The therapeutic effects of saikosaponins on depression through the modulation of neuroplasticity: From molecular mechanisms to potential clinical applications.

Depression is a major global health issue that urgently requires innovative and precise treatment options. In this context, saikosaponin has emerged as a promising candidate, offering a variety of therapeutic benefits that may be effective in combating depression. This review delves into the multifaceted potential of saikosaponins in alleviating depressive symptoms. We summarized the effects of saikosaponins on structural and functional neuroplasticity, elaborated the regulatory mechanism of saikosaponins in modulating key factors that affect neuroplasticity, such as inflammation, the hypothalamic-pituitary-adrenal (HPA) axis, oxidative stress, and the brain-gut axis. Moreover, this paper highlights existing gaps in current researches and outlines directions for future studies. A detailed plan is provided for the future clinical application of saikosaponins, advocating for more targeted researches to speed up its transition from preclinical trials to clinical practice.

Research progress on antidepressant effects and mechanisms of berberine.

Depression, a global health problem with growing prevalence, brings serious impacts on the daily life of patients. However, the antidepressants currently used in clinical are not perfectly effective, which greatly reduces the compliance of patients. Berberine is a natural quaternary alkaloid which has been shown to have a variety of pharmacological effects, such as hypoglycemic, lipid-regulation, anti-cancer, antibacterial, anti-oxidation, anti-inflammatory, and antidepressant. This review summarizes the evidence of pharmacological applications of berberine in treating depression and elucidates the mechanisms of berberine regulating neurotransmitter levels, promoting the regeneration of hippocampal neurons, improving hypothalamic-pituitary-adrenal axis dysfunction, anti-oxidative stress, and suppressing inflammatory status in order to provide a reference for further research and clinical application of berberine.

Jiao-tai-wan and its effective component-berberine improve diabetes and depressive disorder through the cAMP/PKA/CREB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiao-tai-wan (JTW), a classic herbal formula of traditional Chinese medicine recorded in Han Shi Yi Tong, has been used to alleviate sleep disorders since ancient times. In modern pharmacological research, JTW has been adopted for treating diabetes mellitus and even exerts antidepressant effects. However, the potential mechanisms deserve further elucidation. AIM OF THE STUDY: The prevalence of diabetes mellitus combined with depressive disorder (DD) is continuing to increase, yet it is currently under-recognized and its treatment remains inadequate. The present study aims to explore the underlying therapeutics and mechanisms of JTW on DD. MATERIALS AND METHODS: Chronic restraint stress was used on db/db mice to construct a mouse model of DD. The therapeutic effects of JTW were assessed by glucolipid metabolic indexes, behavioral tests, and depression-related neurotransmitter levels. The inflammatory status and cell apoptosis of different mice were investigated and the changes in the cAMP/PKA/CREB pathway were detected. Combining the results of fingerprinting with molecular docking, the active components of JTW were screened. A cellular model was constructed by intervention of glucose combined with corticosterone (CORT). The levels of apoptosis and depression-related neurotransmitters in HT-22 cells were examined, and the changes in the cAMP/PKA/CREB pathway were tested. Finally, the activator and inhibitor of the PKA protein were used for reverse validation experiments. RESULTS: JTW could improve the impaired glucose tolerance, lipid metabolism disorders, and depression-like symptoms in DD mice. Meanwhile, JTW could alleviate the inflammatory status, suppress the microglia activation, and improve hippocampal neuron apoptosis in DD mice. The dual effects of JTW might be associated with the activation of the cAMP/PKA/CREB pathway. Berberine (Ber) was identified for the in vitro experiment, it could reverse the apoptosis of HT-22 cells and up-regulate the depression-related neurotransmitter levels, and the effects of Ber were related to the activation of the cAMP/PKA/CREB pathway as well. CONCLUSION: JTW could exert both hypoglycemic and antidepressant effects through activating the cAMP/PKA/CREB signaling pathway, its active component, Ber, could improve the damage to HT-22 cells induced by glucose combined with CORT via the activation of the cAMP/PKA/CREB pathway. Ber may be one of the effective components of the dual effects of JTW.

Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway.

