lncRNA CERS6-AS1 upregulates the expression of ANLN by sponging miR-424-5p to promote the progression and drug resistance of lung adenocarcinoma.

Long non-coding RNAs (lncRNAs) play a crucial role in tumor generation and progression. However, the exact functional significance and underlying molecular mechanism by which lncRNA CERS6-AS1 operates in the context of lung adenocarcinoma (LUAD) remain unknown. We aimed to evaluate the potential role of the CERS6-AS1/miR-424-5p/ANLN axis in the progression of LUAD through bioinformatics and cytobehavioral experiments, and to provide a new insight into the combined treatment of LUAD. Based on the TCGA database, the expression of CERS6-AS1 in pan-cancer was evaluated, and its prognostic performance in LUAD was evaluated by ROC curve, survival curve and COX analysis. In addition, quantification of CERS6-AS1 expression levels in LUAD patients and lung cancer cells using quantitative real-time polymerase chain reaction (RT-qPCR), and further validate the functional significance of CERS6-AS1 in promoting the proliferation, migration, and invasion abilities of lung cancer cells. The competitive endogenous RNA (ceRNA) network was constructed, and miR-424-5p inhibitors were applied to CERS6-AS1 knockdown cells. The potential downstream genes associated with the regulatory axis of CERS6-AS1/miR-424-5p were analyzed by PPI network and gene enrichment analysis (KEGG). Finally, we evaluated the prognostic value of high expression of ANLN in LUAD and its effects on immune cell infiltration, tumor mutation burden, chemotherapy response, and immunotherapy. CERS6-AS1 expression was significantly elevated in both LUAD patients and lung cancer cells. In the CERS6-AS1 knockdown assay, the proliferation, invasion, migration and epithelial-mesenchymal transformation (EMT) of cancer cells were significantly inhibited. Notably, there was a prominent upregulation of miR-424-5p expression in cells where CERS6-AS1 was knocked down. Co-transfection of siRNA and miR-424-5p inhibitors into lung cancer cells restored the restriction on lung cancer cells. Anillin (ANLN) has been identified as a potential target gene for miR-424-5p and as a prognostic and immune biomarker associated with immune cell infiltration and tumor mutational burden in LUAD. Additionally, ANLN impacts the efficacy of chemotherapy and immunotherapy in LUAD patients. This study reveals a novel regulatory mechanism in which CERS6-AS1 may contribute to the progression of LUAD by influencing the expression of ANLN as a competitive sponge for miR-424-5p.

[Effect of scalp acupuncture stimulation on expression of pentraxin 3 in striatum in acute ische-mic cerebrovascular disease rats].

OBJECTIVE: To observe the influence of scalp-acupuncture on the expression of pentraxin 3 (PTX3), Interleukin (IL)-1ß, zonula occludens-1(ZO-1) mRNA and Occludin mRNA in striatum in acute ischemic cerebrovascular disease (AICD) rats, so as to investigate its mechanisms underlying improvement of AICD. METHODS: Forty-eight male SD rats were randomly allocated to control, model, IL-1Ra and IL-1Ra+scalp-acupuncture groups (n=12 rats in each group). The AICD model was established by occlusion of the middle cerebral artery (MCAO). Rats of the IL-1Ra group and IL-1Ra+scalp-acupuncture group received intraperitoneal injection of IL-1Ra (0.05 mg·kg-1·d-1), once daily for 6 days. Scalp acupuncture stimulation was applied to bilateral "Dingnieqianxiexian" (MS6) once daily for 6 days for rats in IL-1Ra+scalp-acupuncture group. Before and after intervention, the neurologic deficit score (NDS) was evalua-ted according to Longa's method. The expression of striatum PTX3 and IL-1ß was detected by immunohistochemistry, and ZO-1 mRNA and Occludin mRNA in the striatum tissue were detected by fluorescence quantitative real-time PCR. The Evans Blue (EB) tracer method was used to monitor the degree of blood-brain barrier (BBB) damage. RESULTS: Following modeling, the NDS, EB content and the expression of PTX3 and IL-1ß in the striatum tissue were significantly increased, and the ZO-1 mRNA and Occludin mRNA expression was considerably decreased in the model group compared with the control group (P<0.05). After the interventions and compared with the model group, the NDS, EB content in both IL-1Ra and IL-1Ra+scalp acupuncture groups, and PTX3 in the IL-1Ra group were significantly down-regulated (P<0.05), while the striatum ZO-1 mRNA and Occludin mRNA expression in both IL-1Ra and IL-1Ra+scalp acupuncture groups, and PTX3 in the IL-1Ra+scalp acupuncture group were obviously up-regulated (P<0.05), and the expression of IL-1ß was obviously down-regulated in the IL-1Ra+scalp acupuncture group (P<0.05) rather than in the IL-1Ra group (P>0.05). The effects of scalp acupuncture combined with IL-1Ra were obviously superior to that of IL-1Ra in down-regulating NDS, EB content and IL-1ß expression level, and in up-regulating PTX3, ZO-1 mRNA and Occludin mRNA expression levels (P<0.05). CONCLUSION: Scalp acupuncture can improve neurological function and reduce the degree of BBB injury in AICD rats, which may be associated with its function in up-regulating the expression of PTX3 and in promoting the expression of ZO-1 mRNA and Occludin mRNA.

