Electroacupuncture Promotes Motor Function Recovery in MCAO/R Rats by Activating Astrocyte-Related PI3K/AKT Pathway.

Background: Electroacupuncture (EA) is a widely used traditional Chinese medicine method to manage various diseases, including cerebral ischemia-reperfusion injury (CIRI). Objectives: We assessed the neuroprotective effects of EA and examined its mechanism in a rat model of the middle cerebral artery occlusion-reperfusion (MCAO/R). The gait analysis was performed to evaluate the neuroprotective effects. Western blot and immunohistochemistry assays were carried out to determine the molecular mechanisms of EA. Methods: Male SD rats were randomly divided into the sham operation group, right MCAO/R group, and EA group. EA was administered every day (4/20 Hz, 10 min/1 d) at the following acupoints: Baihui (DU20), Yintang (EX-HN3), and Zusanli (ST36). Gait and motor function were analyzed from day 8 onward. Results: The plantar support and balance coordination of MCAO/R rats decreased, and the cellular structure of the ischemic penumbra was unclear. EA improved the gait dynamics of the rats, adjusted the cell structure, further activated astrocytes, and increased the expression and phosphorylation of phosphoinositide 3-kinase/protein kinase B (PI3K/PKB or AKT). Conclusion: EA promoted astrocyte-related effects in the rat model. Our findings suggest that the neuroprotective mechanism of EA may be related to the activation of the PI3K/AKT signaling pathway. The intervention enhanced brain protection and improved motor functions.

[Influence of electroacupuncture of"Zusanli"(ST36)on mast cells/TRPV1 signaling pathway in visceral hypersensitivity rats with functional dyspepsia].

OBJECTIVE: To explore the interventional mechanism of electroacupuncture (EA) of "Zusanli"(ST36)based on the involvement of mast cells/ transient receptor potential vanilloid type1 (TRPV1) signaling pathway in relieving visceral hypersensitivity in functional dyspepsia (FD) rats. METHODS: Sixty SD rats (half male and half female, 10 days in age) were randomly divided into normal control, model, medication (ketotifen) and EA groups, with 15 rats in each group. The FD model was established by gavage of iodoacetamide combined with tail clamping (stress stimulation). Rats of the medication group received intraperitoneal injection of ketotifen (1 mg·kg-1·d-1) for 14 d, and those of the EA group received EA of ST36 for 20 min, once a day for 14 d. An air-balloon was inserted into the rat's stomach for recording changes of the intragastric pressure (mL/mm Hg) via a pressure transducer. The visceral hypersensitivity was assessed using abdominal withdrawal reflex (AWR) score and the number and degranulation of mast cells of gastric mucosa were observed using toluidine blue staining. The expression levels of TRPV1 and proteinase activated receptor 2 (PAR2) in the stomach were observed using immunofluorescence histochemistry and Western blot, separately, and the contents of SP and CGRP in the stomach detected using ELISA. RESULTS: When the intragastric pressure was at 50, 60 and 70 mm Hg, the gastric compliance was significantly decreased (P<0.01), and the levels of visceral sensitivity increased in the model group （P<0.01）。 TRPV1 immunofluorescence tensity, expression of PAR2 and TRPV1 proteins, and contents of SP and CGRP in the stomach were considerably up-regulated in the model group compared with the normal control group (P<0.01). In comparison with the model group, under intragastric pressure of 50，60 and 70 mm Hg, the gastric compliance was obviously increased, and the visceral hypersensitivity decreased in the EA group （P<0.01,P<0.05）. TRPV1 immunofluorescence intensity, expression levels of PAR2 and TRPV1 proteins, and the contents of SP and CGRP in the stomach were considerably down-regulated in both medication and EA groups compared with the model group (P<0.01, P<0.05). The therapeutic effect of EA was significantly superior to that of medication in up-regulating the gastric compliance （at 70 mm Hg）, and down-regulating the contents of SP and CGRP (P<0.05). No significant differences were found between the EA and medication groups in up-regulating gastric compliance at intragastric pressure of 50 and 60 mm Hg, and in down-regulating the visceral sensitivity, TRPV1 fluorescence intensity, and expression of PAR2 and TRPV1 proteins (P>0.05). Toluidine blue staining showed an apparent increase of mast cell number with obvious degranulation in the gastric mucosa of rats in the model group, which was milder in the EA and medication groups. CONCLUSION: EA of ST36 can suppress visceral hypersensitivity and increase the gastric compliance in FD rats, which may be related with its effects in inhibiting the activation of gastric mast cells, and down-regulating the expression of gastric PAR2 and TRPV1 proteins and SP and CGRP contents.