BACKGROUND: Obesity has emerged as a global epidemic. Recent research has indicated that diet-induced obesity can be prevented by promoting lacteal junction zippering. Berberine, which is derived from natural plants, is found to be promising in weight reduction, but the underlying mechanism remains unspecified. PURPOSE: To determine whether berberine protects against obesity by regulating the lacteal junction and to explore potential molecular mechanisms. METHODS: Following the induction of the diet-induced obese (DIO) model, mice were administered low and high doses of berberine for 4 weeks. Indicators associated with insulin resistance and lipid metabolism were examined. Various methods, such as Oil Red O staining, transmission electron microscopy imaging, confocal imaging and others were used to observe the effects of berberine on lipid absorption and the lacteal junction. In vitro, human dermal lymphatic endothelial cells (HDLECs) were used to investigate the effect of berberine on LEC junctions. Western Blot and immunostaining were applied to determine the expression levels of relevant molecules. RESULTS: Both low and high doses of berberine reduced body weight in DIO mice without appetite suppression and ameliorated glucolipid metabolism disorders. We also found that the weight loss effect of berberine might contribute to the inhibition of small intestinal lipid absorption. The possible mechanism was related to the promotion of lacteal junction zippering via suppressing the ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) signaling pathway. In vitro, berberine also promoted the formation of stable mature junctions in HDLECs, involving the same signaling pathway. CONCLUSION: Berberine could promote lacteal junction zippering and ameliorate diet-induced obesity through the RhoA/ROCK signaling pathway.

Berberine exerts antidepressant effects in vivo and in vitro through the PI3K/AKT/CREB/BDNF signaling pathway.

BACKGROUND: Depression, a global neuropsychiatric disorder, brings a serious burden to patients and society as its incidence continues to rise. Berberine is one of the main compounds of a variety of Chinese herbal medicines and has been shown to have multiple pharmacological effects. However, whether berberine can exert antidepressant effects in vivo and in vitro and its related mechanisms remain to be explored. METHODS: The chronic restraint stress (CRS) method and corticosterone (CORT) were applied to simulate depression-like behavior in vivo and neuronal apoptosis in vitro, respectively. The antidepressant effects of berberine were evaluated by behavioral tests and changes in the content of monoamine neurotransmitters. Inflammatory cytokines were detected and immunofluorescence staining was used to observe the expression levels of apoptosis-related proteins. RT-qPCR and Western blot were used to examine the mRNA and protein expression (or phosphorylation) levels of biomarkers of the PI3K/AKT/CREB/BDNF signaling pathways. RESULTS: Behavioral tests and levels of neurotransmitters proved that berberine could effectively ameliorate depression-like symptoms in CRS mice. Meanwhile, the results of ELISA and immunofluorescence staining showed that berberine could alleviate inflammatory status and reduce cell apoptosis in vivo and in vitro. Moreover, the changes of the PI3K/AKT/CREB/BDNF signaling pathway induced by CRS or CORT in mouse hippocampus or HT-22 cells were significantly reversed by berberine. CONCLUSION: Our current study suggested that berberine could exert antidepressant effects in vitro and in vivo, which may be associated with the PI3K/AKT/CREB/BDNF signaling pathway.

Berberine enhances the function of db/db mice islet ß cell through GLP-1/GLP-1R/PKA signaling pathway in intestinal L cell and islet α cell.

Background: The evidence on berberine stimulating the secretion of GLP-1 in intestinal L cell has been studied. However, few research has explored its role on generating GLP-1 of islet α cell. Our experiment aims to clarify the mechanism of berberine promoting the secretion of GLP-1 in intestinal L cell and islet α cell, activating GLP-1R and its downstream molecules through endocrine and paracrine ways, thus improving the function of islet ß cell and treating T2DM. Methods: After confirming that berberine can lower blood glucose and improve insulin resistance in db/db mice, the identity maintenance, proliferation and apoptosis of islet cells were detected by immunohistochemistry and immunofluorescence. Then, the activation of berberine on GLP-1/GLP-1R/PKA signaling pathway was evaluated by Elisa, Western blot and PCR. Finally, this mechanism was verified by in vitro experiments on Min6 cells, STC-1 cells and aTC1/6 cells. Results: Berberine ameliorates glucose metabolism in db/db mice. Additionally, it also increases the number and enhances the function of islet ß cell. This process is closely related to improve the secretion of intestinal L cell and islet α cell, activate GLP-1R/PKA signaling pathway through autocrine and paracrine, and increase the expression of its related molecule such as GLP-1, GLP-1R, PC1/3, PC2, PKA, Pdx1. In vitro, the phenomenon that berberine enhanced the GLP-1/GLP-1R/PKA signal pathway had also been observed, which confirmed the results of animal experiments. Conclusion: Berberine can maintain the identity and normal function of islet ß cell, and its mechanism is related to the activation of GLP-1/GLP-1R/PKA signal pathway in intestinal L cell and islet α cell.

