Extraction and characterization of polysaccharides from Schisandra sphenanthera fruit by Lactobacillus plantarum CICC 23121-assisted fermentation.

Improving the yield of polysaccharides extracted from Schisandra sphenanthera is a major challenge in traditional Chinese medicinal plants. In this study, we investigated the potential of Lactobacillus plantarum CICC 23121-assisted fermentation as an extraction tool for S. sphenanthera polysaccharides (SSP). We observed that 11.12 ± 0.28 % of polysaccharides were extracted from S. sphenanthera using strain CICC 23121 -assisted fermentation (F-SSP), which was 53.38 % higher than that using hot water extraction (NF-SSP). The optimized parameters were a fermentation time of 15.5 h, substrate concentration of 4 %, and inoculum size of 3 %. Lactic acid produced by strain CICC 23121 increased the release of intracellular polysaccharides by breaking down cell walls. Compared to NF-SSP, F-SSP contained higher and lower total carbohydrate and protein contents, respectively, and its monosaccharide composition was the same as that of NF-SSP; however, their distributions were different. F-SSP had a higher molecular weight, better aqueous stability, and looser surface morphology, and strain CICC 23121-assisted fermentation did not change the molecular structure of SSP. Both NF-SSP and F-SSP showed the potential to regulate human intestinal microflora. Our findings revealed that strain CICC 23121-assisted fermentation is an efficient method for extracting S. sphenanthera polysaccharides without affecting their physicochemical and bioactive properties.

Kaempferol attenuates carbon tetrachloride (CCl4)-induced hepatic fibrosis by promoting ASIC1a degradation and suppression of the ASIC1a-mediated ERS.

BACKGROUND: Kaempferol is a flavonoid derived from the herb, Kaempferia galanga L., in addition to exhibiting a wide range of pharmacological properties, kaempferol is also an anti-inflammatory, anti-lipid metabolizing, and anti-oxidative stress agent. The underlying molecular mechanisms of its effects on vascular endothelial growth factor (VEGF) secretion and activation of hepatic stellate cells (HSCs) are yet unknown. Activated HSCs induces VEGF release and extracellular matrix (ECM) accumulation which are important factors in hepatic fibrosis. PURPOSE: Our aim is to explore how kaempferol may affect hepatic fibrosis and the mechanisms behind its effects. METHODS: The in vivo model was Sprague-Dawley rats induced with carbon tetrachloride (CCl4). Histological staining was used to observe histological features of the liver. The levels of (alanine aminotransferase) ALT and (aspartate aminotransferase) AST were detected by the corresponding kits. Platelet-derived growth factor (PDGF) was used to stimulate the HSC-T6 rat hepatic stellate cells. The mechanisms underlying this process were investigated using a variety of molecular approaches, including immunofluorescence, RT-qPCR, and western blotting. Moreover, intracellular Ca2+ were observed by laser confocal microscope. RESULTS: It was found that kaempferol significantly reduced the expression of ASIC1a, VEGF, α-SMA and Collagen-I proteins in a model of CCl4-induced hepatic fibrosis in rats. In HSC-T6, kaempferol inhibits activation of HSCs by decreasing expression of ASIC1a, eIF2α, p-eIF2α and ATF-4. Laser confocal fluorescence showed that kaempferol inhibited Ca2+ influx and reduced Ca2+ concentration around the endoplasmic reticulum. Molecular docking and cellular thermal shift assay (CETSA) results further indicated that kaempferol interacted with ASIC1a. We found that kaempferol may promote the degradation of ASIC1a and inhibited ASIC1a- mediated upregulation of ERS. CONCLUSION: The data from our in vivo experiments demonstrate that kaempferol effectively attenuates hepatic fibrosis. In vitro studies we further propose a novel mechanism of kaempferol against hepatic fibrosis which can interact with ASIC1a and promote ASIC1a degradation while inhibiting the activation and VEGF release of HSCs by suppressing the ASIC1a-eIF2α-ATF-4 signaling pathway.

Ergolide covalently binds NLRP3 and inhibits NLRP3 inflammasome-mediated pyroptosis.

