Electroacupuncture Zusanli (ST36) Relieves Somatic Pain in Colitis Rats by Inhibiting Dorsal Root Ganglion Sympathetic-Sensory Coupling and Neurogenic Inflammation.

Referred somatic pain triggered by hyperalgesia is common in patients with inflammatory bowel disease (IBD). It was reported that sprouting of sympathetic nerve fibers into the dorsal root ganglion (DGR) and neurogenic inflammation were related to neuropathic pain, the excitability of neurons, and afferents. The purpose of the study was to explore the potential and mechanism of electroacupuncture (EA) at Zusanli (ST36) for the intervention of colon inflammation and hyperalgesia. Sprague-Dawley (SD) was randomly divided into four groups, including control, model, EA, and sham-EA. Our results showed EA treatment significantly attenuated dextran sulfate sodium- (DSS-) induced colorectal lesions and inflammatory cytokine secretion, such as TNF-α, IL-1ß, PGE2, and IL-6. EA also inhibited mechanical and thermal pain hypersensitivities of colitis rats. Importantly, EA effectively abrogated the promotion effect of DSS on ipsilateral lumbar 6 (L6) DRG sympathetic-sensory coupling, manifested as the sprouting of tyrosine hydroxylase- (TH-) positive sympathetic fibers into sensory neurons and colocalization of and calcitonin gene-related peptide (CGRP). Furthermore, EA at Zusanli (ST36) activated neurogenic inflammation, characterized by decreased expression of substance P (SP), hyaluronic acid (HA), bradykinin (BK), and prostacyclin (PGI2) in colitis rat skin tissues corresponding to the L6 DRG. Mechanically, EA treatment reduced the activation of the TRPV1/CGRP, ERK, and TLR4 signaling pathways in L6 DRG of colitis rats. Taken together, we presumed that EA treatment improved colon inflammation and hyperalgesia, potentially by suppressing the sprouting of sympathetic nerve fibers into the L6 DGR and neurogenic inflammation via deactivating the TRPV1/CGRP, ERK, and TLR4 signaling pathways.

Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways.

Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of Grb10 expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmt1 overexpression dramatically reversed Grb10 up-regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that Grb10 expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future.

Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways.

Atrial fibrillation (AF) is a complex disease with multiple inter-relating causes culminating in rapid atrial activation and atrial structural remodeling. The contribution of endoplasmic reticulum and mitochondria stress to AF has been highlighted. As the class III antiarrhythmic agent, ibutilide are widely used to AF. This study was designed to explore whether ibutilide could treat AF by inhibiting endoplasmic reticulum stress pathways and mitochondria stress. The neonatal rat cardiomyocytes were isolated and exposed to H2O2, ibutilide was add to the culture medium 12 h. Then the cell viability, oxidative stress levels and apoptotic rate were analyzed. In addition, endoplasmic reticulum stress related protein (GRP78, GRP94, CHOP), mitochondria-dependent protein (Bax, Bcl-2) and caspase-3/9/12 were identified by real-time PCR and western blot analysis. In our results, remarkable decreased cell viability and oxidative stress levels were detected in cardiomyocytes after treating with H2O2. The apoptotic rate and the expression of proteins involved in mitochondrial stress and endoplasmic reticulum stress pathways increased. While ibutilide significantly inhibited these changes. These data suggested that ibutilide serves a protective role against H2O2-induced apoptosis of neonatal rat cardiomyocytes, and the mechanism is related to suppression of mitochondrial stress and endoplasmic reticulum stress.

Electroacupuncture at Sensitized Acupoints Relieves Somatic Referred Pain in Colitis Rats by Inhibiting Sympathetic-Sensory Coupling to Interfere with 5-HT Signaling Pathway.