Diosgenin attenuates non-alcoholic fatty liver disease in type 2 diabetes through regulating SIRT6-related fatty acid uptake.

BACKGROUND: More than 70% of patients with type 2 diabetes (T2DM) concomitantly suffer from Non-alcoholic fatty liver disease (NAFLD), and the coexistence and interaction of them increases the intractability of NAFLD. With the protective effect against hepatic steatosis and liver fibrosis, SIRT6 is becoming a notable target of NAFLD. Diosgenin, an active monomer from Chinese herbs, has been reported to protect against NAFLD. PURPOSE: This study aims to figure out the mechanism how diosgenin alleviate NAFLD in T2DM and the relationship with SIRT6. METHODS: In vivo studies used spontaneous diabetic db/db mice and divided them into two parts. The first part included four groups consisting of control (Con) group, model (Mod) group, low dose of diosgenin (DL) group and high dose of diosgenin (DH) group. The second part included four groups consisting of Con group, Mod group, DH+OSS (OSS_128167, inhibitor of SIRT6) group, MDL (MDL800, agonist of SIRT6) group. HepG2 cell line was selected in study in vitro, which was mainly composed of six groups including Con group, palmitic acid (PA) group, PA+DL group, PA+DH group, PA+DH+OSS group, PA+MDL group. OGTT, Biochemical biomarker (including TG, TC, AST, ALT), inflammatory biomarker (including IL-6 and TNF-α) were measured. HE, Oil Red O, and DHE staining were conducted. Immunohistochemistry, immunofluorescence, mRNA-seq, and qPCR were used to explore the mechanism. RESULTS: Results in the first part of study in vivo indicated that diosgenin protected against lipid accumulation, oxidative stress, cell injury, and light inflammatory of liver in db/db mice and regulated the expression of SIRT6 and fatty acid transporter including CD36, FATP2, FABP1. The effect of diosgenin could be reversed in DH+OSS group and the same effect was observed in MDL group in the second part of study in vivo. The same results were also noted in followed study in vitro. Diosgenin inhibited the fatty acids uptake and regulated the expression of SIRT6 and fatty acid transporter including CD36, FATP2, and FABP1 in PA-induced hepG2 cells, and which was reversed in DH+OSS group and resembled in MDL group. CONCLUSIONS: Diosgenin could attenuate non-alcoholic fatty liver disease in type 2 diabetes through regulating SIRT6-related fatty acid uptake.

Traditional herbal formula Jiao-tai-wan improves chronic restrain stress-induced depression-like behaviors in mice.

OBJECTIVES: Jiao-tai-wan (JTW) has been often used to treat insomnia and diabetes mellitus. Recent studies found its antidepressant activity, but the related mechanism is not clear. This study is to evaluate the therapeutic effects of JTW on chronic restraint stress (CRS)-induced depression mice and explore the potential mechanisms. METHODS: CRS was used to set up a depression model. Mice in different groups were treated with 0.9 % saline, JTW and fluoxetine. After the last day of CRS, the behavioral tests were conducted. The levels of neurotransmitters, inflammatory cytokines and HPA axis index were detected and the protein expressions of NLRP3 inflammasome complex were determined. H&E, NISSL, TUNEL and immunofluorescence staining were used to observe histopathological changes and the activation of microglia and astrocytes. The potential mechanisms were explored via network pharmacology and verified by Western blot. RESULTS: The assessment of liver and kidney function showed that JTW was non-toxic. Behavioral tests proved that JTW can effectively ameliorate depression-like symptoms in CRS mice, which may be related to the inhibition of NLRP3 inflammasome activation. JTW can also improve the inflammatory state and HPA axis hyperactivity in mice, and has a protective effect on CRS-induced hippocampal neurons damage. The network pharmacology analysis and the results of Western blot suggested that the antidepressant effects of JTW may be related to the MAPK signaling pathway. CONCLUSION: Our findings indicated that JTW may exert antidepressant effects in CRS-induced mice by inhibiting NLRP3 inflammasome activation and improving inflammatory state, and MAPK signaling pathway may also be involved.