[Effect of electroacupuncture at "Zusanli" (ST 36) on duodenal mast cells, NGF and NTRK1 in rats with functional dyspepsia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST 36) on duodenal mast cells, nerve growth factor (NGF) and neurotrophic tyrosine kinase receptor type 1 (NTRK1), and to explore the mechanism of electroacupuncture at Zusanli (ST 36) on functional dyspepsia (FD). METHODS: Sixty SPF-grade 10-day-old SD rats were randomly divided into a normal group, a model group, a ketotifen group and an EA group, 15 rats in each group. The FD model was prepared by iodoacetamide combined with rat tail clamping method in the model group, the ketotifen group and the EA group. The rats in the ketotifen group were injected intraperitoneally with ketotifen (1 mg•kg-1•d-1) for 7 days; the rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36), with disperse-dense wave, frequency of 2 Hz/50 Hz and intensity of 0.5 mA, 20 min each time, once a day for 14 days. The gastric emptying rate and small intestinal propulsion rate in each group were observed; the morphology of duodenal mucosa was observed by HE staining; the toluidine blue staining was used to observe the number and degranulation of mast cells in duodenal mucosa; the protein and mRNA expressions of NGF, NTRK1 in duodenum were detected by Western blot and real-time PCR; the level of interleukin-1ß (IL-1ß) in duodenum was measured by ELISA. RESULTS: Compared with the normal group, the gastric emptying rate and small intestinal propulsion rate in the model group were decreased (P<0.01); compared with the model group, the gastric emptying rate and small intestinal propulsion rate in the ketotifen group and the EA group were increased (P<0.01); the small intestinal propulsion rate in the EA group was higher than that in the ketotifen group (P<0.01). In the model group, local defects in duodenal mucosa were observed with a small amount of inflammatory cell infiltration; no obvious abnormality was found in duodenal mucosa of the other groups. Compared with the normal group, the mast cells of duodenal mucosa in the model group were increased significantly with significant degranulation; compared with the model group, the mast cells of duodenal mucosa in the ketotifen group and the EA group were decreased significantly, and the degranulation was not obvious. Compared with the normal group, the protein and mRNA expressions of NGF, NTRK1 as well as the level of IL-1ß in duodenum in the model group were increased (P<0.01); compared with the model group, the protein and mRNA expressions of NGF, NTRK1 as well as the levels of IL-1ß in duodenum in the ketotifen group and the EA group were decreased (P<0.01, P<0.05); compared with the ketotifen group, the mRNA expression of NGF, as well as the protein and mRNA expressions of NTRK1 in duodenum in the EA group were decreased (P<0.05, P<0.01). CONCLUSION: EA at "Zusanli" (ST 36) could inhibit the activation of duodenal mast cells and regulate the expressions of NGF and its receptor to improve the low-grade inflammatory response of duodenum, resulting in treatment effect on FD.

[Electroacupuncture of "Zusanli" (ST 36) Raises Muscular Force by Adjusting AMPK/PGC-1 α Signaling in Rats with Chronic Fatigue Syndrome].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST 36) on mitochondrial oxidative stress of skeletal muscle in rats with chronic fatigue syndrome (CFS) based on adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/ peroxlsome proliferator-activated receptor-γ coactivator-1 α (PGC-1 α) signaling, in order to reveal its mechanism underlying improvement of CFS. METHODS: Forty SD rats were randomly divided into normal control, CFS model, EA-Zusanli (ST 36) and EA-non-acupoint groups (n=10 rats in each group). The CFS model was established by forced exhausted load-bearing swimming (twice daily), chronic constraint (1 h) and sleep deprivation (20 h/day) for 14 days. Following modeling, EA (2 Hz/100 Hz, 2 V) was applied to bilateral Zusanli (ST 36) or non-acupoint (about 10-15 mm superior to the bilateral Iliac creast and about 20 mm lateral to the posterior median line) for 20 min, once a day for 10 days. The expression levels of ATP synthase, AMPK, phosphorylated (p)-AMPK, silent mating type information regulation 2 homolog-1 (SIRT 1) and PGC-1 α proteins, and ATP synthase, SIRT 1 and PGC-1 α mRNAs of the quadriceps femoris muscle were detected by Western blot and fluorescence quantitative PCR, respectively. The rats' grabbing force was detected by using a grabbing-force detector. RESULTS: Compared with the normal group, the grabbing force, and the expression levels of ATP synthase and PGC-1 α proteins and mRNAs were significantly decreased (P<0.05, P<0.01), while the expression of SIRT 1 protein was significantly up-regulated (P<0.05) in the CFS model group. Following EA intervention, the grabbing force and the expression levels of ATP synthase mRNA, SIRT 1 and PGC-1 α proteins and mRNAs, and p-AMPK/AMPK were significantly up-regulated in the EA-Zusanli (ST 36) group (P<0.05, P<0.01). CONCLUSION: EA of ST 36 can raise the grabbing force of CFS rats, which may be related to its effects in up-regulating the expression of ATP synthase mRNA, SIRT 1 and PGC-1 α proteins and mRNAs, and p-AMPK/AMPK to reduce mitochondrial oxidative stress reaction and in increasing ATP synthesis.