OBJECTIVE: To investigate whether electroacupuncture (EA) at sensitized acupoints could reduce sympathetic-sensory coupling (SSC) and neurogenic inflammatory response by interfering with 5-hydroxytryptamine (5-HT)ergic neural pathways to relieve colitis and somatic referred pain, and explore the underlying mechanisms. METHODS: Rats were treated with 5% dextran sodium sulfate (DSS) solution for 7 days to establish a colitis model. Twelve rats were randomly divided into the control and model groups according to a random number table (n=6). According to the "Research on Rat Acupoint Atlas", sensitized acupoints and non-sensitized acupoints were determined. Rats were randomly divided into the control, model, Zusanli-EA (ST 36), Dachangshu-EA (BL 25), and Xinshu (BL 15) groups (n=6), as well as the control, model, EA, and EA + GR113808 (a 5-HT inhibitor) groups (n=6). The rats in the control group received no treatment. Acupuncture was administered on 2 days after modeling using the stimulation pavameters: 1 mA, 2 Hz, for 30 min, with sparse and dense waves, for 14 consecutive days. GR113808 was injected into the tail vein at 5 mg/kg before EA for 10 min for 7 consecutive days. Mechanical sensitivity was assessed with von Frey filaments. Body weight and disease activity index (DAI) scores of rats were determined. Hematoxylin and eosin staining was performed to observe colon histopathology. SSC was analyzed by immunofluorescence staining. Immunohistochemical staining was performed to detect 5-HT and substance P (SP) expressions. The calcitonin gene-related peptide (CGRP) in skin tissue and tyrosine hydroxylase (TH) protein levels in DRG were detected by Western blot. The levels of hyaluronic acid (HA), bradykinin (BK), prostaglandin I2 (PGI2) in skin tissue, 5-HT, tryptophan hydroxylase 1 (TPH1), serotonin transporters (SERT), 5-HT 3 receptor (5-HT3R), and 5-HT 4 receptor (5-HT4R) in colon tissue were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: BL 25 and ST 36 acupoints were determined as sensitized acupoints, and BL 15 acupoint was used as a non-sensitized acupoint. EA at sensitized acupoints improved the DAI score, increased mechanical withdrawal thresholds, and alleviated colonic pathological damage of rats. EA at sensitized acupoints reduced SSC structures and decreased TH and CGRP expression levels (P<0.05). Furthermore, EA at sensitized acupoints reduced BK, PGI2, 5-HT, 5-HT3R and TPH1 levels, and increased HA, 5-HT4R and SERT levels in colitis rats (P<0.05). GR113808 treatment diminished the protective effect of EA at sensitized acupoints in colitis rats (P<0.05). CONCLUSION: EA at sensitized acupoints alleviated DSS-induced somatic referred pain in colitis rats by interfering with 5-HTergic neural pathway, and reducing SSC inflammatory response.

[Tumor infiltrating regulatory T cells in human breast cancer and associated draining lymph nodes: an in-situ analysis].

OBJECTIVE: To retrospectively analyze the quantity and status of the tumor infiltrating regulatory T lymphocytes in breast cancer and the draining lymph nodes, and to elucidate the clinical pathologic significance. METHODS: Seventy-four breast cancer samples with excised axillary lymph nodes were typed and staged histopathologically. The regulatory T lymphocytes were labeled by immunohistochemistry using EnVision method with the monoclonal antibodies against CD25 and Foxp3, and the immunophenotype was analyzed. In addition, the expression of IFN-γ, IL-10 and TGF-ß1 mRNA in lymphocytes of lymph nodes draining the tumors was detected by in situ hybridization with the corresponding specific oligo nucleaic acid probes. RESULTS: The number of CD25(+)Foxp3(+) T cells infiltrating the interstitium was much higher than that in the parenchymal tissue of the cancer. In the tumor draining lymph nodes, CD25(+) cells and Foxp3(+) cells were predominantly distributed in the paracortex with a proliferative pattern. TGF-ß1, INF-γ and IL-10 mRNA positive cells showed a similar distribution pattern in the draining lymph nodes. Among the 39 cases with metastatic disease, the lymph nodes with metastases showed a much higher number of CD25(+)Foxp3(+) cells than that without metastases (23.5 vs 17.3 and 23.8 vs 15.5; P < 0.05). However, there was no difference in the density of Foxp3(+)CD25(+) cells in the draining lymph nodes between the death and survival groups (P > 0.05). Cytokine expression of TGF-ß1, IL-10 and IFN-γ mRNA in the lymphocytes of draining lymph nodes in 24 cases showed that there were more IL-10 mRNA positive cells in the dead patients than that in the survived patients. A similar trend was observed for TGF-ß1 mRNA positive cells but the difference was not statistically significant (P > 0.05). The expression rate of TGF-ß1 and IL-10 mRNA in the draining lymph nodes was proportional to that of CD25(+) and Foxp3(+) cells (P < 0.05), and the expression of TGF-ß1 positive cells was also proportional to that of IL-10 mRNA positive cells (P < 0.01). The expression of IFN-γ mRNA among these groups showed no significance (P > 0.05). CONCLUSIONS: Regulatory T cells may play important roles in inhibiting the host antitumor immunity, and the presence of increased regulatory T cells and Th2-secreting cells in paracortex with a proliferative pattern in the tumor draining lymph nodes implies that the paracortical proliferation of draining lymph nodes may not reflect positive antitumor effects.