Diosgenin protects against podocyte injury in early phase of diabetic nephropathy through regulating SIRT6.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus. DN is the main cause of end-stage renal disease (ESRD). SIRT6 becomes the important target of DN. Diosgenin (a monomer from Chinese herbs) is probable to bind to SIRT6. PURPOSE: Based on studies presented in the literature on kidney injuries plus screening for the binding effects of the drug to Sirt6, we aimed to carry out the study to assess the effects of diosgenin involved in improving podocyte damage in the early phase of DN.. METHODS: DN model was established in spontaneous diabetic db/db mice. Animal experiment was in two parts. The first part includes four groups consisting of control (Con) group, model (Mod) group, low dose of diosgenin (DL) group and high dose of diosgenin (DH) group. The second part includes four groups consisting of control group, model group, DH+OSS_128167 (OSS, inhibitor of SIRT6) group, MDL800 (agonist of SIRT6) group. MPC5 cell line was selected in cell experiment, which was mainly composed of six groups including Con group, palmitic acid (PA) group, PA+DL group, PA+DH group, PA+DH+OSS group, PA+MDL800 group. Some procedures such as transcriptomics, RT-qPCR and so on were used in the study to explore and verify the mechanism. RESULTS: The abnormal changes of mesangial matrix expansion, glomerular basement membrane (GBM) thickness, foot process (FP) width, urine albumin/creatinine (UACR), DESMIN, ADRP, NEPHRIN, PODOCIN, SIRT6 in Mod group were alleviated in DH group rather than DL group in the first part of animal experiment. The effect in DH group could be reversed in DH+OSS group and the same effect was observed in MDL800 group in the second part of animal experiment. The same results were also found in cell experiment. Protein level and mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) and Angiopoietin-like-4 (ANGPTL4) were increased in PA group, which could be alleviated in DH group, MDL800 group rather than DH+OSS group. CONCLUSIONS: Diosgenin could protect against podocyte injury in early phase of diabetic nephropathy by regulating SIRT6.

Antidepressant Potential of Quercetin and its Glycoside Derivatives: A Comprehensive Review and Update.

Depression is a global health problem with growing prevalence rates and serious impacts on the daily life of patients. However, the side effects of currently used antidepressants greatly reduce the compliance of patients. Quercetin is a flavonol present in fruits, vegetables, and Traditional Chinese medicine (TCM) that has been proved to have various pharmacological effects such as anti-depressant, anti-cancer, antibacterial, antioxidant, anti-inflammatory, and neuroprotective. This review summarizes the evidence for the pharmacological application of quercetin to treat depression. We clarified the mechanisms of quercetin regulating the levels of neurotransmitters, promoting the regeneration of hippocampal neurons, improving hypothalamic-pituitary-adrenal (HPA) axis dysfunction, and reducing inflammatory states and anti-oxidative stress. We also summarized the antidepressant effects of some quercetin glycoside derivatives to provide a reference for further research and clinical application.

Identification Prognostic Value and Correlation with Tumor-Infiltrating Immune Cells of Tripartite-Motif Family Genes in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and mortality types of malignant tumors in the world. The Tripartite-Motif (TRIM) protein family consists of more than 80 proteins with E3 ubiquitin ligase activity. Increasing studies have found that TRIM family proteins play an extremely important role in the occurrence and development of tumors. However, the expression and prognostic values of TRIMs in HCC have not been clarified. METHODS: We used bioinformatic methods to explore the potential function of TRIM family genes in the HCC. Web servers ONCOMINE, UALCAN, GEPIA, cBioPortal, STRING, DAVID 6.8 and TIMER were used in this research. RESULTS: We screened TRIM1-76 and found the expressions of TRIM6, TRIM11, TRIM16, TRIM18(MID1), TRIM24, TRIM28, TRIM31, TRIM37, TRIM45, TRIM52, TRIM59, TRIM66 were significantly changed in HCC. Among them, TRIM24, TRIM28, TRIM37, TRIM45 and TRIM59 had significant effects on pathological stages, overall survival and disease free survival. Functions of these genes are primarily related to transcriptional misregulation in cancer, p53 signaling pathway, alcoholism and viral carcinogenesis, FoxO signal pathway, PI3K-AKT pathway, cell cycle, microRNAs in cancer. Our results showed the significant correlation between TRIMs expression and infiltration of innate immune cells (macrophages, neutrophils, and dendritic cells). CONCLUSION: Our result provides novel insights into the function of TRIM family genes, which may be used as potential references for drug targets and accurate survival predictions in patients with HCC.

The effect and mechanism of Jiao-tai-wan in the treatment of diabetes mellitus with depression based on network pharmacology and experimental analysis.

