Withanolides from the active extract of Physalis angulate and their anti-hepatic fibrosis effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalis angulata L., a traditional Chinese medicine called "Kuzhi" in China, was used traditionally to treat liver diseases (eg. icterus, hepatitis) as well as malaria, asthma, and rheumatism. AIM OF THE STUDY: Our study aimed to investigate the withanolides with anti-hepatic fibrosis effect from P. angulate. MATERIALS AND METHODS: Withanolides were obtained from the EtOH extract of P. angulate by bioassay-molecular networking analysis-guided isolation using column chromatography and normal/reversed-phase semipreparative HPLC. The structures of new withanolides were elucidated by combinations of spectroscopic techniques with NMR and ECD calculations. MTT cell viability assay, AO/EB staining method, cell wound healing assay, ELISA and Western blot experiments were employed to evaluate the anti-hepatic fibrosis activity and to uncover related mechanism. Molecular docking analysis and cellular thermal shift assay were used to evaluate and verify the interaction between the active withanolides and their potential targets. RESULTS: Eight unreported withanolides, withagulides A-H (1-8), along with twenty-eight known ones were obtained from P. angulate. Withanolides 6, 9, 10, 24, 27, and 29-32 showed marked anti-hepatic fibrosis effect with COL1A1 expression inhibition above 50 %. Physalin F (9), the main component in the active fraction, significantly decreased the TGF ß1-stimulated expressions of collagen I and α-SMA in LX-2 cells. Mechanism study revealed that physalin F exerted its anti-hepatic fibrosis effect via the PI3K/AKT/mTOR signaling pathway. CONCLUSION: This study suggested that withanolides were an important class of natural products with marked anti-hepatic fibrosis effect. The main withanolide physalin F might be a promising candidate for hepatic fibrosis treatment. The work provided experimental foundation for the use of P. angulate to treat hepatic fibrosis.

Phomopsterone B Alleviates Liver Fibrosis through mTOR-Mediated Autophagy and Apoptosis Pathway.

Liver fibrosis is the initial pathological process of many chronic liver diseases. Targeting hepatic stellate cell (HSC) activation is an available strategy for the therapy of liver fibrosis. We aimed to explore the anti-liver fibrosis activity and potential mechanism of phomopsterone B (PB) in human HSCs. The results showed that PB effectively attenuated the proliferation of TGF-ß1-stimulated LX-2 cells in a concentration-dependent manner at doses of 1, 2, and 4 µM. Quantitative real-time PCR and Western blot assays displayed that PB significantly reduced the expression levels of α-SMA and collagen I/III. AO/EB and Hoechst33342 staining and flow cytometry assays exhibited that PB promoted the cells' apoptosis. Meanwhile, PB diminished the number of autophagic vesicles and vacuolated structures, and the LC3B fluorescent spots indicated that PB could effectively inhibit the accretion of autophagosomes in LX-2 cells. Moreover, rapamycin and MHY1485 were utilized to further investigate the effect of mTOR in autophagy and apoptosis. The results demonstrated that PB regulated autophagy and apoptosis via the mTOR-dependent pathway in LX-2 cells. In summary, this is the first evidence that PB effectively alleviates liver fibrosis in TGF-ß1-stimulated LX-2 cells, and PB may be a promising candidate for the prevention of liver fibrosis.

Advances in the study of exosome-derived miRNAs in the pathogenesis, diagnosis, and treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an inflammatory disease caused by autoantibodies, with high morbidity and mortality. It involves multiple systems, particularly the renal, which can lead to lupus nephritis (LN); its multi-system effects have a significant impact on the physical and mental health of patients. Exosomes are vesicles that are secreted during cell activity and carry a variety of nucleic acids, proteins, and lipids. They are distributed through body fluids for cellular communication. MicroRNAs (miRNAs) are nucleic acids that are packaged within the exosome that are taken up and released in response to changes in plasma membrane structure. MiRNAs are potential participants in immune and inflammatory responses, which are transported to target cells and can inhibit gene expression in receptor cells. It has been suggested that exosomal miRNA can regulate the pathogenesis of SLE and, as such, they are of value in diagnosis and treatment. In this paper, we focus on the research progress into exosomal miRNA in SLE and inspire new directions for SLE related research.