Molecular docking and molecular dynamics study Lianhua Qingwen granules (LHQW) treats COVID-19 by inhibiting inflammatory response and regulating cell survival.

Purpose: 2019 Coronavirus disease (COVID-19) is endangering health of populations worldwide. Latest research has proved that Lianhua Qingwen granules (LHQW) can reduce tissue damage caused by inflammatory reactions and relieve patients' clinical symptoms. However, the mechanism of LHQW treats COVID-19 is currently lacking. Therefore, we employed computer simulations to investigate the mechanism of LHQW treats COVID-19 by modulating inflammatory response. Methods: We employed bioinformatics to screen active ingredients in LHQW and intersection gene targets. PPI, GO and KEGG was used to analyze relationship of intersection gene targets. Molecular dynamics simulations validated the binding stability of active ingredients and target proteins. Binding free energy, radius of gyration and the solvent accessible surface area were analyzed by supercomputer platform. Results: COVID-19 had 4628 gene targets, LHQW had 1409 gene targets, intersection gene targets were 415. Bioinformatics analysis showed that intersection targets were closely related to inflammation and immunomodulatory. Molecular docking suggested that active ingredients (including: licopyranocoumarin, Glycyrol and 3-3-Oxopropanoic acid) in LHQW played a role in treating COVID-19 by acting on CSF2, CXCL8, CCR5, NLRP3, IFNG and TNF. Molecular dynamics was used to prove the binding stability of active ingredients and protein targets. Conclusion: The mechanism of active ingredients in LHQW treats COVID-19 was investigated by computer simulations. We found that active ingredients in LHQW not only reduce cell damage and tissue destruction by inhibiting the inflammatory response through CSF2, CXCL8, CCR5 and IFNG, but also regulate cell survival and growth through NLRP3 and TNF thereby reducing apoptosis.