[Effect of scalp acupuncture stimulation on cerebral infarct volume and expression of IL-10, IL-6 and IL-1ß in para-hippocampal gyrus in acute ischemic cerebrovascular disease rats].

OBJECTIVE: To observe the influence of scalp acupuncture on cerebral infarct size and expression of IL-10, IL-6, and IL-1ß in the para-hippocampal gyrus in acute ischemic cerebrovascular disease(AICD) rats, so as to investigate its mechanisms underlying improvement of AICD. METHODS: Forty-eight male SD rats were randomly allocated to normal control (control), AICD model, medication, and scalp acupuncture groups (n＝12 per group). The AICD model was established by occlusion of the middle cerebral artery (MCAO). Rats of the medication group received intraperitoneal injection of Ammonium 1-Pyrrolidinedithiocarbamate (APDC, 100 mg•kg－1•d－1), once daily for 7 days. Scalp acupuncture stimulation was applied to bilateral "Dingnieqianxiexian" (MS6) once daily for 7 days. Before and after intervention, the neurologic deficit score (NDS) and the neurological score (NS) were evaluated according to Longa's and Schäbitz's methods, respectively. At the end of the intervention, the para-hippocampal gyrus and whole brain were collected respectively. The expression levels of IL-10, IL-6 and IL-1ß in the para-hippocampal gyrus tissue were detected by immunohistochemistry, and the cerebral infarct volume of the brain was detected by triphenyltetrazollium chloride (TTC) staining after sectioning. RESULTS: Following modeling, the NDS, NS and the expression of IL-10, IL-6 and IL-1ß in para-hippocampal gyrus were significantly increased in the model group compared with the control group (P<0.01). After the intervention, the NDS, NS and infarct volume, and the expression levels of IL-6 and IL-1ß in the para-hippocampal gyrus were significantly decreased in both medication and scalp acupuncture groups compared with the model group (P<0.05), and the expression of IL-10 was further obviously up-regulated in the scalp acupuncture group (P<0.05) rather than in the medication group (P>0.05). The effect of scalp acupuncture was obviously superior to that of medication in up-regulating IL-10 expression level (P<0.05). No significant differences were found between the medication and scalp acupuncture groups in the levels of NDS, NS, infarct volume, IL-6 and IL-1ß proteins (P>0.05). CONCLUSION: Scalp acupuncture can improve neurological function and reduce infarct volume in AICD rats, which may be associated with its function in up-regulating the expression of IL-10 and in inhibiting the expression of IL-6 and IL-1ß to reduce inflammation reaction.

[ZHENG' Guo-Yan-Re needling technique for diabetic fundus hemorrhage].

