Sanguinarine modulates microglial function via PPARγ activation and protects against CNS demyelination.

Microglia aggregate in regions of active inflammation and demyelination in the CNS of multiple sclerosis (MS) patients and are considered pivotal in the disease process. Targeting microglia is a promising therapeutic approach for myelin repair. Previously, we identified two candidates for microglial modulation and remyelination using a Connectivity Map (CMAP)-based screening strategy. Interestingly, with results that overlapped, sanguinarine (SAN) emerged as a potential drug candidate to modulate microglial polarization and promote remyelination. In the current study, we demonstrate the efficacy of SAN in mitigating the MS-like experimental autoimmune encephalomyelitis (EAE) in a dose-dependent manner. Meanwhile, prophylactic administration of a medium dose (2.5 mg/kg) significantly reduces disease incidence and ameliorates clinical signs in EAE mice. At the cellular level, SAN reduces the accumulation of microglia in the spinal cord. Morphological analyses and immunophenotyping reveal a less activated state of microglia following SAN administration, supported by decreased inflammatory cytokine production in the spinal cord. Mechanistically, SAN skews primary microglia towards an immunoregulatory state and mitigates proinflammatory response through PPARγ activation. This creates a favorable milieu for the differentiation of oligodendrocyte progenitor cells (OPCs) when OPCs are incubated with conditioned medium from SAN-treated microglia. We further extend our investigation into the cuprizone-induced demyelinating model, confirming that SAN treatment upregulates oligodendrocyte lineage genes and increases myelin content, further suggesting its pro-myelination effect. In conclusion, our data propose SAN as a promising candidate adding to the preclinical therapeutic arsenal for regulating microglial function and promoting myelin repair in CNS demyelinating diseases such as MS.

Activation of TRPV1 receptor facilitates myelin repair following demyelination via the regulation of microglial function.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel that is activated by capsaicin (CAP), the main component of chili pepper. Despite studies in several neurological diseases, the role of TRPV1 in demyelinating diseases remains unknown. Herein, we reported that TRPV1 expression was increased within the corpus callosum during demyelination in a cuprizone (CPZ)-induced demyelination mouse model. TRPV1 deficiency exacerbated motor coordinative dysfunction and demyelination in CPZ-treated mice, whereas the TRPV1 agonist CAP improved the behavioral performance and facilitated remyelination. TRPV1 was predominantly expressed in Iba1+ microglia/macrophages in human brain sections of multiple sclerosis patients and mouse corpus callosum under demyelinating conditions. TRPV1 deficiency decreased microglial recruitment to the corpus callosum, with an associated increase in the accumulation of myelin debris. Conversely, the activation of TRPV1 by CAP enhanced the recruitment of microglia to the corpus callosum and potentiated myelin debris clearance. Using real-time live imaging we confirmed an increased phagocytic function of microglia following CAP treatment. In addition, the expression of the scavenger receptor CD36 was increased, and that of the glycolysis regulators Hif1a and Hk2 was decreased. We conclude that TRPV1 is an important regulator of microglial function in the context of demyelination and may serve as a promising therapeutic target for demyelinating diseases such as multiple sclerosis.

Serum- and glucocorticoid-inducible kinase 1 promotes insulin resistance in adipocytes via degradation of insulin receptor substrate 1.

AIMS: Accumulating evidence indicates that serum- and glucocorticoid-inducible kinase 1 (SGK1) plays a role in the development of metabolic syndrome via a poorly understood mechanism. This study aimed to investigate the direct effect of SGK1 on insulin sensitivity in adipose tissue. MATERIALS AND METHODS: We ectopically expressed or silenced SGK1 in adipocytes via lentiviral transfection, measured glucose uptake and evaluated insulin signalling using western blotting. In vivo insulin resistance was measured at the whole-body and adipose tissue levels in db/db mice treated with an inhibitor of SGK1. RESULTS: After 8 weeks of SGK1 inhibitor treatment, the serum insulin level and homeostasis model assessment of insulin resistance index were significantly decreased, and AKT phosphorylation in adipose tissue was enhanced in db/db mice. Overexpression of constitutively active SGK1 in adipocytes in vitro decreased AKT phosphorylation and insulin-stimulated glucose uptake. Dexamethasone and oleic acid increased SGK1 expression and decreased AKT phosphorylation and insulin receptor substrate expression in adipocytes. Administration of an inhibitor of SGK1 or Lv-shSGK1 reversed the suppression of insulin signalling induced by dexamethasone and oleic acid. SGK1 overexpression increased FoxO1 phosphorylation, and administration of Lv-shSGK1 reversed an increase in FoxO1 phosphorylation induced by dexamethasone and oleic acid. CONCLUSIONS: Thus, SGK1 mediates the effect of glucocorticoids and high-fat feeding and induces insulin resistance in adipocytes. Our data suggest that SGK1 is a possible therapeutic target for metabolic syndrome and related complications.

[Electroacupuncture promotes regeneration and repair of myelin sheath of corpus callosum in demyelination mice].

