Effect of Modified Huangqi Guizhi Wuwutang on Stress-related IRE1α/CHOP Pathway of Sciatic Nerve Endoplasmic Reticulum in Diabetes Rats / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of modified Huangqi Guizhi Wuwutang （MHGW） on endoplasmic reticulum stress in the sciatic nerve of diabetes rats based on the pathways of inositol-requiring enzyme 1α （IRE1α） and CCAAT/enhancer-binding protein homologous protein （CHOP）. MethodSixty rats were fed on a high-sugar and high-fat diet for six weeks， followed by intraperitoneal injection of streptozotocin at a dose of 35 mg·kg-1. Random blood glucose levels were measured three days later and rats with a sustained blood glucose level ≥ 16.7 mmol·L-1 were included in study （n=48）. The rats were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg-1·d-1）， a high-dose MHGW group （2.5 g·kg-1·d-1）， and a low-dose MHGW group （1.25 g·kg-1·d-1）. Another 10 rats were assigned to the normal group. The intervention lasted for 16 weeks. After 16 weeks， the sciatic nerve structure of the rats in each group was observed under light microscopy using Luxol fast blue （LFB） staining. Transmission electron microscopy was used to observe the ultrastructure of the sciatic nerve. Chemiluminescence method was employed to measure the serum reactive oxygen species （ROS） levels. Western blot and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to evaluate the expression of p-IRE1α protein， IRE1α mRNA， CHOP protein， and CHOP mRNA in the sciatic nerve of the rats. ResultCompared with the normal group， the model group showed elevated serum ROS levels （P<0.01）. In contrast， the serum ROS levels were significantly reduced in the treatment groups compared with those in the model group （P<0.01）. The sciatic nerve of the model group showed pathological changes compared with that in the normal group， while the treatment groups exhibited improvement in sciatic nerve pathology compared with the model group. The protein expression of p-IRE1α and CHOP in the sciatic nerve significantly increased in the model group as compared with that in the normal group （P<0.01）. However， the treatment groups showed a significant decrease in the protein expression of p-IRE1α and CHOP in the sciatic nerve compared with the model group （P<0.05， P<0.01）. Furthermore， compared with the normal group， the model group showed upregulated mRNA expression of IRE1α and CHOP in the sciatic nerve （P<0.01）， while the treatment groups exhibited a significant decrease in the mRNA expression of IRE1α and CHOP compared with the model group （P<0.01）. ConclusionMHGW can alleviate endoplasmic reticulum stress-induced cell apoptosis and improve the structure and function of the sciatic nerve in diabetes rats by inhibiting the expression of IRE1α/CHOP pathway-related proteins and mRNA， thereby preventing and treating peripheral neuropathy in diabetes.

Role of IP3 receptor in necroptosis of hippocampal neurons induced by sevoflurane anesthesia in aged rats / 中华麻醉学杂志

Objective:To evaluate the role of 1, 4, 5-inositol triphosphate receptor (IP3R) in necroptosis of hippocampal neurons induced by sevoflurane anesthesia in aged rats.Methods:Sixty healthy male Sprague-Dawley rats, aged 18 months, weighing 500-600 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), sevoflurane anesthesia group (group S) and sevoflurane anesthesia + IP3R antagonist group (group S+ I). S and S+ I groups inhaled 2% sevoflurane for 5 h. In group S+ I, IP3 receptor antagonist 2-APB 3 mg/kg was intraperitoneally injected at 10 min before sevoflurane inhalation, and the equal volume of dimethyl sulfoxide was intraperitoneally injected in group C and group S. Morris water maze test was used to test the cognitive function on the day after the end of sevoflurane anesthesia.Then the animals were sacrificed and the brain tissues were obtained for microscopic examination of the pathological changes after HE staining and Nissl staining (with a light microscope) and for determination of the free calcium concentration ([Ca 2+ ] i) and rate of necroptosis of hippocampal neurons (by flow cytometry) and expression of IP3R, receptor-interacting protein kinase-1 (RIPK1), receptor-interacting protein kinase-3 (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) (by Western blot). Results:Compared with group C, the escape latency was significantly prolonged, the times of crossing the platform were reduced, the time of staying at the target quadrant was shortened, the [Ca 2+ ] i and necroptosis rate of hippocampal neurons were increased, and the expression of IP3R, RIPK1, RIPK3 and p-MLKL in hippocampal neurons was up-regulated in group S and group S+ I ( P<0.05). Compared with group S, the escape latency was significantly shortened, the times of crossing the platform were increased, the time of staying at the target quadrant was prolonged, the [Ca 2+ ] i and necroptosis rate of hippocampal neurons were decreased, and the expression of IP3R, RIPK1, RIPK3 and p-MLKL in hippocampal neurons was down-regulated in group S+ I ( P<0.05). Conclusions:The mechanism by which sevoflurane induces cognitive dysfunction may be related to the imbalance of calcium homeostasis caused by activation of IP3R and thus inducing programmed necrosis in aged rats.

