[Effect of acupuncture at "Houxi" (SI3) and"Huantiao" (GB30) on high mobility group box 1 protein in spinal nerve trunk of rats with lumbar disc herniation].

OBJECTIVE: To observe the effect of acupuncture at "Houxi"(SI3) and "Huantiao"(GB30) on high mobility group box 1(HMGB1) protein and mRNA in spinal nerve trunk(SNT) of rats with lumbar disc herniation(LDH), so as to explore the mechanisms of acupuncture at this paired points on the treatment for LDH. METHODS: SD rats were randomly divided into sham operation, model, conventional acupuncture(CA) and paired points(PP) groups (with 8 rats in each group). The LDH model was established by injection of autologous suspension made from rats' own nucleus pulsus into the epidural space. Rats in the CA group received acupuncture treatment at bilateral "Weizhong"(BL40), "Dachangshu"(BL25) and "Shenshu"(BL23), while rats in the PP group received acupuncture at bilateral SI3 and GB30, 30 min each time, once daily for 14 consecutive days. The thermal pain threshold of bilateral hind feet of rats was detected by thermal pain stimulator. The contents of serum IL-1ß, IL-6 and IL-8 of rats were detected by ELISA. Western blot and immunofluorescence were used to detect the expression of HMGB1 protein in the lumbar(L)5 SNT of rats. The relative expression of HMGB1 mRNA in L5 SNT was determined by qPCR. HE staining was used to observe the morphological changes of L5 SNT. RESULTS: Compared with the sham operation group, the thermal pain threshold of bilateral hind feet in the model group was decreased (P<0.05); compared with the model group, the thermal pain threshold of bilateral hind feet in the CA group and the PP group were increased (P<0.05). The expressions of HMGB1 protein and mRNA in L5 SNT, and the contents of serum IL-1ß, IL-6 and IL-8 of rats in the model group were significantly increased(P<0.000 1, P<0.001) in contrast to the sham operation group. The expressions of HMGB1 protein and mRNA in L5 SNT, and the levels of serum IL-1ß, IL-6 and IL-8 were significantly decreased (P<0.01, P<0.000 1, P<0.001, P<0.05) in the CA and PP group, in comparison with those of the model group. Compared with the CA group, the above indexes of rats in the PP group recovered more significantly (P<0.05,P<0.001, P<0.01,P<0.000 1). The histomorphological results showed scattered and various-sized nerve fibers, vacuolation, a large number of disintegrating myelin sheath and lysed Schwann cells in the model group. Myelin sheaths regeneration, regularly-arranged nerve fibers were seen in the CA group and the PP group, with more obvious histopathological recovery observed in the PP group than the CA group. CONCLUSION: Acupuncture intervention inhibites the expressions of HMGB1 protein and mRNA in rats with LDH, and further reduces the production of IL-1ß, IL-6 and IL-8, which is beneficial to inflammatory response inhibition and pain alleviation. The therapeutic effect of the PP group is more obvious than that of the CA group.

[Effect of deep and shallow electroacupuncture stimulation at "Huantiao"(GB30) on expression of phosphorylated-p38 and phosphorylated-p53 proteins and apoptosis in dorsal root ganglia in sciatic nerve injury rats].

OBJECTIVE: To observe the effect of deep electroacupuncture (EA) stimulation at "Huantiao"(GB30) on hindlimb motor function and expression of p38 mitogen-activated protein kinase (p38 MAPK ) and p53 proteins in dorsal root ganglia (DRG) in rats with chronic constrictive injury (CCI) of sciatic nerve. METHODS: Forty-eight SD rats (half male and half female) were randomly divided into control, model, shallow EA (SEA) stimulation and deep EA (DEA) stimulation groups (n=12 in each group). The CCI model was constructed by implanting a silicone tube close to the sciatic nerve of the left hind limb. For DEA group and SEA group, filiform acupuncture needles were inserted into GB30 about 12-14 mm deep and 5-8 mm deep (monitored by using a high-frequency ultrasound device), respectively, followed by electrical stimulation (2 Hz/100 Hz, 1 mA) using an EA stimulator. The intervention was conducted for 15 min every time, once daily for 14 days. The sciatic nerve function index (SFI) calculated to assess the motor function status. Histopathological changes of the sciatic nerve were displayed by H.E. staining. The expression levels of phosphorylated-p38 MAPK (p-p38) and phosphorylated-tumor protein p53 (p-p53) in DRGs of L4-L5 on the affected side were observed by immunohistochemical staining. RESULTS: Following modeling, the SFI were significantly decreased (P<0.01), and the expression levels of p-p38 and p-p53 proteins of L4-L5 DRGs were considerably increased in the model group (P<0.05). After the intervention, the SFI were obviously increased, and the expression levels of p-p38 and p-p53 proteins notably down-regulated in both DEA and SEA groups relevant to the model group (P<0.01, P<0.05). The therapeutic effect of DEA was significantly superior to that of SEA in raising SFI and down-regulating expression le-vels of p-p38 and p-p53 proteins (P<0.01, P<0.05). H.E. staining showed disordered arrangement of the sciatic nerve fibers and myelin, disaggregation of the myelin and axons with deformity and vacuolation in some of them and with an increase of Schwann cells in the model group, which was relatively milder in both DEA and SEA groups. CONCLUSION: Both DEA and SEA at GB30 can obviously improve the motor function in CCI rats, which may be associated with its function in down-regulating the expression of p-p38 and p-p53 proteins in L4-L5 DRGs, restraining p38 MAPK signaling. The therapeutic effect of DEA is evidently better than that of SEA.