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in accelerating the aggregation of microglia and promoting the remyelination at the location of demyelination. METHODS: C57BL/6 mice were randomly divided into 4 groups: normal, control, model (LPC) and LPC＋EA. The demyelination model was established by microinjection of Lysolecithin (LPC, 1 µL) into the left corpus callosum. EA (2 Hz/15 Hz, 2－4 mA) was applied to "Baihui"(GV20)and "Zhiyang"(GV9)for 30 min，once daily for 3 days, then, once every other day for 18 days. Immuno-fluorescence staining was used to observe the expression of myelin basic protein (MBP) and Axl tyrosine kinase receptor (Axl), Iba1 and numbers of Olig2-positive oligodendrocytes in the corpus callosum. Western blot was employed to detect the expression of MBP in the corpus callosum, and Oil Red O staining was used to observe changes of number of myelin pieces. RESULTS: Following modeling, the expression levels of MBP on day 5 and 10 after modeling were significantly decreased (P<0.05, P<0.01), Iba1 expression and Olig2-positive oligodendrocyte numbers on day 10 apparently increased (P<0.001, P<0.01). On day 21 after modeling, the levels of the above mentioned indexes returned to normal. After EA intervention, the levels of MBP expression on day 5 and 10, Axl, Iba1 protein expression and Olig2-positive oligodendrocyte numbers on day 5 were markedly increased (P<0.001，P<0.01，P<0.05), while Iba1 expression on day 10 was considerably decreased in comparison with the model group (P<0.01)．Oil Red O staining showed that on day 5 after modeling, the number of red lipid droplets were obviously increased in the corpus callosum tissue on the injection side, and apparently reduced in the EA group, suggesting a clearance of the accumulated myelin fragments by EA. CONCLUSION: EA intervention may reduce myelin debris and promote the aggregation of microglial cells and oligodendrocytes to the injured site, accelerate the myelin regeneration and up-regulate the expression of MBP and Axl of corpus callosum in demyelination mice.

miR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation.

Activation of microglia and the subsequently elevated inflammatory cytokine release in the brain during surgery predispose individuals to cognitive dysfunction, also known as postoperative cognitive dysfunction (POCD). miR-124 is one of the most abundant microRNAs in the brain that regulates microglial function. Elucidating the role of miR-124 in microglial activation in the context of surgery may therefore promote understanding of as well as therapeutic development for post-surgical disorders involving microglial activation. The downstream targets of miR-124 were investigated using bioinformatic screening and dual-luciferase reporter assay validation, and vesicle-associated membrane protein 3 (VAMP3) was identified as a potential target. The kinetics of miR-124/VAMP3 expression was first examined in vitro in microglial cells (primary microglia and BV2 microglial cells) following lipopolysaccharide (LPS) stimulation. LPS induced a time-dependent decrease of miR-124 and upregulated the expression of VAMP3. Manipulating miR-124/VAMP3 expression by using miR-124 mimics or VAMP3-specific siRNA in LPS-stimulated BV2 microglial cells inhibited BV2 microglial activation-associated inflammatory cytokine release. To further examine the role of miR-124/VAMP3 in a surgical setting, we employed a rat surgical trauma model. Significant microglial activation and altered miR-124/VAMP3 expression were observed following surgical trauma. We also altered miR-124/VAMP3 expression in the rat surgical trauma model by administration of exogenous miR-124 and by using electroacupuncture, which is a clinically applicable treatment that modulates microglial function and minimizes postoperative disorders. We determined that electroacupuncture treatment specifically increases the expression of miR-124 in the hypothalamus and hippocampus. Increased miR-124 expression with a concomitant decrease in VAMP3 expression resulted in decreased inflammatory cytokine release related to microglial activation post-surgery. Our study indicates that miR-124/VAMP3 is involved in surgery-induced microglial activation and that targeting miR-124/VAMP3 could be a potential therapeutic strategy for postoperative disorders involving microglial activation.

The captopril challenge test for diagnosing primary Aldosteronism in a Chinese population.

BACKGROUND: The Captopril challenge test (CCT) is an easy-conduct confirmatory test for diagnosing primary aldosteronism (PA). Guidelines show that plasma aldosterone is normally suppressed by captopril (> 30%) in primary hypertension (PH) and in healthy people. It is unclear whether this standard is applicable in Chinese subjects. The aim of the present study was to investigate the post-CCT efficacy of plasma aldosterone concentration (PAC) suppression and determine the post-CCT aldosterone renin activity ratio (ARR) and PAC for PA diagnosis. METHODS: We recruited 110 consecutive patients with PA, 163 with primary hypertension (PH), and 40 healthy volunteers (NC). The CCT was conducted in all patients. Total sodium intake was estimated from 24-h urinary excretions. ROC curves were used to analyze the efficiency of different CCT diagnostic criteria for diagnosing PA. RESULTS: In NC and PH patients, PRA was increased and PAC was decreased post-CCT (P < 0.05). The mean degree of PAC decline after CCT was approximately 9.3%, and only 11.7% of the patients with PH showed a greater than 30% suppression of PAC after CCT. In patients with PA, the post-CCT change in PRA and PRC was slight. The post-CCT degree of PAC decline was unrelated to dietary salt intake. The areas under the ROC for the post-CCT ARR, PAC and PAC suppression % were 0.994, 0.754 and 0.606, respectively. The optimal post-CCT cutoff value for ARR for diagnosing PA was 20, which yielded a sensitivity and specificity of 94.0 and 99.4%, respectively. CONCLUSIONS: The PAC suppression percentage after CCT recommended by current clinical guidelines is not applicable when diagnosing Chinese subjects with PA. Compared to post-CCT PAC, post-CCT ARR was a better approach, having an optimal cutoff of 20 when interpreting the results of the CCT in Chinese patients. We found no relationship between high salt intake and low responses of the renin-angiotensin system (RAS) to the CCT.