Engineering Saccharomyces cerevisiae for efficient production of glucaric acid / 生物工程学报

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.

Efficacy of myo-inositol in improving pregnancy rate and regulation of menstrual cycle for patients with polycystic ovarian syndrome: A systematic review and meta-analysis

Background@#Polycystic ovarian syndrome (PCOS) is a common, reproductive endocrinopathy associated with ovarian dysfunction, cardiovascular disorders, obesity, and infertility. Myo-inositol is a novel treatment for women with PCOS that claimed to have improved fertility rate in this population. This systematic review and meta-analysis examined the effect of myo-inositol on pregnancy rate, menstrual cycle, and adverse effects from randomized controlled trials (RCTs). @*Methods@#RCTs that evaluated the efficacy of myo-inositol in improving pregnancy rate and regulation of menstrual cycle in women with PCOS. Electronic databases were searched and studies published up to October 24, 2021 were included in the systematic review and meta-analysis. Study selection and assessment of quality were conducted independently by two review authors. @*Results@#Seven studies with 729 patients treated with myo-inositol and 677 patients treated with placebo and/or metformin were included in the analysis. The research groups did not diverge significantly in terms of basic characteristics, such as age, adnexal or uterine pathology, body mass index, and duration of infertility. In the myo-inositol group, regulation of the normal menstrual cycle is at 20%, significantly higher than the metformin group at 12%, (p<0.001). However, there is no significant difference in the pregnancy rate between myoinositol and placebo (p=0.42) and/or metformin (p=0.17). @*Conclusion@#This systematic review and meta-analysis showed that myo-inositol can be an alternative treatment for PCOS in terms of regulation of menses and may improve the success of spontaneous pregnancies. However, additional randomized, double-blind controlled trials with larger sample sizes, low heterogeneity, and uniform inclusion criteria are recommended to establish the effects of myo-inositol on PCOS treatment and pregnancy rate.

Effects of different doses of fluoride on expression of IRE1α-ASK1-JNK proteins in rat renal cortex / 中华地方病学杂志