Testicular exosomes disturb the immunosuppressive phenotype of testicular macrophages mediated by miR-155-5p in uropathogenic Escherichia coli-induced orchitis.

Male reproductive infections are known to shape the immunological homeostasis of the testes, leading to male infertility. However, the specific pathogenesis of these changes remains poorly understood. Exosomes released in the inflammatory microenvironment are important in communication between the local microenvironment and recipient cells. Here, we aim to identify the immunomodulatory properties of inflammatory testes-derived exosomes (IT-exos) and explore their underlying mechanisms in orchitis. IT-exos were isolated using a uropathogenic Escherichia coli (UPEC)-induced orchitis model and confirmed that IT-exos promoted proinflammatory M1 activation with increasing expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in vitro. We further used small RNA sequencing to identify the differential miRNA profiles in exosomes and primary testicular macrophages (TMs) from normal and UPEC-infected testes, respectively, and identified that miR-155-5p was highly enriched in IT-exos and TMs from inflammatory testes. Further study of bone marrow derived macrophages (BMDMs) transfected with miR-155-5p mimic showed that macrophages polarized to proinflammatory phenotype. In addition, the mice that were administrated IT-exos showed remarkable activation of TM1-like macrophages; however, IT-exos with silencing miR-155-5p showed a decrease in proinflammatory responses. Overall, we demonstrate that miR-155-5p delivered by IT-exos plays an important role in the activation of TM1 in UPEC-induced orchitis. Our study provides a new perspective on the immunological mechanisms underlying inflammation-related male infertility.

Identification of differential metabolites using untargeted metabolomics between gestational diabetes and normal pregnant women.

OBJECTIVE: To study the metabonomics differences between pregnant women with gestational diabetes mellitus (GDM) in the third trimester and those in a group without GDM by screening a group of highly efficient and sensitive markers for GDM and validating previously published early metabolic markers of GDM. METHODS: A cross-sectional cohort study based on ultra performance liquid chromatography tandem mass spectrometry untargeted metabolomics analysis of serum samples collected from 59 pregnant women with GDM and 59 pregnant women without GDM. RESULTS: A total of 121 metabolites were detected, and 27 were identified as differential metabolites between GDM and control. The combination of 27 metabolic peaks had area under curve (AUC) values of 0.90, 0.92, and 0.93 in the prediction models using support vector machine, partial least squares, and random forest, respectively. Finally, five metabolite biomarkers were selected to construct logistic regression models: L-valine, hypoxanthine, eicosapentaenoic acid, 2-amino-1,3,4-octadecanotriol, and choline. The AUC value of these metabolites was 0.769 between the GDM group and the control group. CONCLUSIONS: The discovery of a group of differential metabolites in pregnant women with GDM in the third trimester and in pregnant women without GDM may facilitate the study of the pathologic mechanism of GDM; it may be possible to find an efficient and sensitive alternative GDM detection method.

S100A9 Activates the Immunosuppressive Switch Through the PI3K/Akt Pathway to Maintain the Immune Suppression Function of Testicular Macrophages.

Macrophages are functionally plastic and can thus play different roles in various microenvironments. Testis is an immune privileged organ, and testicular macrophages (TMs) show special immunosuppressive phenotype and low response to various inflammatory stimuli. However, the underlying mechanism to maintain the immunosuppressive function of TMs remains unclear. S100A9, a small molecular Ca2+ binding protein, is associated with the immunosuppressive function of macrophages. However, no related research is available about S100A9 in mouse testis. In the present study, we explored the role of S100A9 in TMs. We found that S100A9 was expressed in TMs from postnatal to adulthood and contributed to maintaining the immunosuppressive phenotype of TMs, which is associated with the activation of PI3K/Akt pathway. S100A9 treatment promotes the polarization of bone marrow-derived macrophages from M0 to M2 in vitro. S100A9 was significantly increased in TMs following UPEC-infection and elevated S100A9 contributed to maintain the M2 polarization of TMs. Treatment with S100A9 and PI3K inhibitor decreased the proportion of M2-type TMs in control and UPEC-infected mouse. Our findings reveal a crucial role of S100A9 in maintaining the immunosuppressive function of TMs through the activation of PI3K/Akt pathway, and provide a reference for further understanding the mechanism of immunosuppressive function of TMs.