Metformin regulates inflammation and fibrosis in diabetic kidney disease through TNC/TLR4/NF-κB/miR-155-5p inflammatory loop.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is significantly increasing worldwide, and the incidence of its complications is also on the rise. One of the main complications of T2DM is diabetic kidney disease (DKD). The glomerular filtration rate (GFR) and urinary albumin creatinine ratio (UACR) increase in the early stage. As the disease progresses, UACR continue to rise and GFR begins to decline until end-stage renal disease appears. At the same time, DKD will also increase the incidence and mortality of cardiovascular and cerebrovascular diseases. At present, the pathogenesis of DKD is not very clear. Therefore, exploration of the pathogenesis of DKD to find a treatment approach, so as to delay the development of DKD, is essential to improve the prognosis of DKD. AIM: To detect the expression of tenascin-C (TNC) in the serum of T2DM patients, observe the content of TNC in the glomerulus of DKD rats, and detect the expression of TNC on inflammatory and fibrotic factors in rat mesangial cells (RMCs) cultured under high glucose condition, in order to explore the specific molecular mechanism of TNC in DKD and bring a new direction for the treatment of DKD. METHODS: The expression level of TNC in the serum of diabetic patients was detected by enzyme-linked immunosorbent assay (ELISA), the protein expression level of TNC in the glomerular area of DKD rats was detected by immunohistochemistry, and the expression level of TNC in the rat serum was detected by ELISA. Rat glomerular mesangial cells were cultured. Following high glucose stimulation, the expression levels of related proteins and mRNA were detected by Western blot and polymerase chain reaction, respectively. RESULTS: ELISA results revealed an increase in the serum TNC level in patients with T2DM. Increasing UACR and hypertension significantly increased the expression of TNC (P < 0.05). TNC expression was positively correlated with glycosylated haemoglobin (HbA1c) level, body mass index, systolic blood pressure, and UACR (P < 0.05). Immunohistochemical staining showed that TNC expression in the glomeruli of rats with streptozotocin-induced diabetes was significantly increased compared with normal controls (P < 0.05). Compared with normal rats, serum level of TNC in diabetic rats was significantly increased (P < 0.05), which was positively correlated with urea nitrogen and urinary creatinine (P < 0.05). The levels of TNC, Toll-like receptor-4 (TLR4), phosphorylated nuclear factor-κB p65 protein (Ser536) (p-NF-κB p65), and miR-155-5p were increased in RMCs treated with high glucose (P < 0.05). The level of TNC protein peaked 24 h after high glucose stimulation (P < 0.05). After TNC knockdown, the levels of TLR4, p-NF-κB p65, miR-155-5p, connective tissue growth factor (CTGF), and fibronectin (FN) were decreased, revealing that TNC regulated miR-155-5p expression through the TLR4/NF-κB p65 pathway, thereby regulating inflammation (NF-κB p65) and fibrosis (CTGF and FN) in individuals with DKD. In addition, metformin treatment may relive the processes of inflammation and fibrosis in individuals with DKD by reducing the levels of the TNC, p-NF-κB p65, CTGF, and FN proteins. CONCLUSION: TNC can promote the occurrence and development of DKD. Interfering with the TNC/TLR4/NF-κB p65/miR-155-5p pathway may become a new target for DKD treatment.

[Expression Level of MiR-146a in Acute Myeloid Leukemia Patients and Its Clinical Significance].

OBJECTIVE: To detect the expression of miR-146a in patients with AML, and to evaluate the relationship between miR-146a expression level and clinical characteristics, treatment response, EFS and OS. METHODS: 154 patients with newly diagnosed AML were enrolled in AML group, 50 controls (patients with thrombocytopenic purpura or voluntary donor of bone marrow) were enrolled in control group. The miR-146a expression levels in bone marrow mononuclear cells was detected by RT-PCR between 2 group. AML patients were treated with chemotherapeutic drugs, and their clinical response and survivals were assessed. RESULTS: The expression level of MiR-146a in AML group was significantly lower than that in control group. The ROC showed that miR-146a could distinguish the patients in AML and control group better (area under curve 0.819 (95%CI: 0.761-0.877). Meanwhile, the proportion of good and moderate good prognosis (P＜0.001), proportion of WBC count ≤15.2×109/L (P＜0.05), CR rate (P＜0.05), EFS (P＜0.01) and OS (P＜0.01) in patients with high miR-146a expression were higher than those in patients with low miR-146a expression. Cox's model showed that miR-146a expression level positively realated with incressed EFS and OS. CONCLUSION: MiR-146a is downregulated in AML patients, which might be served as a biomarker for predicting risk and prognosis of AML patients.

Synaptic remodeling and reduced expression of the transcription factors, HES1 and HES5, in the cortex neurons of cognitively impaired hyperhomocysteinemic mice.

Hyperhomocysteinemia (HHcy) is associated with cognitive impairment and neurodegenerative diseases. The synaptic ultrastructure and the expression of hairy enhancer of split (HES) genes are involved in cognitive impairment induced by HHcy, but their precise role remains unclear. The present study aimed to measure synaptic remodeling and the expression of HES1 and HES5 in the cortex neurons of mice with HHcy to clarify their role in cognitive impairment. Mild HHcy was induced in ApoE-/- mice receiving a high-methionine diet. The correct response percentage, latency, and distance traveled in the mice with HHcy decreased compared with those of non-HHcy control mice (P < 0.05). There was no difference in the neuronal counts and the mean optical density of Nissl bodies in the frontal cortex of HHcy and non-HHcy mice. Increased apoptosis rates and numbers of autophagosomes were observed in the HHcy mice by TUNEL staining and electron microscopy, respectively, compared to those in the control group (P < 0.05). There was a significant increase in the area of postsynaptic density and size variation of synaptic vesicles in the HHcy group compared to that in the control (P < 0.05). Decreased expression of HES1 and HES5 was observed by western blotting and immunostaining in the HHcy group compared to that in the control (P < 0.05). Collectively, these results suggest that increased autophagy, apoptosis, synaptic remodeling, and downregulation of hes1 and hes5 are involved in the cognitive impairment induced by hyperhomocysteinemia.