[Lower frequency electroacupuncture is better in promoting recovery of limb locomotion in rats with sciatic nerve injury by reducing local inflammatory reaction].

OBJECTIVE: To observe the effect of different frequencies (2 Hz, 100 Hz) of electroacupuncture (EA) on limb locomotion and the expression of inflammatory factors IL-1ß, IL-6, TNF-α in sciatic nerve, and nuclear factor kappa B (NF-κB) in lumber(L)4-L5of spinal cord in rats with sciatic nerve injury (SNI), so as to reveal its mechanisms underlying improvement of SNI. METHODS: A total of 48 SD rats (half male and half female) were equally divided into blank control, model, low frequency (2 Hz) EA and high frequency (100 Hz) EA groups. The SNI model was established by clamping the spinal nerve. EA intervention (2 Hz, 100 Hz, 1 mA), starting on the 8th day after modeling, was applied to "Huantiao" (GB30) on the injured side for 15 min, once daily for 14 consecutive days. The sciatic function index (SFI) was calculated to assess the injured hindlimb recovery with reference to BAIN's and colleagues' methods. Histopathological changes of the sciatic nerve were displayed by H.E. staining. The expressions of IL-1ß, IL-6 and TNF-α in the sciatic nerve tissue were detected by immunohistochemistry, and the expression of NF-κB in the spinal cord was detected by using Western blot. RESULTS: After modeling, the SFI level on day 8 was significantly decreased in the model group (P<0.01), and no significant differences were found among the model, low frequency EA and high frequency EA groups before the EA intervention (P>0.05). Following the treatment (at the 22nd day), the SFI values of both low frequency EA and high frequency EA groups were significantly increased (P<0.01), suggesting an improvement of the limb motor function, and the SFI of the low frequency EA group was notably higher than that of the high frequency EA group (P<0.01). In comparison with the blank control group, the expression levels of IL-1ß, IL-6, TNF-α in the sciatic nerve and NF-κB protein in the spinal cord were significantly up-regulated (P<0.05). Following EA intervention, the increased expression levels of IL-1ß, IL-6, TNF-α and NF-κB proteins were significantly down-regulated in both low frequency EA and high frequency EA groups (P<0.05), and the therapeutic effect of low frequency EA was markedly superior to that of high frequency EA in down-regulating the expression levels of IL-1ß, IL-6, TNF-α and NF-κB protein (P<0.05). H.E. staining showed increase of Schwann cells in number, cellular swelling, and disintegration of the axons and myelin sheath, and appearance of vacuolar degeneration in the model group, which was relatively milder in both low frequency EA and high frequency EA groups, particularly in the low frequency EA group. CONCLUSION: EA of GB30 at 2 Hz and 100 Hz can promote the recovery of hindlimb motor function in SNI rats, which is probably related to its function in inhibiting the inflammatory response, and facilitating the repair of the damaged sciatic nerve. 2 Hz EA is better than 100 Hz EA in the therapeutic effect.

[Anti-apoptotic Mechanism of Deep Electroacupuncture at "Huantiao" (GB 30) in Repair of the Injured Sciatic Nerve in Rats].

OBJECTIVE: To observe the effect of deep electroacupuncture (EA) at "Huantiao" (GB 30) on functional and pathological changes of the damaged sciatic nerve and apoptosis-related factors in lumbar dorsal root ganglia (DRGs) in rats, so as to study its mechanisms underlying relieving sciatica. METHODS: Forty-eight SD rats were randomly divided into normal, mo-del, deep EA (DEA) and shallow EA (SEA) groups (n＝12 in each group)．The sciatic nerve injury model was established by silicone tube extrusion of the sciatic nerve stem. For DEA group, acupuncture needles were inserted into GB 30 about 8 mm deep to induce nerve impulse under the guidance of high-frequency ultrasound, and the needles were inserted into GB 30 about 3-5 mm in the SEA group. The EA treatment was applied to bilateral GB 30 for 20 min beginning from the 15th day on after modeling, once daily for 14 days. The sciatic nerve function index (SFI) and motor nerve conduction velocity (MNCV) were recorded and calculated. Pathological changes of the sciatic nerve were displayed by H. E. staining. The cell apoptosis and expression of PI 3 K and AKT proteins in lumbar 4-5 DRGs were detected by TUNEL staining, and immunohistochemistry, respectively. RESULTS: In comparison with the normal group, the SFI and MNCV were significantly decreased (P<0.01，P<0.05), the number of neuronal apoptosis and the expression of PI 3 K and AKT in L 4-L 5 DRGs were significantly increased in the model group (P<0.05). Following the treatment, the SFI, MNCV, and the number of neuronal apoptosis were reversed compared with the model group (P<0.01, P<0.05), both PI 3 K and AKT expression levels were significantly up-regulated in both DEA and SEA groups (P<0.05). The therapeutic effects were significantly better in the DEA group than in the SEA group in down-regulating cell apoptosis number and in up-regulating SFI, MNCV, and PI 3 K and AKT protein expression on day 28 of modeling (P<0.01，P<0.05). CONCLUSION: Deep EA at GB 30 can activate PI 3 K-AKT signaling pathway, inhibit the apoptosis of nerve cells in DRGs after sciatic nerve injury in rats, which may contribute to its effect in improving neurological impairment.