BACKGROUND: The incidence of diabetes mellitus (DM) and depression is increasing year by year around the world, bringing a serious burden to patients and their families. Jiao-tai-wan (JTW), a well-known traditional Chinese medicine (TCM), has been approved to have hypoglycemic and antidepressant effects, respectively, but whether JTW has such dual effects and its potential mechanisms is still unknown. This study is to evaluate the dual therapeutic effects of JTW on chronic restraint stress (CRS)-induced DM combined with depression mice, and to explore the underlying mechanisms through network pharmacology. METHODS: CRS was used on db/db mice for 21 days to induce depression-like behaviors, so as to obtain the DM combined with depression mouse model. Mice were treated with 0.9% saline (0.1 ml/10 g), JTW (3.2 mg/kg) and Fluoxetine (2.0 mg/kg), respectively. The effect of JTW was accessed by measuring fasting blood glucose (FBG) levels, conducting behavioral tests and observing histopathological change. The ELISA assay was used to evaluate the levels of inflammatory cytokines and the UHPLC-MS/MS method was used to determine the depression-related neurotransmitters levels in serum. The mechanism exploration of JTW against DM and depression were performed via a network pharmacological method. RESULTS: The results of blood glucose measurement showed that JTW has a therapeutic effect on db/db mice. Behavioral tests and the levels of depression-related neurotransmitters proved that JTW can effectively ameliorate depression-like symptoms in mice induced by CRS. In addition, JTW can also improve the inflammatory state and reduce the number of apoptotic cells in the hippocampus. According to network pharmacology, 28 active compounds and 484 corresponding targets of JTW, 1407 DM targets and 1842 depression targets were collected by screening the databases, and a total of 117 targets were obtained after taking the intersection. JTW plays a role in reducing blood glucose level and antidepressant mainly through active compounds such as quercetin, styrene, cinnamic acid, ethyl cinnamate, (R)-Canadine, palmatine and berberine, etc., the key targets of its therapeutic effect include INS, AKT1, IL-6, VEGF-A, TNF and so on, mainly involved in HIF-1 signal pathway, pathways in cancer, Hepatitis B, TNF signal pathway, PI3K-Akt signal pathway and MAPK signaling pathway, etc. CONCLUSION: Our experimental study showed that JTW has hypoglycemic and antidepressant effects. The possible mechanism was explored by network pharmacology, reflecting the characteristics of multi-component, multi-target and multi-pathway, which provides a theoretical basis for the experimental research and clinical application of JTW in the future.

Berberine improves ovulation and endometrial receptivity in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a clinical syndrome with reproductive and endocrine disorders. Berberine is a monomer from Chinese herbs such as Coptis chinensis, whose effect on improving ovulation and endometrial receptivity of PCOS is uncertain. PURPOSE: To evaluate the effect of berberine on improving PCOS and explore the mechanism. METHODS: The rat model of PCOS was induced by intraperitoneal injection of testosterone propionate. Then they was divided into model (Mod) group, low-dose of berberine (BL) group, high-dose of berberine (BH) group and metformin (Met) group as well as a control (Con) group was established. Ovary morphology, hormone level, glucolipid metabolism were measured. UID-mRNA-seq of ovary tissue was conducted to seek the mechanism of berberine on improving ovulation. Three biomarkers of endometrial receptivity were also examined in endometrium by immunohistochemistry. RESULTS: The number of cystic follicles was increased while the number of corpus luteum was decreased in the rats of Mod group. These changes could be reversed by high-dose of berberine intervention. Berberine could also decrease the levels of serum luteinizing hormone (LH) and total cholesterol (TC) in PCOS rats. Meanwhile, berberine improved the impairment of abnormal oral glucose tolerance without affecting fasting insulin level and Homeostasis model assessment-insulin resistance (HOMA-IR). Luteinizing hormone/ choriogonadotropin receptor (LHCGR) and cytochrome P450 Family 19 Subfamily A Member 1 (CYP19A1) were focused via RNA-seq of ovary. Protein expression in ovary and mRNA expression in granulosa cell of LHCGR and CYP19A1 were decreased in Mod group and rescued by the intervention of berberine. A decrease of endometrial thickness and an increase of integrin αvß3 and lysophosphatidic acid receptor 3 (LPAR3) protein expression were observed in Mod group, which could be also reversed by berberbine. CONCLUSIONS: Berberine could improve ovulation in PCOS and the mechanism might be associated with up-regulating LHCGR and CYP19A1. Berberine could also improve endometrial receptivity through down-regualting αvß3 and LPAR3.

Impact of montmorillonite clay on the homo- and heteroaggregation of titanium dioxide nanoparticles (nTiO2) in synthetic and natural waters.