LncRNA RHPN1-AS1 Promotes Cell Proliferation, Migration and Invasion Through Targeting miR-7-5p and Activating PI3K/AKT/mTOR Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. Emerging evidence has suggested that lncRNAs play an important role in cancer progression, including HCC. This study aimed to comprehensively investigate the effect of lncRNA RHPN1 antisense RNA 1 (RHPN1-AS1) on HCC and its underlying molecular mechanism. In this study, we evaluated the expressions of lncRNA RHPN1-AS1 and miR-7-5p by qRT-RCR in both HCC tissue and HCC cells. Our findings showed that lncRNA RHPN1-AS1 was upregulated in HCC tissue and HCC cells, while miR-7-5p was downregulated. LncRNA RHPN1-AS1 expression in HCC patients was closely related to vascular invasion, tumor-node-metastasis (TNM) stage and barcelona clinic liver cancer (BCLC) stage. Furthermore, we quantified cell clone-formation ability, proliferation, migration and invasion of HCCLM3 and MHCC97 H cells using several assays (colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and transwell assay, respectively). Functional experiments confirmed that silencing lncRNA RHPN1-AS1 inhibited cell proliferation, migration and invasion in HCCLM3 and MHCC97 H cells. After that, bioinformatics analysis, dual luciferase reporter gene assay, qRT-PCR and western blot were used to investigate the molecular mechanism of lncRNA RHPN1-AS1 on HCC. Mechanistically, the rescue experiments demonstrated that miR-7-5p inhibitor reversed the inhibition effect of silencing lncRNA RHPN1-AS1 on HCCLM3 cells proliferation, migration and invasion. Moreover, silencing lncRNA RHPN1-AS1 also inhibited the activation of PI3K/AKT/mTOR pathway. Taken together our findings demonstrated that lncRNA RHPN1-AS1 could facilitate cell proliferation, migration and invasion via targeting miR-7-5p and activating PI3K/AKT/mTOR pathway in HCC.

LncRNA ANCR Suppresses the Progression of Hepatocellular Carcinoma Through the Inhibition of Wnt/ß-Catenin Signaling Pathway.

OBJECTIVE: Our study aimed to investigate the effect of anti-differentiation noncoding RNA (ANCR) on hepatocellular carcinoma (HCC) and its potential molecular mechanisms. METHODS: The expression of ANCR was detected by qRT-RCR in both HCC tissues and HCC cells. Moreover, the relationship between ANCR expression and clinical parameters in HCC patients was investigated. The proliferation, cell clones, migration, invasion and apoptosis of MHCC97H and HCCLM3 cells were measured by MTT assay, colony formation assay, transwell assay and flow cytometry, respectively. The expressions of N-cadherin, vimentin, E-cadherin, cleaved caspase-3, Bax, Bcl-2, Wnt1, ß-catenin and GSK-3ß in MHCC97H and HCCLM3 cells were measured by Western blot. RESULTS: Our results showed that ANCR was lowly expressed in both HCC tissues and HCC cells. ANCR expression was closely associated with tumor size, tumor-node-metastasis (TNM) stages and vascular invasion in HCC. ANCR could dramatically inhibit cell proliferation, migration and invasion, as well as promote apoptosis in MHCC97H and HCCLM3 cells. ANCR could significantly increase the expression of cleaved caspase-3, Bax, E-cadherin and GSK-3ß but reduce the expression of Bcl-2, N-cadherin, vimentin, Wnt1 and ß-catenin in MHCC97H and HCCLM3 cells. In addition, Wnt/ß-catenin pathway inhibitor (IWP-2) partially reversed the effects of silencing ANCR on the proliferation, migration, invasion and apoptosis of HCCLM3 cells. CONCLUSION: Our study demonstrated that ANCR can suppress cell proliferation, migration and invasion, as well as promote apoptosis of HCC cells via modulation of the Wnt/ß-catenin signaling pathway.