Objective:To investigate the expression and significance of endoplasmic reticulum stress apoptosis pathway related proteins in renal cortex of rats with chronic fluorosis.Methods:Twenty four healthy SD rats were divided into 4 groups (6 rats/group, half male and half female) according to their body mass (100 - 120 g) by random number table method, rats in control group drank tap water (fluoride content < 0.5 mg/L), and in low, medium and high fluoride groups drank tap water with fluoride content (sodium fluoride) of 10, 50 and 100 mg/L, respectively. After 180 days of feeding, dental fluorosis was examined, 24-hour urine sample was collected and the content of fluoride in urine was detected by fluoride ion selective electrode method. Renal tissue was taken after anesthesia, and the pathological changes of renal cortex were observed by hematoxylin-eosin (HE) staining. The expressions of endoplasmic reticulum stress apoptosis pathway related proteins [inositol-requiring enzyme 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), C-Jun N-terminal kinase (JNK) and phosphorylated JNK (P-JNK)] were determined by immunohistochemical staining in rat renal cortex.Results:No dental fluorosis was found in control group. The incidence of dental fluorosis in low, medium and high fluoride groups were 2/6, 5/6 and 6/6, respectively. Compared with control group [(5.707 ± 1.190) mg/L], the urinary fluoride in low, medium and high fluoride groups [(17.028 ± 3.006), (34.378 ± 12.045), (94.759 ± 31.773) mg/L] was significantly higher ( P < 0.05), and the urinary fluoride in high fluoride group was higher than that in low and medium fluoride groups ( P < 0.05). HE staining showed that, compared with control group, the cell volume of renal tubules and glomeruli in medium and high fluoride groups increased, the cells arranged closely, and the eosinophilia of the cytoplasm increased. The immunohistochemical staining results showed that there was no significant difference in the expression of JNK protein in rat renal cortex between control group and low, medium and high fluoride groups ( F = 0.07, P > 0.05). The expressions of IRE1α, ASK1 and P-JNK proteins in rat renal cortex in high fluoride group were higher than those of control, low and medium fluoride groups ( P < 0.05), and the expressions of IRE1α and ASK1 proteins in medium fluoride group were significantly higher than those in control and low fluoride groups ( P < 0.05). Conclusion:Long-term excessive fluoride intake can lead to renal cortex injury in rats, and the mechanism of injury may be related to the activation of IRE1α-ASK1-JNK endoplasmic reticulum stress apoptosis pathway.

Effect of Fuzitang on Proliferation of Human Rheumatoid Arthritis Synovial Fibroblast Cell Line MH7A and Expression of miR-155 / 中国实验方剂学杂志

ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.

Effect of Huangqi Guizhi Wuwutang on Diabetic Peripheral Neuropathy in MKR Mice via Regulating Endoplasmic Reticulum Stress / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Huangqi Guizhi Wuwutang （HQGZWWT） in the treatment of diabetic peripheral neuropathy （DPN） in MKR mice via regulating endoplasmic reticulum （ER） stress. MethodThirty-two 8-week-old MKR mice （half were male and half were female） were fed with a high-fat diet for four weeks， and then 1% streptozotocin （STZ） was injected intraperitoneally for five days. After the blood glucose was stabilized， the mice were housed in the cage covered with ice bags for another one hour stimulation per day for four weeks. Mice with fasting blood glucose （FBG） value ≥11.1 mmol·L-1 were randomly divided into model group ， Huangqi Guizhi Wuwutang in original dosage group （30 g·kg-1·d-1）， Huangqi Guizhi Wuwutang in formula dosage group （6.25 g·kg-1·d-1）， and positive drug group （mecobalamin tablets， 0.17 mg·kg-1·d-1）. Another eight MKR mice of the same age were set as blank group and eight FVB mice were normal group. After four weeks of intragastric administration in each group， the change in FBG was tested， and hematoxylin and eosin （HE） staining and transmission electron microscope were used for observing the morphology of sciatic nerve tissue. In addition， the expression of c-Jun N-terminal kinase （JNK）， phosphorylated c-Jun N-terminal kinase （p-JNK） and inositol requiring enzyme 1α （IRE1α） proteins was determined by immunohistochemical test and Western blot （WB）. ResultCompared with the conditions in the normal group and blank group， the time of paw withdrawal， paw licking and tail flick in the model group was shortened （P<0.01）， and the conduction velocity of sciatic nerve was decreased （P<0.01）. Compared with the conditions in the model group， the behavioral and functional indicators were improved by HQGZWWT （P<0.05，P<0.01）. The immunohistochemical test revealed the JNK expression was elevated in the model group compared with the conditions in the normal group and blank group （P<0.05）， while that was lowered by HQGZWWT compared with the condition in the model group （P<0.05）. However， there was no difference among the treatment groups. According to the WB， the expression of IRE1α and p-JNK in the model group was enhanced compared with the conditions in the normal group and blank group （P<0.05，P<0.01）， while that was decreased by HQGZWWT compared with the condition in the model group （P<0.05，P<0.01）. No difference was observed between the HQGZWWTO and HQGZWWTF groups. ConclusionHQGZWWT can improve the neurophysiological function and pathological damage of sciatic nerve， which may be related to its delaying the ER stress response of sciatic nerve.