[Effects of electroacupuncture at "Zusanli" (ST 36) on expression of mitophagy-related proteins in skeletal muscle in rats with spleen deficiency syndrome].

OBJECTIVE: To observe the expression change of mitophagy-related proteins in skeletal muscle in rats with spleen deficiency syndrome and to explain the partial action mechanism of acupuncture at Zusanli (ST 36) for spleen deficiency syndrome. METHODS: Forty male SD rats, after normal feeding, were randomly divided into a normal group, a spleen deficiency group, a Zusanli group and a non-acupoint group, ten rats in each group. Except the normal group, the three factors modeling method was used for 14 days to establish the model of spleen deficiency syndrome on the other 3 groups. The rats in the Zusanli group were treated with EA at bilateral "Zusanli" (ST 36), while the rats in the non-acupoint group were treated with EA at bilateral non acupoint (dense-sparse wave, frequency of 2 Hz/100 Hz, 20 min per treatment, once a day for 10 days). The rats in the normal group and spleen deficiency group were treated with immobilization for 20 min per day, and no EA was given. The HPLC method was applied to measure the content of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) in skeletal muscle. The Western blotting method was applied to measure the expression of adenosine monophosphate activated protein kinase (AMPK), p-AMPK, ULK1, p-ULK1，LC3-Ⅰand LC3-Ⅱ in skeletal muscle. RESULTS: The ATP content in the spleen deficiency group was significantly lower than that in the normal group (P<0.01); the ATP content in the Zusanli group was significantly higher than that in the spleen deficiency group (P<0.05) but lower than that in the normal group (P<0.05), there was no significant difference between the non-acupoint group and the spleen deficiency group (P>0.05). Compared with the normal group, the AMP/ATP in the spleen deficiency group and the Zusanli group were significantly up-regulated (P<0.01, P<0.05). The differences of p-AMPK/AMPK between the spleen deficiency group and the normal group was not significant (P>0.05). Compared with the normal group and spleen deficiency group, the p-AMPK/AMPK in the Zusanli group was significantly up-regulated (both P<0.05). The p-ULK1/ULK1 and LC3-Ⅱ/LC3-Ⅰin the Zusanli group was higher than those in the normal group and spleen deficiency group (all P<0.01). CONCLUSION: EA at "Zusanli" (ST 36) might activate AMPK and produce stable ULK1/AMPK compound and increase the mitochondrial autophagy, which could regulate spleen-stomach and treat spleen deficiency.

Semen Astragali Complanati- and Rhizoma Cibotii-enhanced bone formation in osteoporosis rats.