BACKGROUND: NLR family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis plays a key role in various acute and chronic inflammatory diseases. Targeted inhibition of NLRP3-mediated pyroptosis may be a potential therapeutic strategy for various inflammatory diseases. Ergolide (ERG) is a sesquiterpene lactone natural product derived from the traditional Chinese medicinal herb, Inula britannica. ERG has been shown to have anti-inflammatory and anti-cancer activities, but the target is remains unknown. HYPOTHESIS/PURPOSE: This study performed an in-depth investigation of the anti-inflammatory mechanism of ERG in NLRP3-mediated pyroptosis and NLPR3 inflammasome related sepsis and acute lung injury model. METHODS: ELISA and Western blot were used to determine the IL-1ß and P20 levels. Co-immunoprecipitation assays were used to detect the interaction between proteins. Drug affinity response target stability (DARTS) assays were used to explore the potential target of ERG. C57BL/6J mice were intraperitoneally injected with E. coli DH5α (2 × 109 CFU/mouse) to establish a sepsis model. Acute lung injury was induced by intratracheal administrationof lipopolysaccharide in wild-type mice and NLRP3 knockout mice with or without ERG treatment. RESULTS: We showed that ERG is an efficient inhibitor of NLRP3-mediated pyroptosis in the first and second signals of NLRP3 inflammasome activation. Furthermore, we demonstrated that ERG irreversibly bound to the NACHT domain of NLRP3 to prevent the assembly and activation of the NLRP3 inflammasome. ERG remarkably improved the survival rate of wild-type septic mice. In lipopolysaccharide-induced acute lung injury model, ERG alleviated acute lung injury of wild-type mice but not NLRP3 knockout mice. CONCLUSION: Our results revealed that the anti-pyroptosis effect of ERG are dependent on NLRP3 and NLRP3 NACHT domain is ERG's direct target. Therefore, ERG can serve as a precursor drug for the development of novel NLRP3 inhibitors to treat NLRP3 inflammasome mediated inflammatory diseases.

Ginseng-Containing Sijunzi Decoction Ameliorates Ulcerative Colitis by Orchestrating Gut Homeostasis in Microbial Modulation and Intestinal Barrier Integrity.

Ulcerative colitis (UC) has become a global epidemic, and the lack of an effective cure highlights the necessity and urgency to explore novel therapies. Sijunzi Decoction (SJZD), a classical Chinese herbal formula, has been comprehensively applied and clinically proven effective in treating UC; however, the pharmacological mechanism behind its therapeutic benefits is largely obscure. Here, we find that SJZD can restore microbiota homeostasis and intestinal barrier integrity in DSS-induced colitis. SJZD significantly alleviated the colonic tissue damage and improved the goblet cell count, MUC2 secretion, and tight junction protein expressions, which indicated enhanced intestinal barrier integrity. SJZD remarkedly suppressed the abundance of phylum Proteobacteria and genus Escherichia-Shigella, which are typical features of microbial dysbiosis. Escherichia-Shigella was negatively correlated with body weight and colon length, and positively correlated with disease activity index and IL-1[Formula: see text]. Furthermore, through gut microbiota depletion, we confirmed that SJZD exerted anti-inflammatory activities in a gut microbiota-dependent manner, and fecal microbiota transplantation (FMT) validated the mediating role of gut microbiota in the SJZD treatment of UC. Through gut microbiota, SJZD modulates the biosynthesis of bile acids (BAs), especially tauroursodeoxycholic acid (TUDCA), which has been identified as the signature BA during SJZD treatment. Cumulatively, our findings disclose that SJZD attenuates UC via orchestrating gut homeostasis in microbial modulation and intestinal barrier integrity, thus offering a promising alternative approach to the clinical management of UC.

Electroacupuncture prevents LPS- induced neuroinflammation via upregulation of PICK-TLR4 complexes in the microglia of hippocampus.