[Clinicopathological significance of cytotoxic lymphocytes in breast cancer and draining lymph nodes].

OBJECTIVE: To analyze retrospectively the quantity and activation status of the tumor infiltrating cytotoxic lymphocytes in breast cancer and the draining lymph nodes, and its relation to the clinical pathological significance. METHODS: Seventy-four breast cancer samples with their corresponding axillary lymph nodes were histologically typed and staged. Cytotxic lymphocytes were analyzed by immunohistochemistry with the monoclonal antibodies against CD8, CD56, granzyme B and perforin. RESULTS: The number of infiltrating CD8(+) T cells in the cancerous interstitial tissue were much higher than that in the tumor parenchyma. Compared with the metastatic tumor samples, the CD8(+) T cells were more intensive in the primary tumors (35.7 +/- 16.0 vs. 23.7 +/- 9.6). The tumor infiltrating CD8(+) T cells of patients with 5 years survivals were more than that of the dead cases in this follow-up series death (32.9 +/- 14.1 vs. 20.1 +/- 9.9). There was no significant difference of activated tumor infiltrating cytotoxic T cell analyzed by using the activation marker granzyme B(+) and there was also no significant correlation between the intensity of CD8(+), CD56(+) cells and the clinicopathological stages. However, percentages of the activated cytotoxic lymphocytes in Stage I groups were significantly higher than those in stage III and IV. Moreover, the number of perforin(+) cells was significantly less than that of granzyme B(+) cells, particularly in the cancerous tissue, indicating a dysfunctional status of tumor infiltrating cytotoxic lymphocytes. CONCLUSIONS: Activated cytotoxic lymphocytes may play a significant role against the tumor progression and is associated with a favorable prognosis to some extent. However, a putative dysfunctional status of cytotoxic lymphocytes at tumor site may compromise the host immunity against cancer.

Electroacupuncture relieved visceral and referred hindpaw hypersensitivity in colitis rats by inhibiting tyrosine hydroxylase expression in the sixth lumbar dorsal root ganglia.

Irritable bowel syndrome patients frequently complain of pain in body regions somatotopically distinct from the gut, suggesting the involvement of an exaggerated signaling process in both visceral and somatic sensory pathways. Increasing evidence has shown that sprouting of tyrosine hydroxylase immunoreactive (TH-IR) fibers toward sensory neurons in dorsal root ganglia maintains and exacerbates the neuropathic and inflammatory pain, as well as colonic inflammation. The aim of the present study was to determine whether electroacupuncture could alleviate the visceral and secondary somatic hyperalgesia in colitis rats by suppressing the TH-IR expression in related dorsal root ganglia. After trinitrobenzene sulfonic acid irritation, rats developed inflammatory tissue damage in the distal colon, which was accompanied by visceral hypersensitivity and secondary hind paw hyperalgesia, as indicated by enhanced visceromotor response to colorectal distension and decreased mechanical and thermal withdrawal latency of the hind paw. Additionally, excessive TH-IR fibers sprouted toward calcitonin gene-related peptide immunoreactive sensory neurons, and TH-IR neurons also increased in the sixth lumbar dorsal root ganglia of colitis rats. Both electroacupuncture and guanethidine attenuated visceral and referred hind paw hyperalgesia by inhibiting the over-expression of TH-IR neurons and fibers in the sixth lumbar dorsal root ganglia. Moreover local inflammatory damage in the distal colon was restored after 7 days of electroacupuncture intervention. These results suggest that electroacupuncture relieved visceral and referred hind paw hypersensitivity in colitis rats by inhibiting TH expression in the sixth lumbar dorsal root ganglia.

[From molecular pharmacy to electroceuticals: SPARC program and acupuncture research].