Significance of phosphoinositol metabolism by DNA methylation may contribute in oral leukoplakia carcinogenesis / 口腔疾病防治

Objective @# To study the role of DNA methylation in oral leukoplakia carcinogenesis.@*Methods@# DNA methylation was detected in forty cases of oral squamous cell carcinoma (OSCC), twenty-eight cases of oral leukoplakia (OLK) and forty cases of healthy oral mucosa. Download the expression profile data of OSCC, OLK and healthy oral mucosa from Gene Expression Omnibus (GEO) database. DNA methylation data and expression profile data were compared for repeatability, DNA methylation data for difference analysis and corresponding expression profile data for IPA pathway analysis.@*Results @#The data analysis showed that DNA methylation had greater flexibility and instability. Ingenuity Pathway Analysis (IPA) analysis showed that genes related to OLK differential methylation sites were mainly concentrated in the process of cell movement and differentiation. Genes related to differential methylation sites of OSCC are mainly enriched in cell proliferation, migration, oxidation regulation, and anti-apoptosis processes. The genes associated with OLK and OSCC differential methylation sites are co-enriched in phosphoinositol metabolism and phospholipase C signaling pathway.@* Conclusion@#DNA methylation is involved in the formation of oral squamous cell carcinoma, and the activation of phosphoinositol metabolism may promote oral leukinoma.

Efficacy of antihyperglycemic drugs and inositol on polycystic ovary syndrome: A network meta-analysis / 中华内分泌代谢杂志

Objective:The study was designed to evaluate the efficacy in polycystic ovary syndrome (PCOS) of glucagon-like peptide-1 receptor agonists (GLP-1RA), metformin combined with thiazolidinediones, α-glucosidase inhibitor, and inositol.Methods:Eligible studies were searched in databases of PubMed, EMBase, Cochrane Library, Wanfang data, and CNKI based on population, interventions, comparisons, outcomes, and study design (PICOS) principle (inception to Nov 2020). Two researchers independently screened randomized controlled trials in strict accordance with the inclusion and exclusion criteria, extracted basic information and outcomes of included studies, and used Cochrane risk of bias tool to evaluate the methodological quality of the literature. Network meta-analysis was conducted by STATA 14.0. Continuous variables without dimensional differences were calculated by weighted mean difference and 95% CI, and continuous variables with dimensional differences were calculated using standardized mean difference and 95% CI. Results:A total of 27 studies with 1 445 patients were included in this study. Network meta-analysis showed that acarbose presented a better efficacy than other interventions in reducing total testosterone [surface under the cumulative ranking curve (SUCRA): 89.4%]. GLP-1RAs may have the best efficacy in reducing body mass index and homeostasis model assessment for insulin resistance (HOMA-IR; SUCRA: 99.1%, 89.2%, respectively), while using inositol may be a good choice to reduce serum fasting insulin, HOMA-IR, blood total cholesterol, and blood triglycerides (SUCRA: 94.5%, 85.4%, 96.6%, and 82.8%, respectively).Conclusions:Acarbose may have advantages over other antihyperglycemic drugs in lowering blood testosterone. GLP-1RAs are more helpful to improve body mass index and HOMA-IR in PCOS patients. Inositol, as an insulin sensitizer, has a favorable effect on reducing fasting insulin, HOMA-IR, blood total cholesterol, and blood triglycerides, and there are no reports of side effects in current researches. Further study is still needed to confirm its efficacy.

Expression of 1L-myo-Inositol -1-Phosphate Synthase (EC 5.5.1.4) is Deregulated in the Cerebellum of Curly Tail Mutant Mice