OBJECTIVE: To observe the clinical effect of ZHENG ' Guo-Yan-Re needling technique for diabetic fundus hemorrhage. METHODS: With before-after study design, 34 patients with diabetic eyeground hemorrhage were treated with basic treatment (oral administration of antidiabetic medication or insulin injections to ensure blood glucose in the normal range); in addition, acupuncture was given at bilateral Fengchi (GB 20), Taiyang (EX-HN 5), Jingming (BL 1), Cuanzhu (BL 2), Sanyinjiao (SP 6) and Hegu (LI 4). The ZHENG ' Guo-Yan-Re needling technique was applied at Fengchi (GB 20); the heat reinforcing needling technique was applied at Taiyang (EX-HN 5); the slow needle insertion technique was applied at Jingming (BL 1); and the Xique-Dengmei needling technique was applied at Cuanzhu (BL 2); the neutral supplementation and draining method was applied at remaining acupoints. The acupuncture was given once a day, 6 times as one course, and totally 4 courses were given with an interval of 1 day between courses. The follow-up visit was 6 months after treatment. The TCM symptom scores, fundus examination results and vision improvement were observed before and after treatment, and the effect was observed. RESULTS: Compared before treatment, the visual acuity, TCM symptom scores, fundus microaneurysm and hemorrhage points in 34 patients (68 eyes) were significantly improved after treatment (P<0.05). The total effective rate was 88.2% (60/68) after treatment; at follow-up visit, the visual acuity, TCM symptom scores and fundus pathological changes were all improved (P<0.05) and stable at the post-treatment level (P>0.05). CONCLUSION: ZHENG ' Guo-Yan-Re needling technique could improve symptoms, promote the absorption of fundus hemorrhage, and improve vision in patients with diabetic fundus hemorrhage.

[Scalp-acupuncture improves neurological function by regulating expression of IL-10 mRNA, IL-6 mRNA and TNF-α of parahippocampal gyrus in cerebral ischemic rats].

OBJECTIVE: To observe the effect of scalp-acupuncture intervention on the expression of Interleukin (IL)-10 mRNA, IL-6 mRNA and tumor necrosis factor (TNF) - α in the parahippocampal gyrus of cerebral ischemia (CI) rats, so as to explore its molecular mechanisms underlying improvement of CI. METHODS: A total of 64 male SD rats were randomized into normal control, model, medication and scalp-acupuncture groups (n＝16 rats in each group). The focal CI model was established by middle cerebral artery occlusion. Intraperitoneal injection of Ammonium Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS6), followed by twirling the needles at 100 cycles/min for 1 min, once again every 10 min during 20 min' needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－4 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schabitz's (2004) methods, respectively. The expression levels of IL-10 mRNA and IL-6 mRNA were detected by fluorescence quantitative PCR, and the expression of TNF-α was detected by immunohistochemistry. RESULTS: After modeling, the neurologic deficit and neurological function scores and the expression levels of IL-10 mRNA, IL-6 mRNA and TNF-α protein in the parahippocampus were significantly increased in the model group than in the normal control group (P<0.01). Following the intervention, the neurologic deficit and neurological function scores as well as IL-6 mRNA and TNF-α protein expression were significantly down-regulated in both scalp-acupuncture and medication groups (P<0.05), and the expression of IL-10 mRNA was obviously increased (P<0.05) relevant to the model group. CONCLUSION: Scalp-acupuncture can improve neurologic function in CI rats, which is related to its effects in up-regulating the expression of IL-10, then down-regulating the expression of IL-6 and TNF-α (reducing inflammatory response) in the parahippocampal gyrus.

[Scalp-acupuncture Improves Neurological Function by Regulating Expression of NF-κB p 65 mRNA, IκB mRNA and IL-1 ß and TNF-α in Parahippocampal Gyrus of Cerebral Ischemia Rats].