The homoaggregation of titanium dioxide nanoparticles (nTiO2) and their heteroaggregation with ubiquitous natural clay colloids are crucial processes affecting the environmental transport and fate of nTiO2, whereas the latter has received less attention. In this study, the effects of pH, electrolytes, natural organic matter (NOM), and montmorillonite on the homo- and heteroaggregation of nTiO2 were systematically investigated. The isoelectric point of bare nTiO2 was 6.98, whereas TiO2-montmorillonite mixtures remained negative charged due to the reduced particle surface potential by heteroaggregation. Homoaggregation of nTiO2 was mainly affected by anions, whereas heteroaggregation in TiO2-montmorillonite mixtures was mainly affected by cations. Heteroaggregation between nTiO2 and montmorillonite involved the adsorption of CC/CH. Intensive aggregation of nTiO2 was observed with 4 mg/L montmorillonite, whereas with 20 mg/L montmorillonite, the aggregation was significantly inhibited by the over-coverage of montmorillonite. NOM was attached to the surface of nTiO2 with the adsorption of functional groups involving CC/CH and OCO. The addition of NOM effectively reduced the homo- and heteroaggregation of nTiO2, and the stabilizing effect was enhanced with the increased molecular weight and aromatic/aliphatic fraction in NOM. Besides electrostatic repulsion, steric repulsion could also be one of the main stabilization mechanisms of NOM. With Ca2+ in the solutions, the stabilizing effect of NOM was significantly weakened through cation bridging. The addition of montmorillonite could facilitate the aggregation of nTiO2 in the presence of NOM. The homo- and heteroaggregation of nTiO2 were also observed in 7 different types of natural waters. Homoaggregation predominated in waters with high ionic strength and low NOM contents (seawater and groundwater), whereas heteroaggregation predominated in surface freshwater and wastewater systems. The results reflect the instability of nTiO2 in natural aquatic environments and the potential risk they pose to benthic organisms.

Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes.

Keratitis-ichthyosis-deafness (KID) syndrome is a severe, untreatable condition characterized by ocular, auditory, and cutaneous abnormalities, with major complications of infection and skin cancer. Most cases of KID syndrome (86%) are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in the GJB2 gene, encoding gap junction protein Cx26, which alters gating properties of Cx26 channels in a dominant manner. We hypothesized that a mutant allele-specific small interfering RNA could rescue the cellular phenotype in patient keratinocytes (KCs). A KID syndrome cell line (KID-KC) was established from primary patient KCs with a heterozygous p.D50N mutation. This cell line displayed impaired gap junction communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp, and neurobiotin uptake assays. A human-murine chimeric skin graft model constructed with KID-KCs mimicked patient skin in vivo, further confirming the validity of these cells as a model. In vitro treatment with allele-specific small interfering RNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wild-type allele. This corrected both gap junction and hemichannel activity. Notably, allele-specific small interfering RNA treatment caused only low-level off-target effects in KID-KCs, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept and model system for the potential use of allele-specific small interfering RNA in treating KID syndrome and other dominant genetic conditions.

Differential regulation of Connexin50 and Connexin46 by PI3K signaling.

Gap junction channels can modify their activity in response to cell signaling pathways. Here, we demonstrate that Connexin50 (Cx50) coupling, but not Connexin46 (Cx46), increased when co-expressed with a constitutively active p110α subunit of PI3K in Xenopus oocytes. In addition, inhibition of PI3K signaling by blocking p110α, or Akt, significantly decreased gap junctional conductance in Cx50 transfected HeLa cells, with no effect on Cx46. Alterations in coupling levels were not a result of reduced Cx50 unitary conductance, suggesting that changes in the number of active channels were responsible. These data indicate that Cx50 is specifically regulated by the PI3K signaling pathway.

Aberrant connexin26 hemichannels underlying keratitis-ichthyosis-deafness syndrome are potently inhibited by mefloquine.

Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes nonsyndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small-molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine (MFQ) inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50∼16 µM), using an extracellular divalent cation, zinc (Zn(++)), as a nonspecific positive control for comparison (IC50∼3 µM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity.

Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome.

Improving cardiac conduction with a skeletal muscle sodium channel by gene and cell therapy.

The voltage-gated Na+ channel is a critical determinant of the action potential (AP) upstroke. Increasing Na+ conductance may speed AP propagation. In this study, we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells that electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. Human embryonic kidney 293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18 mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the AP. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro reentry model induced by high-frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced reentry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues.

The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome.

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.