Expression and purification of GRIK3 intracellular region protein in prokaryotic cell system / 实用医学杂志

Objective To express and purify GRIK3 intracellular region protein in prokaryotic cell system. Methods The histamine(His)tagged GRIK3 intracellular region recombinant plasmid pCzn1-GRIK3-in-tra was constructed and transformed into Escherichia coli(E.coli). The His-GRIK3-intra recombinant protein was expressed after the induction with IPTG. The target protein was purified by Ni-NTA affinity chromatography column. Results The prokaryotic expression plasmid pCzn1-GRIK3- intra was successfully constructed and the GRIK3 intracellular region protein in E.coli were efficiently expressed after the induction with IPTG. The purified target pro-tein was successfully obtained by Ni-NTA affinity chromatography column. Conclusion The successful construc-tion of prokaryotic expression plasmid expressing GRIK3 intracellular region gene and preparation of high purity GRIK3 intracellular region protein have paved the way for further exploration of the intracellular signal transduction mechanism of membrane protein GRIK3.

High Mobility Group Box 1/ CD 24 Receptor/β-EP Signaling in "Zusanli" (ST 36) Region Contributes to Electroacupuncture Analgesia in Rats with Neuropathic Pain / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of high mobility group box 1 (HMGB 1) and its receptor CD 24 proteins and β-endorphin (β-EP) content in "Zusanli" (ST 36) region in rats with chronic constriction injury (CCI), so as to explore its mechanisms underlying pain relief. METHODS: Thirty male Wistar rats were rando-mized into control, CCI model and EA groups (n＝ 10 rats in each). The neuropathic pain model was established by ligature of the left sciatic nerve to induce CCI in the model and EA groups, and sham operation was performed in rats of the control group. Paw with drawal latency (PWL, thermal pain threshold) of the bilateral hind-limbs was detected by using an algesia-detector. Eight days after CCI operation, EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 5 days. The acetylated-HMGB 1 expression was determined by immunoprecipitation, and the expression of HMGB 1 and toll like receptor 4 (TLR 4) proteins and CD 24 mRNA were detected using Western blot and fluorescent quantitative real time-PCR, respectively, and the content of β-EP in the acupoint region was assayed by enzyme linked immunosorbent assay (ELISA). Anti-CD 24 neutralizing antibody (200 µL, 100 µg/mL) was injected into ST 36 region once daily for 3 days for verifying the involvement of HMGB 1/CD 24 signaling in EA analgesia. RESULTS: Compared with the control group, the bilateral PWL difference values in the other two groups were significantly increased (P0.05). After local injection of anti-CD 24 antibody, EA-induced increases of β-EP content and reduction of thermal pain threshold were significantly suppressed (P<0.05). CONCLUSION: EA of ST 36 and GB 34 can alleviate neuropathic pain in CCI rats, which is associated with its effects in up-regulating β-EP content, and HMGB 1 protein and CD 24 mRNA expression levels in ST 36 region. The activated HMGB 1/CD 24/β-EP signaling contributes to EA-ST 36 induced analgesia.

Analysis on interrelation between electroacupuncture-induced cumulative analgesic effect and hypothalamic cholinergic activities in chronic neuropathic pain rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36)- Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury (CCI) and/or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia.</p><p><b>METHODS</b>A total of 103 female Wistar rats were randomly divided into normal control (n =15), CCI (n =15), CCI+EA2d (n =15), CCI+EA2W (n =15), OVX+CCI =13), OVX+CCI+EA2d (n =15), and OVX+CCI+EA2W groups (n =15). CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats' memory learning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>In comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and SON regions of OVX+CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably (P <0.05). In comparison with the OVX+CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05). Similar situations were found in OVX+CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P <0.05), suggesting a reduction of EA2W effects after OVX.</p><p><b>CONCLUSION</b>Repeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals' neuromemory ability.</p>