OBJECTIVE: To observe the effect of scalp-acupuncture intervention on the expression of parahippocampal factor-κB p 65 mRNA (NF-κB p 65 mRNA), IκB mRNA, interleukin-1 ß (IL-1 ß) and tumor necrosis factor-α (TNF-α) in rats with cerebral ischemia (CI), so as to investigate its molecular mechanisms underlying improving CI by reducing inflammatory response. METHODS: A total of 64 SD rats were randomized into normal control, model, medication and scalp-acupuncture groups, with 16 rats in each group. The focal CI model was established by middle cerebral artery occlusion (MCAO). Intraperitoneal injection of Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), followed by twirling the needles at 200 cycles/min for 1 min, once again every 10 min during 30 min's needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－5 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schäbitz's (2004) methods, respectively. The expression levels of NF-κB p 65 mRNA and IκB mRNA in the parahippocampus gyrus tissue were detected by fluorescence quantitative reverse transcription-PCR, and IL-1 ß and TNF-α proteins in the parahippocampus gyrus tissue were detected by immunohistochemistry. RESULTS: After modeling, the neurologic deficit and neurological function scores and the expression levels of NF-κB p 65 mRNA, IL-1 ß and TNF-α in the parahip-pocampus were significantly increased in the model group than in the normal group (P<0.01), while the expression of IκB mRNA was considerably down-regulated (P<0.01). Following treatment intervention, the neurologic deficit and neurological function scores as well as NF-κB p 65 mRNA, and IL-1 ß and TNF-α protein expression were significantly decreased in both scalp-acupuncture and medication groups compared with the model group (P<0.05, P<0.01), and the decreased expression of IκB mRNA was obviously increased (P<0.05)．. CONCLUSION: Scalp-acupuncture can improve neurologic function in cerebral ischemic rats, which is related with its effects in up-regulating the expression of IκB to inhibit the dissociation of NF-κB, then decreasing the expression of IL-1 ß and TNF-α (reducing inflammatory response) in the parahippocampal gyrus tissue.

[Mechanism of Heat-reinforcing Needling for Rheumatoid Arthritis Rabbits with Cold Syndrome Based on Metabonomics].

OBJECTIVE: To explore the specific mechanism of heat-reinforcing needling (HRN) for rheumatoid arthritis (RA) rabbits with cold syndrome based on metabolomics. METHODS: A total of 40 healthy rabbits were randomly divided into a normal group, a model group, a reinforcing-reducing needling(RRN) group, a twirling-reinforcing needling (TRN) group and a HRN group (n=8 rabbits/group). The RA model with cold syndrome was established with ovalbumin and freezing. Except the normal and model groups, RRN, TRN and HRN were given at "Zusanli" (ST 36) in the corresponding acupuncture groups for 30 min, once a day for 7 days. After the interventions, the pain threshold and the local skin temperature of each group were observed. The endogenous metabolites in the serum were analyzed by the gas chromatography mass spectrometry (GC-MS) technology combined with the pattern-recognition method. RESULTS: The pain threshold and the local skin temperature in the model group were lower than those in the normal group (P<0.05). The pain thresholds and the local skin temperatures in the three acupuncture groups were higher than those in the model group after interventions (P<0.05), and those in the HRN group were obviously higher than those in the RRN and TRN groups (P<0.05). The serum metabolites of leucine, valine, isoleucine, and arachidonic acid in the model group were increased compared with those in the normal group (P<0.05), and the metabolites of α-ketoglutaric acid, citric acid, succinic acid, glucose, inositol, palmitic acid, stearic acid, lactose, d-ribose, and D-mannose were decreased (P<0.05). The above-mentioned metabolites in the three acupuncture groups were significantly reversed relevant to the model group (P<0.05), and the effect of HRN group was significantly superior to those of the RRN and TRN groups in regulating α-ketoglutaric acid, citric acid, succinic acid, glucose, inositol, d-ribose, and D-mannose (P<0.05). CONCLUSIONS: HRN for RA with cold syndrome is effective, which may be related to the specific regulation for the krebs cycle and glycometabolism.

Gambogic acid suppresses inflammation in rheumatoid arthritis rats via PI3K/Akt/mTOR signaling pathway.

