5-Hydroxytryptamine 4 Receptor Agonist Attenuates Diabetic Enteric Neuropathy through Inhibition of the Receptor-Interacting Protein Kinase 3 Pathway.

Necroptosis, considered as a form of programmed cell death, contributes to neural loss. The 5-hydroxytryptamine 4 receptor (5-HT4R) is involved in neurogenesis in the enteric nervous system. However, whether the activation of 5-HT4R can alleviate diabetic enteric neuropathy by inhibiting receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is unclear. This study aimed to explore the beneficial effects of 5-HT4R agonist on enteric neuropathy in a mouse model of diabetes and the mechanisms underlying these effects. Diabetes developed neural loss in the colon of mice. 5-HT4Rs localized in submucosal and myenteric plexuses were confirmed. Administration of 5-HT4R agonist attenuated diabetes-induced colonic hypomotility and neural loss of the colon in mice. Remarkably, RIPK3, phosphorylated RIPK3, and its downstream target mixed lineage kinase domain-like protein (MLKL), two key proteins regulating necroptosis, were significantly up-regulated in the colon of diabetic mice. Treatment with 5-HT4R agonist appeared to inhibit diabetes-induced elevation of RIPK3, phosphorylated RIPK3, and MLKL in the colon of mice. Diabetes-induced up-regulation of MLKL in both the mucosa and the muscularis of the colon was prevented by Ripk3 deletion. Moreover, diabetes-evoked neural loss and delayed colonic transit were significantly inhibited by Ripk3 removal. These findings suggest that activation of 5-HT4Rs could potentially provide a protective effect against diabetic enteric neuropathy by suppressing RIPK3-mediated necroptosis.

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

Vascular Endothelial Growth Factor Mediates the Sprouted Axonogenesis of Breast Cancer in Rat.

Nerve infiltration into the tumor is a common feature of the tumor microenvironment. The mechanisms of axonogenesis in breast cancer remain unclear. We hypothesized that vascular endothelial growth factor (VEGF), as well as nerve growth factor (NGF), is involved in the axonogenesis of breast cancer. A N-methyl-N-nitrosourea (MNU)-induced rat model of breast cancer was used to explore the presence of axonogenesis in breast tumor and the involvement of VEGF, as well as NGF, in the axonogenesis of breast tumor. Nerve infiltration into the tumor was found in MNU-induced rat model of breast cancer including the sensory and sympathetic nerve fibers. Nerve density was increased following the growth of tumor. The sensory neurons innervating the thoracic and abdominal mammary tumors peaked at T5 to T6 and L1 to L2 dorsal root ganglions, respectively. Either VEGF receptor inhibitor or antibody against VEGF receptor 2, as well as NGF receptor inhibitor, apparently decreased both the nerve density and vascular density of breast tumor. The reduced nerve density was correlated with the decreased vascular density induced by these treatments. In cultured dorsal root ganglion neurons, phosphatidylinositol 3 (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK), and p38 inhibitors significantly attenuated VEGF-induced neurite elongation. These findings provide direct evidence that VEGF, as well as NGF, may control the axonogenesis of breast cancer.

Salicylate sensitizes oral squamous cell carcinoma to chemotherapy through targeting mTOR pathway.