Neuronal degeneration in autonomic nervous system of Dystonia musculorum mice.

BACKGROUND: Dystonia musculorum (dt) is an autosomal recessive hereditary neuropathy with a characteristic uncoordinated movement and is caused by a defect in the bullous pemphigoid antigen 1 (BPAG1) gene. The neural isoform of BPAG1 is expressed in various neurons, including those in the central and peripheral nerve systems of mice. However, most previous studies on neuronal degeneration in BPAG1-deficient mice focused on peripheral sensory neurons and only limited investigation of the autonomic system has been conducted. METHODS: In this study, patterns of nerve innervation in cutaneous and iridial tissues were examined using general neuronal marker protein gene product 9.5 via immunohistochemistry. To perform quantitative analysis of the autonomic neuronal number, neurons within the lumbar sympathetic and parasympathetic ciliary ganglia were calculated. In addition, autonomic neurons were cultured from embryonic dt/dt mutants to elucidate degenerative patterns in vitro. Distribution patterns of neuronal intermediate filaments in cultured autonomic neurons were thoroughly studied under immunocytochemistry and conventional electron microscopy. RESULTS: Our immunohistochemistry results indicate that peripheral sensory nerves and autonomic innervation of sweat glands and irises dominated degeneration in dt/dt mice. Quantitative results confirmed that the number of neurons was significantly decreased in the lumbar sympathetic ganglia as well as in the parasympathetic ciliary ganglia of dt/dt mice compared with those of wild-type mice. We also observed that the neuronal intermediate filaments were aggregated abnormally in cultured autonomic neurons from dt/dt embryos. CONCLUSIONS: These results suggest that a deficiency in the cytoskeletal linker BPAG1 is responsible for dominant sensory nerve degeneration and severe autonomic degeneration in dt/dt mice. Additionally, abnormally aggregated neuronal intermediate filaments may participate in neuronal death of cultured autonomic neurons from dt/dt mutants.

Pressor effects on blood pressure induced by isovolumic bladder distension and electro-acupuncture stimulations in anesthetized rats.

The pressor effects on blood pressure (BP) elicited by electro-acupuncture (Ea) stimulations and vesico-vascular reflex (VVR) were investigated in anesthetized rats. Twenty adult female Sprague-Dawley rats were used throughout this study. Two acupoints, the Hoku (Li-4) and the Tsusanli (St-36), were tested by a low frequency Ea (LFEa, 2 Hz) and a high frequency Ea (HFEa, 20 Hz) with intensities 20 times that of the motor threshold. Ea at Tsusanli elicited no pressor effects (98.15 +/- 4.10% and 101.43 +/- 3.96% of pre-stimulation control in LFEa and HFEa, respectively) whereas pressor effects could be induced by Ea stimulations at Hoku (126.3 +/- 3.3% and 136.3 +/- 3.8% of pre-stimulation control in LFEa and HFEa, respectively, P < 0.01, n=10). In addition, the patterns of pressor effects elicited by LFEa and HFEa at Hoku were different, i.e., LFEa at Hoku elicited a tonic pressor effect, while HFEa elicited a phasic one. The VVR induced by bladder isovolumic saline distension also elicited a pressor effect on BP (119.2 +/- 2.2%, P < 0.01, n=10) in the same preparations during bladder contractions. The VVR did not modify the Ea-induced pressor responses, and vice versa, when both of them were superimposed. All the results suggested that the pressor effects elicited by the VVR and the Ea stimulation were additive responses. In addition, for future clinical application, our findings may imply that patients should be carefully monitored for signs and symptoms of autonomic dysfunction during clinical acupuncture treatments.