Effects of repeated electroacupuncture on beta-endorphin and adrencorticotropic hormone levels in the hypothalamus and pituitary in rats with chronic pain and ovariectomy / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of electroacupuncture (EA)-induced cumulative analgesic effects on chronic pain in rats with or without ovariectomy (OVX).</p><p><b>METHODS</b>A total of 110 female Wistar rats were randomized into normal control (n=10), chronic constrictive injury (CCI, n=10), CCI+EA (n=30), OVX+CCI (n=30), and OVX+CCI+EA (n=30) groups. Each of the latter 3 groups was further divided into 2 days (2 d), 2 weeks (2 W) and 3 weeks (3 W) subgroups, respectively (n=10 in each subgroup). The CCI pain model was established by ligature of the right sciatic nerve, and the memory impairment model duplicated by OVX. The paw withdrawal latency (PWL, pain threshold) of the bilateral footplates was detected by radiant heat irradiation, and the bilateral difference in PWL (PWLD) was used to evaluate changes in the pain reaction. Morris water maze test was conducted for evaluating the rats' learning-memory ability. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 2 d, 2 W and 3 W, respectively. Pituitary and hypothalamic beta-endorphin (EP) and adrenocorticotrophic hormone (ACTH) contents were detected by immunoradioassay.</p><p><b>RESULTS</b>Compared with the CCI group, PWLD of the CCI+EA-3 W group decreased significantly (P<0.05). Compared with the OVX+CCI group, PWLD of the OVX+CCI+EA-3 W group was lowered considerably (P<0.05), but the value was markedly higher than its basal value and those of the normal control and CCI+EA groups (P<0.05). In comparison with the sham-OVX group, the escape latency, swimming distance (SD) in the target quadrant and total SD were increased remarkably in the OVX group (P<0.05), while the number of target platform crossings was decreased significantly (P<0.05), suggesting an impairment of the OVX rats' learning-memory ability. In simple CCI rats, both beta-EP and ACTH contents of the pituitary increased markedly (P<0.05), and those of the hypothalamus decreased obviously compared to the normal control group (P<0.05). After EA, pituitary and hypothalamic ACTH levels were significantly lowered at 2 d and hypothalamic ACTH and beta-EP contents increased obviously at 3 W in comparison with the CCI group (P<0.05). In OVX+CCI rats, following EA, pituitary beta-EP contents at 2 d, 2 W and 3 W, and hypothalamic beta-EP and ACTH contents at 2 W and hypothalamic ACTH levels at 3 W increased significantly (P<0.05), but hypothalamic beta-EP level at 3W decreased markedly (P<0.05). The effects of repeated EA in lowering pituitary ACTH and raising hypothalamic beta-EP and ACTH levels disappeared after OVX+CCI.</p><p><b>CONCLUSIONS</b>Repeated EA has a cumulative analgesic effect, which is closely associated with its effects in regulating pituitary and hypothalamic beta-EP and ACTH levels. OVX may weaken the analgesic effect of EA by affecting hypothalamic-pituitary axis activity.</p>

Dynamic contrast-enhanced MRI of vertebral metastatic tumors' early diagnosis / 中国基层医药

Objective To discuss the diagnostic value of dynamic contrast-enhanced MRI in differentiating vertebral metastatic tumors from benign deseases after studying the early changes of signal intensity.Methods Twenty patients were scanned by dynamic contrast-enhanced MRI.The parameters of signal intensity were measured and calculated,and time-intensity cures(TICs)were drawn.Then,they were compared with those of normal verte- braes,the characteristics were analyzed.Results The parameters of vertebral metastatic tumors:peak signal intensi- ty,signal intensity minimum signal intensity of post-peak,peak slope,initial ratio of enhancement,maximum ratio of enhancement was respectively(50.61?11.38),(46.75?10.23),(0.73?0.12),(0.33?0.08),(1.03?0.31); that of normal vertebraes was respectively(40.53?12.12),(34.72?14.06),(0.31?0.11),(-0.25?0.05), (0.69?0.28);and ten normal cases showed negative in initial percent of enhancement,but only two abnormal ver- tebraes showed that.All of the above had significant statistical difference.Four types of TICs were concluded:early rapid rise and early rapid descent followed by steady phase(type A),persistently rise(type B),rise phase followed by steady phase(type C),rise phase followed by rapid descent phase(type D).TICs of abnormal vertebraes were respec- tively 6,5,5,5 cases;types of normal vertebraes were mainly type C,15 cases.Type A and type B and the type with the characteristic of early rapid descent phase were features of abnormal vertebraes;type C was the feature of normal ones.Conclusions Dynamic contrast-enhanced MRI could quantitatively show the characteristics of early changes of signal intensity of vertebral metastatic tumors,which were significant statistically different from those of normal ver- tebraes.Dynamic contrast-enhanced MRI was worth performing in the early diagnosis of vertebral metastatic tu- mors.