Oral squamous cell carcinoma (OSCC) is an extremely aggressive neoplasm, which is usually diagnosed in the advanced stage of the disease. Extensive studies have shown a link between chronic inflammation and various types of cancer, including OSCC. Salicylate is a biotransformation product of aspirin, with similar anti-inflammatory ability to aspirin but lacks aspirin's inhibitory effect on the isolated cyclooxygenase activity. Our study indicates that salicylate sensitizes OSCC to anti-cancer drugs, but the mechanisms of its action are unclear. Here, OSCC cells were used to evaluate the cytotoxicity of salicylate alone or in combination with cisplatin (CDDP). RPPA proteomic array and Western blotting were employed to determine the signaling pathways affected by salicylate. Salicylate decreased cell survival rate and induced cell apoptosis in OSCC cells but not human normal oral mucosal epithelial cells (hTERT-OME). The use of sodium salicylate (SS) dramatically sensitized OSCC cells to CDDP. RPPA array showed that SS reduced many oncogenes such as PI3K/mTOR signaling and cancer stem cell (CSC)-related genes versus control. Western and transcriptional analyses substantiated that salicylate down-regulated these CSC-associated genes and the mTOR pathway dose dependently. Salicylate preferentially repressed the ability of sorted ALDH1+ cells to form tumor spheres. Finally, salicylate suppressed tumor growth in vivo, and the combination of salicylate and CDDP further synergistically reduced the growth of tumors. Salicylate hinders OSCC cell growth and sensitizes OSCC cells to CDDP through targeting CSCs and the mTOR signaling pathway. We propose that salicylate is beneficial for OSCC patients, and salicylate may be combined with chemotherapies to effectively treat OSCC patients.

Oral administration of curcumin attenuates visceral hyperalgesia through inhibiting phosphorylation of TRPV1 in rat model of ulcerative colitis.

Background Curcumin has been reported to have anti-inflammatory and anti-nociceptive effects. The present study was designed to explore the potential therapeutic effects of curcumin on visceral hyperalgesia and inflammation in a rat model of ulcerative colitis. We observed the effects of orally administered curcumin on the disease activity index, histological change in colon, colorectal distension-induced abdominal withdrawal reflex, the expression of transient receptor potential vanilloid 1 (TRPV1) and phosphorylated TRPV1 in dextran sulfate sodium-induced colitis rats. In addition, a HEK293 cell line stably expressing human TRPV1 (hTRPV1) was used to examine the effects of curcumin on the change in membrane expression of TRPV1 induced by phorbol myristate acetate (a protein kinase C activator). Results Repeated oral administration of curcumin inhibited the increase in abdominal withdrawal reflex score induced by dextran sulfate sodium without affecting dextran sulfate sodium-induced histological change of colon and the disease activity index. A significant increase in colonic expression of TRPV1 and pTRPV1 was observed in dextran sulfate sodium-treated rats and this was reversed by oral administration of curcumin. TRPV1 expression in L6-S1 dorsal root ganglion was increased in the small- to medium-sized isolectin B4-positive non-peptidergic and calcitonin gene-related peptide-positive peptidergic neurons in dextran sulfate sodium-treated rats and oral administration of curcumin mitigated such changes. In the HEK293 cell line stably expressing hTRPV1, curcumin (1, 3 µm) inhibited phorbol myristate acetate-induced upregulation of membrane TRPV1. Conclusion Oral administration of curcumin alleviates visceral hyperalgesia in dextran sulfate sodium-induced colitis rats. The anti-hyperalgesic effect is partially through downregulating the colonic expression and phosphorylation of TRPV1 on the afferent fibers projected from peptidergic and non-peptidergic nociceptive neurons of dorsal root ganglion.

CircRNA Expression Profile in Early-Stage Lung Adenocarcinoma Patients.

BACKGROUND/AIMS: Lung adenocarcinoma, a form of non-small cell lung cancer with high lethality at an advanced stage, is becoming more common in women, non- or never-smokers, and even young adults. At present, there are still no effective early diagnosis methods for patients to be cured in a timely manner. Circular RNAs (circRNAs), which are stable and conserved non-coding RNA in mammalian cells, have been reported to be widely involved in the processes of cancer disease. However, it is still a puzzle as to which specific circRNAs are involved in the development of early-stage lung adenocarcinoma. METHODS: Tumor samples and paired adjacent normal tissues from 4 patients with early-stage lung adenocarcinoma were selected to investigate the expression profile of circRNAs by using a high-throughput circRNA microarray. Bioinformatic analyses were conducted to screen those differentially expressed circRNAs. qRT-PCR and sequencing were performed to assure the microarray data. RESULTS: A total of 357 circRNAs were dysregulated in the tumor samples, which suggests potential roles in lung cancer. qRT-PCR detection showed that five selected circRNAs were identical to the microarray data, and the potential circRNA-miRNA interactions were predicted. CONCLUSION: This work illustrates that clusters of circRNAs are aberrantly expressed in early-stage lung adenocarcinoma, which might offer potential targets for the early diagnosis of this disease and new genetic insights into lung cancer.