BACKGROUND: Growing evidence shows that herb medicines have some anti-osteoporotic effects, the mechanism underlying is unknown. This study aims to investigate the therapeutic effect of Chinese herb supplements on rats that had osteoporosis-like symptom induced by ovariectomy (OVX). METHODS: OVX or sham operations were performed on virgin Wistar rats at three-month old, which were randomly divided into eight groups: sham (sham); OVX control group (OVX); OVX rats with treatments [either diethylstilbestrol (DES) or Semen Astragali Complanati decoction (SACD) or Rhizoma Cibotii decoction (RCD) or Herba Cistanches decoction (HCD) or Semen Allii Tuberosi decoction (SATD)]. Non-surgical rats were served as a normal control (NC). The treatments began 4 weeks after surgery, and lasted for 12 weeks. Bone mass and its turnover were analyzed by histomorphometry. Levels of protein and mRNA of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in osteoblasts (OB) and bone marrow stromal cells (bMSC) were evaluated by immunohistochemistry and in situ hybridization. RESULTS: Compared to OVX control, TBV% in both SACD and RCD groups was increased significantly, while TRS%, TFS%, MAR, and mAR were decreased remarkably in the SACD group, only TRS% decreased dramatically in the RCD group. No significant changes in bone formation were observed in either HCD or SATD groups. OPG levels in both protein and mRNA were reduced consistantly in OB and bMSC from OVX control rats, in contrast, RANKL levels in both protein and mRNA were increased significantly. These effects were substantially reversed by treatments with either DES or SACD or RCD. No significant changes in both OPG and RANKL expression were observed in OB and bMSC from OVX rats treated with SATD and HCD. CONCLUSIONS: Our study showed that SACD and RCD increased bone formation by stimulating OPG expression and downregulating RANKL expression in OB and bMSC. This suggests that SACD and RCD may be developed as alternative anti-osteoporotic agents for therapy of postmenopausal osteoporosis.

Therapeutic Effects of Cortex acanthopanacis Aqueous Extract on Bone Metabolism of Ovariectomized Rats.

The aim of this study was to evaluate effects of aqueous extract from Cortex acanthopanacis (CAE) on osteoporosis rats induced by ovariectomy (OVX) using aqueous extract from Folium Epimedii (FEE) as positive control agent. Three-month-old female rats that underwent OVX were treated with CAE. After 12 weeks, bone mineral density (BMD) and indices of bone histomorphometry of tibia were measured. Levels of protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) in tibia were evaluated. In addition, the serum concentrations of osteocalcin (OC), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), calcitonin (CT), and parathyroid hormone (PTH) were determined. Administration of CAE significantly prevented OVX-induced rats from gain of the body weight. Treatment with CAE increased bone mass remarkably and showed a significant inhibitory effect on bone resorption by downregulating significantly the expression of RANKL in tibia of OVX rats. Meanwhile, treatment of CAE significantly reduced serum level of IL-1ß and increased level of CT in OVX rats. This suggests that CAE has the potential to be used as an alternative therapeutic agent for postmenopausal osteoporosis.

Therapeutic effects of radix dipsaci, pyrola herb, and Cynomorium songaricum on bone metabolism of ovariectomized rats.

BACKGROUND: The objective of this study was to evaluate the effects of herbal medicines, such as Radix Dipsaci (RDD), Pyrola Herb (PHD), and Cynomorium songaricum decoction (CSD), on osteoporotic rats induced by ovariectomy (OVX). METHODS: OVX or sham operations were performed on 69 virgin Wistar rats that were divided into six groups: sham (sham, n = 12), OVX control group (OVX, n = 12), and OVX rats with treatments (diethylstilbestrol, E2, n = 12; RDD, n = 11, PHD, n = 11, and CSD, n = 11). Non-surgical rats served as normal control (NC, n = 12). The treatments began four weeks after surgery and lasted for 12 weeks. Bone mass and bone turnover were analyzed by histomorphometry. Levels of protein expression and mRNA of OPG and RANKL in osteoblasts (OB) and bone marrow stromal cells (bMSC) were evaluated by immunohistochemistry and in situ hybridization. RESULTS: Compared to NC and sham rats, trabecular bone formation was significantly reduced in OVX rats, but restored in E2-treated rats. Treatment with either RDD or PHD enhanced trabecular bone formation remarkably. No significant change of bone formation was observed in CSD-treated rats. OPG expression of protein and mRNA was reduced significantly in OB and bMSC of OVX control rats. RANKL expression of protein and mRNA was increased significantly in OB and bMSC of OVX control rats. These effects were substantially reversed (increased in OPG and decreased in RANKL) by treatment with E2, RDD, or PHD in OB and bMSC of OVX rats. No significant changes in either OPG or RANKL expression were observed in OB and bMSC of OVX rats treated with CSD. CONCLUSIONS: Our study showed that RDD and PHD increased bone formation by stimulating overexpression of OPG and downregulation of RANKL in OB and bMSC. This suggests that RDD and PHD may be used as alternative therapeutic agents for postmenopausal osteoporosis.