Previous research, defining spatial control of inositol phosphate biosynthesis in the developing brain of CBA (normal) and CT [curly tail (ct-CT) and straight tail (st-CT)] mutant mice implicated a role for 1l-myo-inositol 1-phosphate synthase (MIP) in normal functioning of the central nervous system. Biochemical research indicated that MIP enzymatic activity, conversion of glucose 6-phosphate into inositol phosphate, is highest in the cerebellum of ct-CT and lowest in st-CT, when compared to that of CBA mice.Here, we utilized microscopic and biochemical investigations to analyze and extend previous findings of MIP expression in the cerebellum. Results of this research indicated that MIP expression correlates, well, with its enzymatic activity in the cerebellum of CBA and CT mutantmice. Statistical analyses of fluorescent micrographs detected a significant difference in fluorescence intensity between MIP from ct-CT, st-CT, and CBA mice.These data support vitallinks between inositol phosphate biosynthesis, MIP expression, and normal functioning of the cerebellum. Moreover, published data, identifying significant behavioral differences in the CT mutant, as well as data linking motor and non-motor cerebellar functions to abnormal levels of inositol, support the conclusion that aspects of normal cerebellar functions require temporal and spatial control of inositol phosphate biosynthesis, MIP expression.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.(AU)

Banxia Xiexin Decoction () Treats Diabetic Gastroparesis through PLC-IP / 中国结合医学杂志

OBJECTIVE@#To test the effect of Banxia Xiexin Decoction (, BXD) on the contraction and relaxation of gastric smooth muscle (SM) in diabetic gastroparesis (DGP) model rats, and to explore the mechanism of BXD in the prevention and treatment of DGP through experiments of signal pathway both in vivo and in vitro.@*METHODS@#Sixty Sprague-Dawley rats were divided into 6 groups according to a random number table: control group, model group, high-, medium- and low-dose BXD groups (9.2, 4.6 and 1.8 g/(kg·d), respectively), and domperidone group (10 mg/(kg·d)), 10 rats per group. DGP model was established initially by a single intraperitoneal injection of streptozotocin (STZ), and was confirmed by recording gastric emptying, intestinal transport velocity and gastric myoelectric activity of rats after 2 months. Each group was treated with a corresponding drug for 4 weeks. The mRNA and protein expressions of phospholipase C (PLC), inositol triphosphate (IP@*RESULTS@#Compared with the model group, high- and medium-dose BXD and domperidone significantly increased the expressions of PLC, IP@*CONCLUSIONS@#Treatment with high- and medium-dose BXD significantly attenuated STZ-induced experimental DGP in rats. The therapeutic effect of BXD on DGP rats might be associated with the PLC-IP

Resveratrol inhibits Ca

BACKGROUND@#Resveratrol has been shown to inhibit platelet aggregation. However, the mechanism for this action of resveratrol remains to be clarified. The purpose of this study was to elucidate the Ca@*METHODS@#Ca@*RESULTS@#Thapsigargin-induced Ca@*CONCLUSIONS@#The results suggest that resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca

Effects of N-acetylcysteine on oxidative stress in lung tissue of smoked rats / 中国组织工程研究

BACKGROUND: N-Acetyl-L-cysteine is a precursor of glutathione, which can directly scavenge oxygen free radicals. However, it is not entirely clear whether N-acetyl-L-cysteine can alleviate smoking-induced lung injury by reducing oxidative stress. OBJECTIVE: To investigate the effects of N-acetylcysteine on oxidative stress in lung tissue of smoked rats, and to clarify its possible mechanism of action. METHODS: Thirty male rats were randomly divided into control, smoked and smoked+N-acetylcysteine groups. The rats in the smoked and smoked+N-acetylcysteine groups were placed in the passive smoking animal exposure system, with smoking of 20 cigarettes, twice/d, 1 hour/times, for 8 consecutive weeks. The rats in the smoked+N-acetylcysteine group were given 200 mg/kg N-acetylcysteine via gavage before passive smoking daily, for 8 consecutive weeks. The control rats were fed normally for 8 consecutive weeks. The study was approved by the Laboratory Animal Ethical Committee of Shenyang Medical College in October 2018, approval No. (2018)85. RESULTS AND CONCLUSION: (1) Pathological observation showed that the pulmonary tissue was disordered, alveolar septal thickening, inflammatory cell infiltration and interstitial fibrosis in the smoking group, while the pulmonary hemorrhage, the number of inflammatory cells and the degree of fibrosis in the smoking+N-acetylcysteine group were significantly reduced. (2) Compared with the control group, the expression levels of malondialdehyde-5 and inositol-alpha genes were significantly increased, and superoxide dismutase-1 gene expression was significantly decreased in the smoked group, while N-acetylcysteine could inhibit the above changes. (3) Immunofluorescence staining and western blot assay results revealed that the expression levels of malondialdehyde-5 and inositol-alpha in lung tissue of rats in the smoked+N-acetylcysteine group was significantly lower than those in the smoked group, and the expression level of superoxide dismutase-1 was significantly higher than that in the smoked group. In addition, the expression levels of Nrf2 and Keap1 mRNA and protein were significantly increased, and the expression levels of Bach1 mRNA and protein were significantly decreased in the smoking+N-acetylcysteine group. (4) These results suggest that N-acetylcysteine can protect smoking-induced lung injury by reducing oxidative stress, and which might be through activating Nrf2/Keap1 signaling pathway.

Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa / 亚洲男科学杂志(英文版)

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

A comparative study of combination of Myo-inositol and D-chiro-inositol versus Metformin in the management of polycystic ovary syndrome in obese women with infertility

Background: Polycystic ovary syndrome (PCOS) is a symptom complex associated with increased amounts of circulating androgens in females, increased insulin resistance and obesity. The drugs, Myo-inositol, D-chiro-inositol and Metformin, which are insulin sensitizers, are very helpful in taking care of one of the key components of PCOS that is insulin resistance. This study was done to compare the effects of combination of Myo-inositol and D-chiro-inositol with the use of metformin on clinical and biochemical profile in PCOS.Methods: A prospective, randomized, comparative study was conducted on 200 patients. The patients were randomly assigned into the two groups of 100 each. Group A receiving Tab. Myoinositol 550mg twice daily and Tab. D-chiro-inositol 13.8mg twice daily and Group B receiving Tab. Metformin 500mg thrice daily. The patients were assessed by menstrual cycle regulation, hirsutism score (Ferriman Gallwey), fasting and post prandial glucose and insulin levels, serum DHEA levels, serum free testosterone levels and fasting day 3 serum LH and FSH ratio.Results: In both the groups there was significant improvement in all the above mentioned parameters, however the group with Combination of Myo-inositol and D-chiro-inositol had statistically significant improvement over the Metformin group.Conclusions: Combination of Myo-inositol and D-chiro-inositol and use of metformin, significantly improved insulin sensitivity in PCOS women. But combination of Myo-inositol and D-chiro-inositol was effective in controlling the hormonal profiles (LH/FSH ration and free testosterone) when compared to Metformin.

Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

Screening of lung cancer tumor-associated suppressor genes using shRNA library targeting human transcriptome / 实用肿瘤学杂志

Objective The shRNA library was used to screen the tumor suppressor genes related to lung epithelial cells,and its function of inhibiting malignant transformation in lung cells was preliminarily verified,which provided a theoretical basis and a new therapeutic target for tumor prevention and treatment. Methods The shRNA retrovirus library was constructed by GP2 -293 virus packaging cell line to infect the immortalized lung bronchial epithelial BEAS-2B cells. The transfected epithelial cell clones were screened by soft agar colony formation,and a single transformed cell clone with a diameter greater than 1. 0 mm was selected. The in-serted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conven-tional DNA sequencing and blast alignment. A candidate tumor suppressor gene INPP4B was verified by soft agar cloning and tumor formation in nude mice. MTT assay was used to detect the cell proliferation. Results Six lung epithelial malignant transformation in-hibitory factors were screened by soft agar colony formation. The candidate INPP4B gene was selected for functional experiments. Si-lencing INPP4B gene in BEAS-2B cells promoted the formation of clones in the soft agar plates,and the cell proliferation rate was accelerated. The silencing cell line showed the enhanced tumorigenicity in nude mice,indicating that INPP4B was involved in tumor formation. Conclusion shRNA library and soft agar colony formation assays are a powerful tool for screening tumor suppressor genes, and INPP4B is a malignant transformation inhibitor of lung epithelial cells.