Role of MRI in the early diagnosis of tubal ectopic pregnancy.

OBJECTIVE: To determine the role of MRI in the early diagnosis of tubal ectopic pregnancy (EP). METHODS: Clinical and MRI features of 27 cases of tubal pregnancy were reviewed. RESULTS: A thick-walled gestational sac (GS)-like structure was demonstrated lateral to the uterus in all cases. On T2-weighted images, the thick wall typically exhibited 3 discrete rings in 22 cases (81 %), among which 17 cases (63 %) displayed small vessels and 6 cases (33 %) exhibited small areas of fresh haemorrhage inside the thick wall. The contents demonstrated non-specific liquid in 26 %, papillary solid components in 56 %, and fresh blood or fluid-fluid level in 19 % of the cases. Dilatation of the affected fallopian tube associated with hematosalpinx was demonstrated in 18 cases (67 %) and marked enhancement of the tubal wall was observed in 22 cases (81 %). No correlation was found between the size of the GS and the estimated gestational age (r = 0.056). CONCLUSION: MRI plays an important role in the early diagnosis and management of tubal pregnancy. The characteristic MRI features include a GS-like structure with a "three rings" appearance on T2-weighted images, presence of solid components in the sac, dilatation of the affected fallopian tube with hematosalpinx, and tubal wall enhancement. KEY POINTS: • MR imaging has served as a problem-solving procedure in ectopic pregnancy. • MR imaging features can be criteria for early diagnosis of tubal pregnancy. • Detailed assessment of ectopic implantation is necessary for management decision-making.

Impairments of the primary afferent nerves in a rat model of diabetic visceral hyposensitivity.

BACKGROUND: Diabetic neuropathy in visceral organs such as the gastrointestinal (GI) tract is still poorly understood, despite that GI symptoms are among the most common diabetic complications. The present study was designed to explore the changes in visceral sensitivity and the underlying functional and morphological deficits of the sensory nerves in short-term diabetic rats. Here, we compared the colorectal distension (CRD)-induced visceromotor response (VMR, an index of visceral pain) in vivo, the mechanosensitivity of colonic afferents ex vivo as well as the expression of protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP) in colon between diabetic (3-6 weeks after streptozotocin injection) and control (age-matched vehicle injection) rats. RESULTS: VMR was markedly decreased in the diabetic compared to the control rats. There was a significant decrease in multiunit pelvic afferent nerve responses to ramp distension of the ex vivo colon and single unit analysis indicated that an impaired mechanosensitivity of low-threshold and wide dynamic range fibers may underlie the afferent hyposensitivity in the diabetic colon. Fewer PGP 9.5- or CGRP-immunoreactive fibers and lower protein level of PGP 9.5 were found in the colon of diabetic rats. CONCLUSIONS: These observations revealed the distinctive feature of colonic neuropathy in short-term diabetic rats that is characterized by a diminished sensory innervation and a blunted mechanosensitivity of the remnant sensory nerves.

The clinicopathological significance of neurogenesis in breast cancer.