Sepsis-associated encephalopathy (SAE) is a common complication of sepsis caused by neuroinflammation. Electroacupuncture (EA) can be used to treat SAE, but the underlying mechanism is not clear. Lack of PICK1 further aggravates the inflammatory response in mice with sepsis. Therefore, we sought to investigate whether PICK1 is involved in the protective effects of electroacupuncture to SAE. In this study, mice were treated with EA after lipopolysaccharide (LPS) treatment. Behavioral tests; microglial activity of hippocampus; neuron survival and the inflammatory factors PICK1 and TLR4, as well as TLR4-related proteins, such as ERK, JNK, and P38, were assessed after EA treatment. PICK1, TLR4, and TLR4-related proteins, as well as PICK1-TLR4 complex levels were assessed in BV2 cells treated with LPS, PICK1 siRNA, or PICK1 polypeptide. The results indicated that EA could improve neurological assessment and reduce activation of microglial and TLR4 and expression of proinflammatory cytokines. EA also reduced the expression of TLR4 and phosphorylation of ERK/JNK/P38 while, increased the expression of PICK1 and TLR4 complexes. PICK1 knockdown further promoted the expression of TLR4 and phosphorylation of ERK/JNK/P38 in BV2 cells, but this effect was reversed by PICK1 polypeptides. These results suggest that EA may reduce neuroinflammation responses, decrease inflammatory factors, and finally, protect SAE by increasing the formation of PICK1-TLR4 complexes in microglia.

[Effect of Yes-associated protein in regulation of electroacupuncture pretreatment on cerebral ischemia reperfusion injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) pretreatment on inflammatory reaction, apoptosis and expression of Yes-associated protein (YAP) of ischemic penumbra of cerebral cortex in cerebral ischemia reperfusion injury rats, and to explore the possible mechanism of its neuroprotection effect. METHODS: A total of 84 SD rats were randomized into a sham operation group (12 rats), a model group (18 rats), an EA group (18 rats), an EA+YAP virus transfection group (18 rats) and an EA+virus control group (18 rats). Except for the sham operation group, thread embolization method was adopted to establish the middle cerebral artery occlusion (MCAO) model in rats of the other groups. EA was applied at "Baihui" (GV 20) and "Dazhui" (GV 14) for 30 min in the 3 EA intervention groups 2 h before model establishment, disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in intensity. Adenovirus transfection technique was used to induce gene silencing of YAP in the EA+YAP virus transfection group, and adenovirus vectors was injected as negative control in the EA+virus control group 4 d before model establishment. Twenty-four hours after model establishment, neurological function score was evaluated, the relative cerebral infarction area was observed by TTC staining, the apoptosis in the ischemic penumbra of cerebral cortex was detected by TUNEL staining, the levels of inflammatory factors IL-1ß, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex was detected by ELISA method, the expression of YAP was detected by Western blot and immunofluorescence. RESULTS: Compared with the sham operation group, the expression of YAP was increased in the model group (P<0.05); compared with the model group, the expression of YAP in the ischemic penumbra of cerebral cortex was increased in the EA group (P<0.05). Compared with the sham operation group, the neurological function score, the percentage of TUNEL positive cells and the levels of IL-1ß, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were increased in the model group (P<0.001, P<0.01); compared with the model group, the neurological function score, the relative cerebral infarction area, the percentage of TUNEL positive cells and the levels of IL-1ß, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were decreased in the EA group (P<0.05, P<0.01); compared with the EA group, the neurological function score, the relative cerebral infarction area, the percentage of TUNEL positive cells and the levels of IL-1ß, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were increased in the EA+YAP virus transfection group (P<0.01, P<0.05); compared with the EA+YAP virus transfection group, the neurological function score, the relative cerebral infarction area, the percentage of TUNEL positive cells and the levels of IL-1ß, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were decreased in the EA+virus control group (P<0.01, P<0.05). CONCLUSION: Electroacupuncture pretreatment can effectively improve the ischemia reperfusion injury, its mechanism may be related to up-regulating the expression of YAP in the ischemic penumbra of cerebral cortex and relieving the apoptosis and inflammatory reaction.

Effect of traditional Chinese medicine (TCM) on survival, quality of life, and immune function in patients with ovarian carcinoma: A protocol for systematic review and meta analysis.