Gamboge is the dried resin secreted by the Garcinia maingayi gambogic tree and is a substance that may be used to treat a variety of diseases, exhibits anti­tumor and detoxification effects and prevents bleeding. The primary active constituent is gambogic acid. The present study aimed to investigate the anti­inflammatory effects of gambogic acid in rheumatoid arthritis (RA) rats and to elucidate the mechanisms by which these effects occur. The swelling degree, the clinical arthritic scoring and pain threshold measurements were used to evaluate the effects of gambogic acid on RA. ELISA kits and western blot analysis were used to investigate inflammatory processes and the expression of RA­associated proteins, respectively. The present results demonstrated that gambogic acid significantly inhibited the degree of right foot swelling, increased pain thresholds and reduced clinical arthritic scores of RA rats. Treatment with gambogic acid suppressed the activities of interleukin (IL)­1ß and IL­6, promoted the protein expression of phosphorylated (p)­Akt serine/threonine kinase (Akt), p­mammalian target protein of rapamycin (mTOR) and inhibited hypoxia­inducible factor­1α and vascular endothelial growth factor expression in RA rats. The results of the present study therefore suggest that the anti­inflammatory effects of gambogic acid in RA rats occur via regulation of the phosphoinositide 3­kinase/Akt/mTOR signaling pathway.

[Effect of Scalp-acupuncture Stimulation on Neurological Function and Expression of ASIC 1 a and ASIC 2 b of Hippocampal CA 1 Region in Cerebral Ischemia Rats].

OBJECTIVE: To observe the influence of scalp-acupuncture on the expression of acid-sensing ion channels (ASICs) 1 a and 2 b of hippocampal CA 1 region in cerebral ischemia (CI) rats, so as to investigate its mechanism underlying improvement of ischemic stroke. METHODS: Thirty-two male SD rats were randomly allocated to normal control, model, scalp-acupuncture and Amiloride group (n=8 in each group). The model of focal CI was established by middle cerebral artery occlusion (MCAO). Scalp acupuncture stimulation was applied to bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), once daily for 7 days. Rats of the Amiloride group were fed with Amiloride solution, twice a day for 7 days, and those of the normal control and model groups were grabbled and fixed in the same way with the acupuncture and Amiloride groups. The neurological deficit score was given according to Longa's method. The expression of hippocampal ASIC 1 a and ASIC 2 b was detected by immunohistochemistry, and the Ca2+ concentration in the hippocampal tissue assayed using flowing cytometry. RESULTS: After the intervention, the neurological deficit score of both the scalp-acupuncture and Amiloride groups were significantly decreased in comparison with pre-treatment (P<0.01), and the effect of scalp-acupuncture was markedly superior to that of Amiloride in reducing neurological deficit score (P<0.05). The expression of ASIC 1 a and ASIC 2 b in the hippocampal CA 1 region and hip-pocampal Ca2+ concentration were significantly up-regulated in the model group compared with the normal control group (P<0.01), and obviously down-regulated in both scalp-acupuncture and Amiloride groups (P<0.01, P<0.05),without significant differences between the two treatment groups in the ASIC 1 a and ASIC 2 b expression and Ca2+ concentration (P>0.05). CONCLUSIONS: Scalp-acupuncture stimulation can improve neurological function in CI rats, which may be related to its effects in suppressing the increased expression of hippocampal ASIC 1 a and ASIC 2 b proteins and in reducing calcium overload in hip-pocampal neurocytes.

Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins.

Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera. This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins.

A new choice for the treatment of segmental tibial bone defects: intramedullary nail internal fixation combined adipose-derived stem cells technique.

The repair of various segmental tibial bone defects continues to be a challenging part of many reconstructive procedures. Many methods have been tried to repair the defects, followed by many complications and the results may be unsatisfied. Since 2001 Zuk et al. established human adipose-derived stem cells (hASCs) as a multipotent stem cell population with the ability to assume osteogenic phenotypes through chemically induced differentiation, hASCs represent a valuable tool for pharmacological and biological studies of osteoblast differentiation in vitro and bone development in vivo, and have been proved to be a useful source of stem cells in bone repair. Recently, hASCs have been found to repair both animals and human calvarial defects. In this paper, we hypothesize that hASCs cultured on custom scaffolds can be used to repair of tibial segmental bone defects with intramedullary nail internal fixed. Unlike current treatment modalities, it would promote the regeneration of tibial defects, provide structural support and allow for weight bearing and bony substitution over time.