Effect of Epimedium flavonoids on decreasing secretion function of Sertoli cells in testis of natural aging rats / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of total flavonoids of Epimedium (TFE) on the decline of secretion function of senile testis-supporting cells (Sertoli cells), and to explore their molecular mechanism from inositol-requiring enzyme 1α(IRE1α)/X-box- binding protein 1(XBP1) signaling pathway. METHODS Thirty-six SPF 18-month-old SD male rats were randomly divided into natural aging group, low and high dose TFE groups, with 12 rats in each group. Another ten 2-month-old rats were used as a youth control group. TFE was ig given once a day, and the ig administration was continued every five days after a two-day interval, and the administration lasted for 4 months. Then, the testicular index was calculated. The testicular histomorphology was observed by HE staining; while quantitative-PCR (qPCR) and Western blotting were used to detect the mRNA and protein expression levels of glial cell derived neurotrophic factor (GDNF), bone morphogenetic protein 4 (BMP4) and stem cell factor (SCF), and Western blotting protein expression levels of p-IRE1α and XBP1 in testes. The number of Sertoli cells in the testis and the localization of p-IRE1α in the testis were detected by immunofluorescence. RESULTS Compared with the young control group, the testicular index decreased significantly in the natural aging group (P<0.01), but was significantly up-regulated after TFE 40 m g · k g- 1 (P<0.05). The results of HE showed that the morphological structure of the testicular seminiferous tubules in the natural aging group were disorderly, and some spermatogenic cells were shed. The TFE group could improve the morphological structure of the seminiferous tubules of the testis. qPCR and Western blotting results showed that compared with the young control group, the mRNA and protein expression levels of GDNF, BMP4 and SCF in Sertoli cells in the natural aging group were significantly down-regulated (P< 0.01). However, the expression of secretory factors in TFE group was significantly up-regulated (P< 0.05, P<0.01). Western blotting results showed that compared with the young control group, the expression of p-IRE1α and XBP1 protein in the testis of the natural aging group was significantly down-regulated (P<0.01), but the expression of p-IRE1α and XBP1 protein in the TFE group was significantly up-regulated (P<0.01). The results of immunofluorescence also showed that there was no significant difference in the number of Sertoli cells between the natural aging group and the TFE group compared with the young control group, while the expression of p-IRE1α was localized both in cytoplasm of Sertoli cells and spermatogenic cells. CONCLUSION TFE can significantly improve the decline of secretion function of testicular Sertoli cells induced by aging, and the mechanism may be related to the regulation of IRE1α/XBP1 signaling pathway.

Role of inositol-requiring kinase 1α/X-box binding protein 1 in airway mucus secretion induced by neutrophil elastase / 上海交通大学学报（医学版）

Objective: To explore the effect of endoplasmic reticulum stress (ERS) on neutrophil elastase (NE) induced mucin 5AC (MUC5AC) production in human airway epithelial cells. Methods: HBE16 airway epithelial cells were cultured and pretreated with reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) or ERS inhibitor 4-phenylbutyrate (4-PBA), or transfected with small interfering RNA (siRNA) against inositolrequiring kinase 1α (IRE-1α) or X-box binding protein 1 (XBP-1), respectively before incubation with NE. NE group and blank control group were also set up. ROS production was assayed by detection kit; expression of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK), activating transcription factor 6 (ATF6), phosphorylated IRE-1α (pIRE-1α), and XBP-1 protein was detected by Western blotting; spliced XBP-1 (XBP-1s) mRNA was measured by real-time PCR; levels of MUC5AC protein in culture supernatant and cytoplasm were assayed by ELISA and immunofluorescence. Results: There was an obvious increase of ROS production with strong elevation of GRP78, ATF6, pPERK, and pIRE-1α protein in NE group cells after 24 h, compared with blank control group (P<0.05). The protein and mRNA of XBP-1s, and MUC5AC production also increased obviously (P<0.05). NAC and 4-PBA reduced ERS-related protein expression and MUC5AC production and secretion (P<0.05). Further studies showed that MUC5AC secretion was also blunted by IRE-1α siRNA or XBP-1 siRNA, accompanied with decreased expression of XBP-1s mRNA and protein (P<0.05). Conclusion: NE induces ERS by producing ROS, and increases MUC5AC protein production and secretion; IRE-1α/XBP-1 play a certain role in this process.