BACKGROUND: Traditional Chinese medicine (TCM) has been widely applied as promising adjunctive drugs for ovarian carcinoma (OC) in China and other Asian countries. However, its exact clinical efficacy and safety is still not well investigated. In this study, we aimed to summarize the efficacy of TCM on survival, quality of life (QoL), and immune function in patients with OC through the meta-analysis. METHODS: Relevant clinical trials of TCM for the treatment OC patients will be searched in Cochrane Library, Web of Science, Google Scholar, PubMed, Medline, Embase, China Scientific Journal Database, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, and Wanfang Database from their inception to November 2020. Two researchers will perform data extraction and risk of bias assessment independently. The clinical outcomes, including overall survival (OS), QoL, immune function, tumor markers, and adverse events, were systematically evaluated by using Review Manager 5.3 and Stata 14.0 statistical software. RESULTS: The results of this study will provide high-quality evidence for the effect of TCM on survival, QoL and immune function in patients with OC. CONCLUSION: The conclusions of this meta-analysis will be published in a peer-reviewed journal, and draw an objective conclusion of the efficacy of TCM on survival, QoL, and immune function in patients with OC. TRIAL REGISTRATION NUMBER: INPLASY2020110104.

Electroacupuncture pretreatment prevents ischemic stroke and inhibits Wnt signaling-mediated autophagy through the regulation of GSK-3ß phosphorylation.

Electroacupuncture (EA), a traditional Chinese replacement therapy, is widely accepted to treat ischemic stroke. Increasing evidence show that autophagy is involved in the process of cerebral ischemia injury and the Wnt/GSK3ß pathway, playing an important role in protecting central nervous system. In this study, rats were treated with EA prior to focal ischemia by middle cerebral artery occlusion (MCAO). Deficit score, infarct volumes and levels of autophagy markers, such as LC3I, LC3II and p62, were assessed with either PI3K inhibitor wortmannin or a GSK-3ß inhibitor LiCl. Oxygen-glucose deprivation/re-oxygenation (OGD/R) was made in the primitive neuron in vitro, and was respectively treated with autophagy inhibitors 3-MA, LiCl, GSK3ß siRNA, or mTOR inhibitor rapamycin. The results indicated that EA pretreatment increased the levels of autophagy marker LC3-II and reduced the levels of p62. Meanwhile, deficit outcome was improved, and infarct volumes were reduced by EA pretreatment. Furthermore, the beneficial effects of EA pretreatment were reversed by wortmannin. LiCl and GSK3ß siRNA can mimic the neuroprotective effects of EA pretreatment by downregulating autophagy, and increasing protein levels of p-mTOR, p-GSK3ß and ß-catenin in OGD/R neurons. However, the protective effects of GSK3ß siRNA were blocked by rapamycin. These results suggest that EA pretreatment induces tolerance to cerebral ischemia by inhibiting autophagy via the Wnt pathway through the inhibition of GSK3ß.

Role of spinal adenosine A1 receptors in the analgesic effect of electroacupuncture in a rat model of neuropathic pain.

OBJECTIVE: The aim of this study was to determine the role of spinal adenosine A1 receptors (A1Rs) in the analgesic effects of electroacupuncture (EA) for neuropathic pain. METHODS: We performed EA for 30 minutes at the zusanli acupoint in the legs of rats with previously induced chronic constriction injuries and observed the mechanical and thermal pain thresholds 1 hour later. We also examined adenosine levels by high-performance liquid chromatography and A1R expression in the L4-6 spinal cord by western blot analysis. We then injected A1R short interfering RNA (AV-shA1RNA) into the L4-6 spinal cord to downregulate A1R expression and re-examined the mechanical and thermal pain thresholds. RESULTS: Adenosine levels and A1R expression in the L4-6 spinal cord were increased at 1 hour after EA. In addition, EA exhibited an analgesic effect that was reversed by AV-shA1RNA. CONCLUSIONS: Our results suggest that EA at the zusanli acupoint elicits an analgesic effect against neuropathic pain, mediated by A1Rs in the spinal cord.

Effect of Ultrasonic Treatment on Freeze-thaw Stability of Soy Protein Isolate Gel.

This study investigated the impact of ultrasonic modification at various ultrasonic power extents on the freeze-thaw stability of soy protein isolate (SPI) gel. The freeze-thaw stability of the gel was evaluated by examining the changes in texture, water holding capacity, microstructure and soluble protein content during the process of 5 freeze-thaw cycles. In addition, effects on particle size, surface hydrophobicity and structure were also explored. The results showed that within a certain range, the average particle size of the protein gradually decreased, and the particle size distribution was narrower with ultrasonic intensity, it may be due to the high shear and cavitation effects of sonication that reduce the degree of protein aggregation. Furthermore, we also detected that treated proteins had lower fluorescence intensity, higher surface hydrophobicity and more flexible molecular structure with the reduction of α-helical structure as well as the rise of random coil. In terms of gel freeze-thaw stability, moderate ultrasound treatment made the water holding capacity and soluble protein content of SPI gel reduce by 38.27% and 3.58%, whereas the hardness and elasticity increased by 510.23g and 0.06mm after 5 FTC. The corresponding changes of indexes of the control group were 75.05%, 51%, 1062.75g and 0.11mm, respectively. It can be observed that the change range of treated SPI gel properties was smaller than that of natural SPI gel, indicating that ultrasonic treatment can remarkably improve the freeze-thaw stability of the gel which might have something to do with changes of protein structure.