BACKGROUND: Recent reports support a novel biological phenomenon about cancer related neurogenesis. However, little is known about the clinicopathological significance of neurogenesis in breast cancer. METHODS: A total of 196 cases, including 20 of normal tissue, 14 of fibroadenoma, 18 of ductal carcinoma in situ (DCIS) and 144 of invasive ductal carcinoma (IDC) of the breast were used. The tissue slides were immunostained for protein gene product (PGP) 9.5 and S 100 to identify nerves. The correlation between the expression of PGP 9.5 and clinicopathological characteristics in IDC of the breast was assessed. RESULTS: While the PGP 9.5 positive nerve fibers are identified in all cases of normal breast tissue controls and in the tumor stroma of 61% (89/144) cases of invasive ductal carcinomas, PGP 9.5 positive nerve fibers are not seen in the tumor stroma of cases of fibroadenoma and DCIS. The percentage of tumors that exhibited neurogenesis increased from tumor grade I to tumor grade II and III (29.4% vs 71.8%, p < 0.0001). In addition, patients with less than 3 years of disease-free survival tended to have a higher positive expression of PGP 9.5 compared to patients with an equal or more than 3 years of disease-free survival (64.8% vs 46.7%, p = 0.035). Furthermore, moderate/strong expression of PGP 9.5 was found to be significantly related to microvessel density (MVD, p = 0.014). Interestingly, PGP 9.5 expression was significantly associated with higher MVD in the ER-negative (p = 0.045) and node-negative (p = 0.039) subgroups of IDC of the breast. CONCLUSIONS: This data indicates that neurogenesis is associated with some aggressive features of IDC including tumor grade and patient survival as well as angiogenesis, especially in ER-negative and node-negative subtypes of IDC of the breast. Thus, neurogenesis appears to be associated with breast cancer progression and may play a role in therapeutic guidance for patients with ER-negative and node-negative invasive breast cancer.

[Cannabinoid receptor 1 controls nerve growth in ectopic cyst in a rat endometriosis model].

OBJECTIVE: To investigate whether cannabinoid receptor 1 (CB1R) is involved in nerve growth in endometriosis-associated ectopic cyst. METHODS: The effect of CB1R agonist and antagonist on the expression of pan-neuronal marker protein gene product (PGP) 9.5 in ectopic cyst was examined by immunofluorescence and Western blot in endometriosis model of 18 rats. RESULTS: Immunofluorescence revealed that PGP 9.5 was expressed in the nerve fibers and was mainly distributed in the cyst hilum. Western blot revealed that the protein density of either PGP 9.5 (2 week: 0.38 ± 0.05; 4 week: 0.63 ± 0.03; 8 week: 0.80 ± 0.07, P < 0.01) or CB1R (2 week: 0.48 ± 0.04; 4 week: 0.68 ± 0.01; 8 week: 0.80 ± 0.03, P < 0.01) in the ectopic cyst increased with cyst size. In addition, compared to control group (0.75 ± 0.01), PGP 9.5 expression in the ectopic cyst was promoted by CB1R agonist ACPA (0.81 ± 0.01, P < 0.05), and inhibited by CB1R antagonist AM251 (0.67 ± 0.03, P < 0.01). CONCLUSIONS: CB1R was involved in the nerve growth of ectopic cyst associated with endometriosis.

[Prognostic significance of NADPH quinine oxidoreductase 1 overexpression in head and neck squamous cell carcinoma].

OBJECTIVE: To investigate the significance of NADPH quinine oxidoreductase 1 (NQO1) protein overexpression on prognostic evaluation of head and neck squamous cell carcinoma (HNSCC). METHODS: NQO1 protein was detected in 162 of HNSCC, 45 cases of adjacent nontumor tissues and 26 samples of normal head and neck epithelia using EnVision immunohistochemical. Correlation between NQO1 overexpression and patients prognosis was also analyzed. RESULTS: The positive rate and strongly positive rate of NQO1 protein were 84.0% (136/162) and 69.8% (113/162) in HNSCC, respectively, and both of which were significantly higher than either those in adjacent nontumor tissues and normal head and neck epithelia (both P < 0.01). NQO1 expression was significantly correlated with the clinical stage, pT and chemoradiotherapy of HNSCC (P < 0.01). Kaplan-Meier survival analysis showed that overall survival and disease-free survival rates were significantly higher in HNSCC patients with high level NQO1 expression than that those with low level of NQO1 expression (Log-rank = 6.625 , P = 0.010;Log-rank = 6.234 , P = 0.013). Additional analysis by Cox proportional hazard regression model showed that high level of NQO1 expression was an independent hazard predictor for overall survival of patients with HNSCC (Wald = 6.626, P = 0.008). CONCLUSIONS: NQO1 expression level is closely correlated with the progression and prognosis of patients with HNSCC. High level of NQO1 expression may be used as an important indicator for patients with poor prognostic HNSCC.