The NIH-funded "Stimulating Peripheral Activity to Relieve Conditions (SPARC)" program has been initiated in Octomber 2016, aiming at developing high resolution neural circuit maps and next generation neural modulation devices. This program has brought great stimulus to acupuncturists and acupuncture researchers both at home and abroad. Is the SPARC program a driving force or a challenge of acupuncture research? In the present study, we introduced the SPARC program and compared it with current acupuncture research. The first step of SPARC is to better map neural circuits associated with disease on the anatomical level so as to identify the best points for intervention, and to decode the neural language at these intervention points for developing a dictionary of patterns associated with health and disease states on the signaling level. Similarly, our acupuncture research also focuses on revealing the neural encoding of acupuncture stimulation and its effect on visceral function, seeking suitable stimulation parameters to regulate the abnormal visceral activity precisely. Therefore, the common point of SPARC program and acupuncture research is the scientific basis of peripheral somatic neuronal regulation, and their difference is that acupuncture regulates the visceral function through multiple neural circuits and neural feedbacks by stimulating the sensitized points or acupoints, achieving homeostasis at last. Acupuncture-induced regulation effect is limited and the therapy is safe. Whereas, "stimulating periphe-ral activity (SPA)" can regulate the visceral organs precisely but without neural feedback. Inevitably, SPA will produce tolerance or side effects. Therefore, there is still much work to be done in terms of the initiation of trigger stimulation and the feedback inhibition of target organ effects. The SPARC program is definitely a powerful force in revealing the mechanisms by which acupuncture works.

Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells.

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.

Heparin chromatography to deplete high-abundance proteins for serum proteomics.

BACKGROUND: Serum is a very informative sample for disease diagnosis. However, a few of the high-abundance proteins existing in serum make the identification of disease-specific serum biomarkers extremely challenging using currently available technologies. A highly promising first step for most analytical approaches of serum is to deplete as many of the high-abundance proteins as possible. METHODS: We introduced the traditional method of heparin chromatography coupled with protein G sepharose to deplete the high-abundance proteins for serum proteomics. RESULTS: Compared with the multiple affinity removal system (MARS) column (a commercial version to deplete 6 major proteins in serum), heparin chromatography can deplete more high-abundance proteins in a single step, especially many high molecular-weight proteins. Using this simple and inexpensive method to pretreat serum for 2-DE analysis, more protein spots can be visualized. IgGs depletion by protein G sepharose can further enhance the resolution of the resulting serum proteome. CONCLUSIONS: Heparin chromatography coupled with protein G appears to be an efficient and economical strategy to pretreat serum for serum proteomics.

Hyperhomocysteinemia induces injury in olfactory bulb neurons by downregulating Hes1 and Hes5 expression.

Hyperhomocysteinemia has been shown to be associated with neurodegenerative diseases; however, lesions or histological changes and mechanisms underlying homocysteine-induced injury in olfactory bulb neurons remain unclear. In this study, hyperhomocysteinemia was induced in apolipoprotein E-deficient mice with 1.7% methionine. Pathological changes in the olfactory bulb were observed through hematoxylin-eosin and Pischingert staining. Cell apoptosis in the olfactory bulb was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Transmission electron microscopy revealed an abnormal ultrastructure of neurons. Furthermore, immunoreactivity and expression of the hairy enhancer of the split 1 (Hes1) and Hes5 were measured using immunohistochemistry, immunofluorescence, and western blot assay. Our results revealed no significant structural abnormality in the olfactory bulb of hyperhomocysteinemic mice. However, the number of TUNEL-positive cells increased in the olfactory bulb, lipofuscin and vacuolization were visible in mitochondria, and the expression of Hes1 and Hes5 decreased. These findings confirm that hyperhomocysteinemia induces injury in olfactory bulb neurons by downregulating Hes1 and Hes5 expression.

Regulating Gut Flora Dysbiosis in Obese Mice by Electroacupuncture.