Polyphyllin I Overcomes EMT-Associated Resistance to Erlotinib in Lung Cancer Cells via IL-6/STAT3 Pathway Inhibition.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is the most important limiting factor for treatment efficiency in EGFR-mutant non-small cell lung cancer (NSCLC). Much work has linked the epithelial-mesenchymal transition (EMT) to the emergence of drug resistance, consequently, ongoing research has been focused on exploring the therapeutic options to reverse EMT for delaying or preventing drug resistance. Polyphyllin I (PPI) is a natural compound isolated from Paris polyphylla rhizomes and displayed anti-cancer properties. In the current work, we aimed to testify whether PPI could reverse EMT and overcome acquired EGFR-TKI resistance. We exposed HCC827 lung adenocarcinoma cells to erlotinib which resulted in acquired resistance with strong features of EMT. PPI effectively restored drug sensitivity of cells that obtained acquired resistance. PPI reversed EMT and decreased interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway activation in erlotinib-resistant cells. Moreover, addition of IL-6 partially abolished the sensitization response of PPI. Furthermore, co-treatment of erlotinib and PPI completed abrogation of tumor growth in xenografts, which was associated with EMT reversal. In conclusion, PPI serves as a novel solution to conquer the EGFR-TKI resistance of NSCLC via reversing EMT by modulating IL-6/STAT3 signaling pathway. Combined PPI and erlotinib treatment provides a promising future for lung cancer patients to strengthen drug response and prolong survival.

[Electroacupuncture Intervention Enhances Splenic Natural Killer Cell Activity via Inhibiting Phosphorylation of ERK 5 in the Hypothalamus of Surgically Traumatized Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on cytotoxic activity of splenic natural killer (NK) cells after surgical trauma via extracellular signal-regulated kinase (ERK) 5 pathway in the rats' hypothalamus, so as to explore its mechanism underlying improving immune disorders after surgery. METHODS: Sprague-Dawley rats were randomly divided into the following 6 groups: control, trauma model, EA, sham EA, 4 nmol-BIX 02188 (an inhibitor for ERK 5 catalytic activity) and 20 nmol-BIX 02188 (n = 6 rats per group). The surgical trauma model was established by making a longitudinal incision (6 cm in length) along the median line of the back to expose the spinal column and another longitudinal incision along the abdominal median line. EA (2 Hz/15 Hz, 1 - 2 mA) was applied to bilateral "Zusanli" (ST 36) for 30 min immediately after surgery. For rats of the BIX groups, intra-lateral ventricular microinjection of BIX 02188 (10 µL, 4 nmol or 20 nmol, or saline for control rats) was conducted 30 min before the surgery. The expression level and protein of phosphorylated ERK 5 (p-ERK 5) and corticotropin-releasing factor (CRF) protein were measured by immunohistochemistry and Western blot, respectively. The cytotoxicity of splenic NK cells and the expression of splenic Perforin and Granzyme-B genes were measured by lactate dehydrogenase (LDH) release assay and real-time PCR, respectively. RESULTS: In comparison with the control group, hypothalamic p-ERK 5 immunoactivity, p-ERK 5 protein and CRF protein expression levels were significantly up-regulated in the model group (P<0. 01, P<0. 05), while splenic NK cell cytotoxicity and Perforin mRNA and Granzyme-B mRNA expression levels were notably down-regulated in the model group (P <0. 05, P < 0. 01). Following EA and administration of ERK 5 antagonist, the increased expression levels of p-ERK 5 immunoactivity in the EA group, and p-ERK 5 and CRF proteins in both EA and 20 nmol-BIX 02188 groups were obviously down-regulated (P<0. 05, P<0. 01), without changes in the sham EA and 4 nmol-BIX 02188 groups (P>0. 05) except the increased p-ERK 5 protein in the 4 nmol-BIX 02188 group. In addition, the down-regulated NK cell activity, Perforin mRNA and Granzyme-B mRNA expression levels were significantly reversed in the EA and 20 nmol-BIX 02188 groups (P<0. 05, P<0. 01). No significant differences were found between the EA group and 20 nmol-BIX 02188 group in down-regulating hypothalamic p-ERK 5 and CRF protein expression and up-regulating splenic NK cytotoxicity and Perforin and Granzyme-B gene expression (P>0. 05). CONCLUSION: EA can promote the cytotoxicity of splenic NK cells in surgical trauma rats, which may be closely associated with its functions in down-regulating trauma-induced activation of ERK 5 pathway and production of CRF in the hypothalamus.