment strategy for OKCs. However, studies on the molecular mechanisms associated with OKCs have been hampered by limited epithelial cell yields during OKC explant culture. Here, we constructed an isogenic PTCH1 cellular model of PTCH1 inactivation by introducing a heterozygous mutation, namely, c.403C>T (p.R135X), which has been identified in OKC patients, into a human embryonic stem cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This was followed by the induction of epithelial differentiation. Using this in vitro isogenic cellular model, we verified that the PTCH1 heterozygous mutation causes ligand-independent activation of SHH signalling due to PTCH1 haploinsufficiency. This activation was found to be downregulated in a dose-dependent manner by the SHH pathway inhibitor GDC-0449. In addition, through inhibition of activated SHH signalling, the enhanced proliferation observed in these induced cells was suppressed, suggesting that GDC-0449 might represent an effective inhibitor of the SHH pathway for use during OKC treatment.

Effects of PTCH1 mutations on the epithelial proliferation derived from keratocystic odontogenic tumour / 北京大学学报(医学版)

Objective: To explore the relationship between the PTCH1 mutation and the expression of bcl-2, filaggrin, and loricrin in the keratocystic odontogenic tumour (KCOT), as well as the effects of the mutated PTCH1 on the epithelial proliferation and differentiation.Methods: The samples were collected from 20 cases of KCOT with mutated PTCH1, as well as 20 cases without mutation.All the samples were analyzed with immunohistochemical staining, for the purpose of investigating the expression of bcl-2, filaggrin, and loricrin.Results: In the samples with mutated PTCH1, the epithelia of 60% (12/20) cases expressed intensively positive bcl-2 staining, 20% (4/20) expressed moderate staining, and 20% (4/20) weak staining, but no negative bcl-2 staining samples were investigated;it was significantly different from the samples without PTCH1 mutation, in which 20% (4/20) expressed intensive staining, no moderate staining, 50% (10/20) weak staining, and 30% (6/20) negative staining were investigated (U=72, P=0.001).For the expression of filaggrin, 55% (11/20) of samples with PTCH1 mutations were stained weakly and 45% (9/20) showed negative staining, while in the samples not harboring PTCH1 mutations, 30% (6/20) cases showed moderate positive staining, 40% (8/20) weak staining and 30% (6/20) negative staining, no intensive staining was investigated (U=182, P=0.48).The loricrin expressed in all the layer of the epithelia in all the samples, while the filaggrin was mainly loca-lized within 1-4 layer cells of the epithelia.The differences of the expression of filaggrin and loricrin between the samples with mutated PTCH1 and without mutated PTCH1 displayed no significance.Conclusion: In the epithelia of KCOT, the bcl-2 expression was significantly associated with the PTCH1 mutation, which suggested that the mutated PTCH1 gene perhaps promotes the proliferation of KCOT epithelium.