Recently, gut flora has been linked to the onset of obesity and has been shown to influence the host's metabolism. Acupuncture is a well-known agent used for the treatment of numerous diseases such as obesity. This study aimed to explore the impacts of electroacupuncture treatment on gut microbiota composition and function in obese mice. Pyrosequencing of 16S rRNA genes and Metagenomic analysis of the fecal microbiota were used for this purpose. The basic parameters of body weight, Lee's index, serum lipid and epididymal adipose weight were ameliorated significantly after introducing an electroacupuncture intervention. Acidobacteria, Cyanobacteria and Basidiomycota (Normal group) and Fusobacteria, Firmicutes and Spirochmycetes (Model group) were remarkably affluent at the phylum level. Bacteroides sp. CAG: 927 and Prevotella sp. CAG: 1031 (Normal group), Lachnospiraceae bacterium and Helicobacter rodentium (Model group) at the species level were distinctly enriched. The structures and functions of the intestinal flora were significantly different between healthy and obese mice, and animals in the acupuncture group gradually tended towards healthy controls. Moreover, electroacupuncture altered the bacterial diversity and metabolic genes to establish new balance, observed the obvious change from 7[Formula: see text]d and stabilized gradually through 21[Formula: see text]d. These findings suggested gut flora could be a novel target of electroacupuncture treatment against obesity.

A PEG-based method for the isolation of urinary exosomes and its application in renal fibrosis diagnostics using cargo miR-29c and miR-21 analysis.

PURPOSE: To assess a new and highly specific, but low-cost, easily performed and suitable for large-scale applications method for renal fibrosis (RF) diagnostics. METHODS: Thirty-five RF and twenty non-RF patients were enrolled in the study. An appropriate polyethylene glycol (PEG) was used to isolate urinary exosomes. The efficiency of isolation process was evaluated by the morphology and size observation, as well as the detection of specific markers (CD63, CD9). The expression level of exosomal miR-29c, miR-21 and the endogenous control snRNA-U6 were detected by qRT-PCR. The diagnostic potency of urinary exosomal miR-29c and miR-21 was estimated by the ROC method. Spearman's rank-order correlations analysis was used to assess the correlation between the miRNAs and clinical parameters, including pathological index. RESULTS: PEG-based method for isolation urinary exosome was effective and could be completed with a relatively low-speed centrifugal machine. Exosomal miR-29c and miR-21 were detected in all samples. The analysis of miRNAs in urinary exosomes revealed significant dys-regulation of miR-29c and miR-21 associated with RF. Exosomal miR-29c and miR-21 could predict degree of RF with AUC of 0.8333 and 0.7639 (P < 0.05). Correlation analysis showed that the level of miR-29c had a significant negative relationship with eGFR and the interstitial relative area. CONCLUSIONS: The PEG-based method for isolation urinary exosome is an inexpensive and easily performed approach. The application for cargo miRNA analysis is feasible. Urinary exosomal miR-29c may present a promising diagnostic approach.

Neuropeptide Initiated Mast Cell Activation by Transcutaneous Electrical Acupoint Stimulation of Acupoint LI4 in Rats.

Transcutaneous electrical acupoint stimulation (TEAS) has been consistently used clinically for its ease of operation, non-invasiveness and painlessness, in contrast to the characteristics of inserted needles. However, the mechanism remains unknown. The aim of this study was to investigate the local response of TEAS at Hegu acupoint (LI4). Immunohistochemistry was used to measure the expression of tryptase-positive mast cells, neuropeptides of the calcitonin gene-related peptide (CGRP) and substance P (SP) in LI4. Mast cells were also labelled with serotonin (5-HT), neurokinin-1 receptor (NK-1R) and toluidine blue. The results showed that cutaneous CGRP and SP immune-positive (CGRP-IP or SP-IP) nerve fibres in LI4 were more highly expressed. There were high degrees of mast cell aggregation and degranulation with release of 5-HT near the CGRP-IP or SP-IP nerve fibres and blood vessels after TEAS. The degranulation of mast cells (MCs) was accompanied by expression of NK-1R after TEAS. Either mast cell membrane stabilizer (Disodium cromoglycate) or NK-1R antagonist (RP 67580) diminished the accumulation and degranulation of MCs induced by TEAS. Taken together, the findings demonstrated that TEAS induced sensory nerve fibres to express CGRP and SP, which then bound to the NK-1R on MCs, after which MCs degranulated and released 5-HT, resulting in TEAS-initiated acupuncture-like signals.