[Expression of neuronal marker protein gene product 9.5 and its clinicopathologic significance in breast cancer].

OBJECTIVE: To detect the expression of pan-neuronal marker protein gene product (PGP)9.5 and its clinicopathologic significance in breast cancer. METHODS: The expression of PGP9.5 was examined by immunohistochemistry EnVision method in 196 cases during 2007 to 2011, including 20 normal tissues, 14 cases of fibroadenoma, 18 cases of ductal carcinoma in situ (DCIS) and 144 cases of invasive ductal carcinoma (IDC) of the breast. The relationship between PGP9.5 expression and clinicopathologic characteristics of IDC was assessed. RESULTS: PGP9.5 expression was localized in the stroma of all normal breast tissues, but there was no expression observed in all fibroadenomas and DCIS. Overall, the expression rate of PGP9.5 in IDC was 61.8% (89/144). PGP9.5 expression increased from grade 1 tumors (29.4%, 10/34) to grade 2-3 tumors (71.8%, 79/110; P = 0.000). In addition, patients with less than 3 years disease-free survival tended to show higher PGP9.5 expression (64.8%, 35/54), compared to patients with equal to and/or more than 3 years disease-free survival (46.7%, 42/90; P = 0.035). However, there was no correlation between PGP9.5 expression and tumor size, tumor stage, lymph metastasis, hormone receptor expression. CONCLUSION: PGP9.5 expression is correlated with tumor grade and prognosis in IDC of the breast.

Different roles of peripheral mitogen-activated protein kinases in carrageenan-induced arthritic pain and arthritis in rats.

BACKGROUND: Accumulating evidence suggests that extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) might be involved in hypersensitivity of various pain models. However, there is a lack of direct evidence for actual involvement of peripheral ERK, p38, and JNK in induction and maintenance of arthritic pain and the development of arthritis. METHODS: We evaluated the effects of preemptive and therapeutic intra-articular administration of selective inhibitors of p38 (SB203580) and JNK (SP600125), and indirect inhibition of ERK with a blocker (PD98059) of the kinase that activates ERK (i.e., MEK, the mitogen-activated protein kinase [MAPK]/ERK kinase), on arthritic pain-related behavior such as reduction of weight load and the inflammatory responses such as neutrophil infiltration into the synovium and knee joint diameter in rats. In addition, arthritis-induced phosphorylation of ERK, p38, and JNK in synovium of knee joint was examined. RESULTS: Pretreatments with PD98059, SB203580, and SP600125 prevented the reduction of weight load induced by the carrageenan injected into the knee joint cavity, but their effects showed different time course patterns. Therapeutic administration of PD98059 and SB203580 partially reversed carrageen-induced reduction of weight load, and their effects showed a similar time course pattern. However, therapeutic administration of SP600125 had no effect on the reduction of weight load. Hematoxylin and eosin staining revealed that carrageenan-induced neutrophil infiltration into the synovium was inhibited by pretreatment with SB203580 or SP600125, but not PD98059. Western blot measurements showed distinct expression of phosphorylated ERK, p38, and JNK in the synovium at different time points after carrageenan injection. CONCLUSION: These results suggest that ERK, p38, and JNK signaling pathways at the peripheral level may play different roles in arthritic pain and arthritis of the knee joint.

GPRC5A reduction contributes to pollutant benzo[a]pyrene injury via aggravating murine fibrosis, leading to poor prognosis of IIP patients.