Calcium/calmodulin-dependent kinase II mediates NO-elicited PKG activation to participate in spinal reflex potentiation in anesthetized rats.

Calcium/calmodulin protein kinase (CaMK)-dependent nitric oxide (NO) and the downstream intracellular messenger cGMP, which is activated by soluble guanylate cyclase (sGC), are believed to induce long-term changes in efficacy of synapses through the activation of protein kinase G (PKG). The aim of this study was to examine the involvement of the CaMKII-dependent NO/sGC/PKG pathway in a novel form of repetitive stimulation-induced spinal reflex potentiation (SRP). A single-pulse test stimulation (TS; 1/30 Hz) on the afferent nerve evoked a single action potential, while repetitive stimulation (RS; 1 Hz) induced a long-lasting SRP that was abolished by a selective Ca(2+)/CaMKII inhibitor, autocamtide 2-related inhibitory peptide (AIP). Such an inhibitory effect was reversed by a relative excess of nitric oxide synthase (NOS) substrate, L-arginine. In addition, the RS-induced SRP was abolished by pretreatment with the NOS inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME). The sGC activator, protoporphyrin IX (PPIX), reversed the blocking effect caused by L-NAME. On the other hand, a sGC blocker, 1H-[1, 2, 4]oxadiazolo[4, 3-alpha]quinoxalin-1-one (ODQ), abolished the RS-induced SRP. Intrathecal applications of the membrane-permeable cGMP analog, 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt monohydrate (8-Br-cGMP), reversed the blocking effect on the RS-induced SRP elicited by the ODQ. Our findings suggest that a CaMKII-dependent NO/sGC/PKG pathway is involved in the RS-induced SRP, which has pathological relevance to hyperalgesia and allodynia.

Electroacupuncture-induced pressor and chronotropic effects in anesthetized rats.

The effects of electroacupuncture (Ea) on circulatory dynamics were investigated in anesthetized rats. The arterial blood pressure (BP) and the heart rate (HR) in response to Ea stimulations at the Tsusanli point (St-36) and the Hoku point (Li-4) were tested by a low frequency Ea (2 Hz; LFEa) and a high frequency Ea (20 Hz; HFEa) with stimulation intensities 20 times the motor threshold. Neither the HR nor the BP was affected when the Tsusanli point was stimulated. Whereas, Ea stimulations at the Hoku point elicit chronotropic and pressor effects. The patterns of pressor responses caused by the LFEa were different from that of an HFEa, i.e., the LFEa elicited a tonic effect, while an HFEa had a phasic one. The HFEa-induced pressor and chronotropic effects were attenuated, while the LFEa induced effects were completely blocked by an intravenous infusion of an alpha-adrenergic blocker (moxisylyte 0.2 mg/min/kg, i.v., for 20 min). A co-infusion with alpha-and beta-adrenergic blockers (propanolol 0.2 mg/min/kg, i.v., for 20 min) completely blocked the HFEa-induced pressor and chronotropic effects. We concluded that Ea stimulations, at the Hoku acupoint, with appropriate stimulation parameters can increase and maintain BP. Furthermore, the LFEa stimulation activates sympathetic vasomotor tone, whereas the HFEa stimulation causes an additional potentiation on the sympathetic drive to the heart.

Leber's hereditary opric neuropathy: a case report.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disease that primarily affects the optic nerve, causing bilateral vision loss in juveniles and young adults. A 12-year-old boy had complained of blurred vision in both eyes for more than 1 year. His best-corrected visual acuity was 0.08 in the right eye and 0.1 in the left. Ophthalmologic examination showed bilateral optic disc hyperemia and margin blurring, peripapillary telangiectasis, and a relative afferent pupil defect in his right eye. Fluorescein angiography showed no stain or leakage around the optic disc in the late phase. Visual field analysis showed central scotoma in the left eye and a near-total defect in the right. Upon examination of the patient's mitochondrial DNA, a point mutation at nucleotide position 11778 was found, and the diagnosis of LHON was confirmed. Coenzyme Q10 was used to treat the patient.