Islet-cell autoantigen 69 mediates the antihyperalgesic effects of electroacupuncture on inflammatory pain by regulating spinal glutamate receptor subunit 2 phosphorylation through protein interacting with C-kinase 1 in mice.

Electroacupuncture (EA) is widely used in clinical settings to reduce inflammatory pain. Islet-cell autoantigen 69 (ICA69) has been reported to regulate long-lasting hyperalgesia in mice. ICA69 knockout led to reduced protein interacting with C-kinase 1 (PICK1) expression and increased glutamate receptor subunit 2 (GluR2) phosphorylation at Ser880 in spinal dorsal horn. In this study, we evaluated the role of ICA69 in the antihyperalgesic effects of EA and the underlying mechanism through regulation of GluR2 and PICK1 in spinal dorsal horn. Hyperalgesia was induced in mice with subcutaneous plantar injection of complete Freund adjuvant (CFA) to cause inflammatory pain. Electroacupuncture was then applied for 30 minutes every other day after CFA injection. When compared with CFA group, paw withdrawal frequency of CFA+EA group was significantly decreased. Remarkable increases in Ica1 mRNA expression and ICA69 protein levels on the ipsilateral side were detected in the CFA+EA group. ICA69 expression reached the peak value around day 3. More importantly, ICA69 deletion impaired the antihyperalgesic effects of EA on GluR2-p, but PICK1 deletion could not. Injecting ICA69 peptide into the intrathecal space of ICA69-knockout mice mimicked the effects of EA analgesic and inhibited GluR2-p. Electroacupuncture had no effects on the total protein of PICK1 and GluR2. And, EA could increase the formation of ICA69-PICK1 complexes and decrease the amount of PICK1-GluR2 complexes. Our findings indicate that ICA69 mediates the antihyperalgesic effects of EA on CFA-induced inflammatory pain by regulating spinal GluR2 through PICK1 in mice.

[Effect of transcutaneous electrical acupoint stimulation on gastric emptying in patients undergoing surgery].

OBJECTIVE: To observe the effects of transcutaneous electrical acupoint stimulation (TEAS) on gastric emptying in patients undergoing selective surgery based on velocity of gastric emptying by ultrasonography. METHODS: A total of 75 patients with selective operation of subarachnoid block at lower limb in the afternoon were randomly assigned to a TEAS group, a sham group and a control group, 25 patients in each one. All the patients were provided with semi-fluid diet at 8 a.m. The TEAS group was treated with TEAS 5 min after semi-fluid diets at bilateral Zusanli (ST 36) and Neiguan (PC 6) for 30 min, with frequency of 5 Hz and intensity which was 1 mA lower than the tolerance threshold. The sham group patients were stimulated at the same acupoints with current intensity which was 1 mA lower than the sensory threshold. The control group received no treatment. On the day of operation, and ultrasonography was given at time of empty stomach (T0), immediately after the semi-fluid diets (T1), and every 30 min after diets (T2-T6), respectively, to measure the gastric content and emptying time at semire-clining position and right lateral position. RESULTS: The volume of gastric content in the three groups at T3-T6 was significantly less than that at T1 (all P<0.05). The volume of gastric content at T4-T6 at semire-clining position in the TEAS group was significantly less than that in the control group and sham group (all P<0.05). The volume of gastric content at T5-T6 at right lateral position in the TEAS group was significantly less than that in the control group and sham group (all P<0.05). The gastric emptying time in the TEAS group was significantly less than that in the control group and sham group (both P<0.05). CONCLUSION: The gastric emptying velocity could be evaluated by ultrasonography. TEAS could improve the velocity of gastric emptying and reduce the gastric emptying time.