Air pollution exposure is recently reported to be one of the drivers of exacerbation in idiopathic pulmonary fibrosis (IPF). But there was a lack of direct evidence between pollution and lung fibrosis. Here, our data show effects of pollutant benzo[a]pyrene (BaP) and protein G-protein-coupled receptor family C group 5 type A (GPRC5A) on pulmonary fibrosis, which might help limit potential pollutant injury and disease progression. We cross-referenced epithelial differentially-expressed-genes (DEGs) from pollutant injury and published experimental fibrosis and IPF patients' data, top common-DEG (CO-DEG) GPRC5A was identified as a potential link between exposure-damage and fibrogenesis. The role of GPRC5A was evaluated under BaP exposure, in idiopathic interstitial pneumonia (IIP) tissue-array and via CRISPR/Cas9 knockout mice (Gprc5a-/-). BaP exposure enhanced bleomycin (BLM)-induced murine pulmonary fibrosis with increased Fibronectin and α-SMA expression in primary fibroblasts, thickened respiratory membrane and damaged alveolar type II cell, combined with Gprc5a decline in fibrotic mass. GPRC5A mRNA reduced after 10-14 days' BaP exposure in human epithelial cell A549. GPRC5A protein was further found to decrease in IIP epithelium, especially hyperplastic regions. A high epithelial GPRC5A expression score was positively associated with long survival time (R = 0.34) while negatively with high age (R = -0.4) and IIP type IPF (R = -0.5). Low GPRC5A expression predicts poor prognosis (HR = 4.5). Gprc5a depletion aggravated mortality rate (50%) with increased collagen deposition and myofibroblast activation under BLM treatment and exacerbated BaP injury in lung remodeling. Vitamin metabolic imbalance and Mitofusion2 (Mfn2) or Opa1-regulated mitochondrial dynamics were deduced to contribute to Gprc5a depletion and fibrogenesis. Pollutant BaP exposure worsens murine fibrosis and myofibroblast activation via GPRC5A reduction in the damaged epithelium. GPRC5A deficiency was first confirmed to contribute to both poor prognosis of IIP patients and fibrogenesis in murine model; thus, GPRC5A could serve as a novel therapeutic target in pollutant injury and pulmonary fibrosis.

Performance Evaluation of SHOX2 and RASSF1A Methylation for the Aid in Diagnosis of Lung Cancer Based on the Analysis of FFPE Specimen.

Emerging molecular diagnostic methods are more sensitive and objective, which can overcome the intrinsic failings of morphological diagnosis. Here, a RT-PCR-based in vitro diagnostic test kit (LungMe®) was developed and characterized to simultaneously quantify the DNA methylation of SHOX2 and RASSF1A in FFPE tissue specimens. The clinical manifestations were evaluated in 251 FFPE samples with specificity and sensitivity of 90.4 and 89.8%, respectively. Furthermore, the quantitative analysis shows that the degree of SHOX2 methylation was correlated with the stages of lung cancer, but not in the case of RASSF1A. Our observation indicated that the DNA methylation of SHOX2 and RASSF1A may play different roles in cancer development. Comparison of the methylation levels of SHOX2 and RASSF1A between cancer and cancer-adjacent specimens (n = 30), showed they have "epigenetic field defect". As additional clinical validation, the hypermethylation of SHOX2 and RASSF1A was detected not only in surgical operative specimens, but also in histopathological negative puncture biopsies. SHOX2 and RASSF1A methylation detection can be used to increase sensitivity and NPV, which provide us with a more accurate method of differential diagnosis and are likely to be rapidly applied in clinical examinations.

Vaccarin ameliorates insulin resistance and steatosis by activating the AMPK signaling pathway.

Destructive glucose and lipid metabolism in the liver is a crucial characteristic of type 2 diabetes mellitus (T2DM), eventually leading to insulin resistance and subsequent hyperglycemia and hyperlipidemia. Vaccarin is an active flavonoid glycoside associated with various biological functions, but its effects on glucose and lipid metabolic disorder in T2DM are still unclear. In this study, glucosamine (GlcN) and free fatty acids (FFAs, oleate/palmitate, 2:1 ratio) were applied to mimic insulin resistance and lipid deposition in HepG2 cells, respectively. Type 2 diabetic mice were induced using a high-fat diet (HFD) together with streptozotocin (STZ). GlcN stimulation was found to upregulate glucose production and gluconeogenesis but downregulate glycogen synthesis and phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in HepG2 cells, abnormal changes that were reversed by vaccarin. Furthermore, lipid accumulation was enhanced, but AMPK was inactivated in FFAs-exposed HepG2 cells, issues that were rectified by vaccarin treatment. These ameliorative effects of vaccarin on insulin resistance and steatosis were eliminated by AMPK inhibitor Compound C. In type 2 diabetic mice, chronic vaccarin administration decreased fasting blood glucose and lipid levels in both serum and the liver, and improved insulin sensitivity and steatosis in the liver. Taken together, these results suggest that vaccarin attenuates insulin resistance and steatosis by activating the AMPK signaling pathway.

Vaccarin prevents ox-LDL-induced HUVEC EndMT, inflammation and apoptosis by suppressing ROS/p38 MAPK signaling.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial-mesenchymal transition (EndMT), inflammation and apoptosis in endothelial cells play crucial roles in the progression of cardiovascular diseases including atherosclerosis. Vaccarin is a flavonoid glycoside from vaccariae semen associated with powerful cardiovascular protective effects. However, the effects of vaccarin on human umbilical vein endothelial cells (HUVEC) injury in response to ox-LDL remain unknown. Herein, we showed that treatment with vaccarin significantly suppressed ox-LDL-induced HUVEC inflammation, EndMT and apoptosis. Mechanistically, the HUVECs exposed to ox-LDL exhibited enlarged reactive oxygen species (ROS) production and p38 MAPK phosphorylation, which was counteracted by vaccarin. Importantly, ROS activator hydrogen peroxide (H2O2) and p38 MAPK activator anisomycin pretreatment prevent the protective effect of vaccarin on endothelial injury induced by ox-LDL. Our study suggested that vaccarin impeded ox-LDL-triggered HUVEC inflammation, EndMT and apoptosis via inhibition of ROS/p38 MAPK signaling pathway. Vaccarin may have a therapeutic effect on endothelial injury-related disorders.

Cannabinoid receptor 1 contributes to sprouted innervation in endometrial ectopic growth through mitogen-activated protein kinase activation.

The endocannabinoid system regulates neurite outgrowth and neurogenesis during development of the central nervous system. Cannabinoid receptor 1 (CB1R) is expressed in neurons, including the somata and fibers, that innervate the endometrial ectopic cyst in rats. Here, we investigated the contribution of CB1R and its downstream signaling to the innervation of endometrial ectopic growth. We found that intrathecal injection of a CB1R agonist enhanced both the density of protein gene product (PGP) 9.5-immunoreactive sprouted nerve fibers and the protein level of PGP 9.5 of the ectopic cyst, and the CB1R antagonist induced opposite effects. The CB1R agonist increased the expression of phosphorylated extracellular signal-regulated kinase (pERK) and c-Jun N-terminal kinase (pJNK), but not pp38, in dorsal root ganglion (DRG), whereas the CB1R antagonist only decreased the expression of pERK. In cultured DRG neurons, CB1R agonists dose-dependently increased neurite elongation. The mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) and JNK inhibitors, but not the p38 inhibitor, attenuated CB1R agonist-induced neurite elongation. The inhibitions of CB1R and its downstream ERK and JNK signaling pathways may alleviate the sprouted innervation that has been involved in ENDO-associated pain. This finding may provide a new therapeutic target for patients with endometriosis.