Efficacy of Sijunzi decoction on limb weakness in spleen Qi deficiency model rats through adenosine monophosphate-activated protein kinase/unc-51 like autophagy activating kinase 1 signaling.

OBJECTIVE: To investigate the efficacy of Sijunzi decoction () on limb weakness in a rat model of spleen Qi deficiency (SQD), and to study its effect on mitophagy in skeletal muscle through adenosine monophosphate-activated protein kinase (AMPK) / unc-51 like autophagy activating kinase 1 (ULK1) signaling. METHODS: SQD model rats were produced by fasting combined with forced swimming method for 15 d. After model assessment, rats were randomly divided into four groups of 10 [low/middle/high (L/M/H) Sijunzi decoction dose groups and a normal saline (S) group]. Limb holding power (HP) and body mass (BM) were measured after 2 weeks of treatment. Following euthanasia, quadriceps femoris were dissected and myofiber and mitochondrial morphology were observed by transmission electron microscopy (TEM). Mitochondrial membrane potential (MMP), adenosine triphosphatase (ATP) and reactive oxygen species (ROS) levels were determined using colorimetric methods, and immunoblot analysis of Microtubule-associated protein light chain 3 (LC3) and Sequestosome 1 (p62) was performed to monitor mitophagy and AMPK/ULK1 signaling. RESULTS: Compared with control (C) group rats, in the S group, HP was reduced, the myofiber Z line was disordered, mitochondria were scattered, and numerous vacuoles and mitophagy were observed. MMP and ATP levels were reduced, ROS levels were elevated, and LC3B expression, and p-AMPKα (Thr172)/AMPKα, p-ULK1 (Ser555)/ULK1, and p-Raptor (Ser792)/Raptor ratios were increased, while p62 expression and p-mTOR (Ser2448)/mTOR and p-ULK1 (Ser757)/ULK1 ratios were decreased. After treatment, compared with the S group, HP was improved in M and H groups but not in the L group. Mitophagy was reduced in M, H and L groups but the Z line was disordered and vacuolization remained in the L group. ATP levels were elevated in M, H and L groups, and MMPs were elevat-ed in M and H groups but not in the L group. ROS levels were decreased in M, H and L groups, as were LC3B expression and p-Raptor (Ser792)/Raptor ratios, while p62 expression and p-mTOR (Ser2448)/mTOR and p-ULK1 (Ser757)/ULK1 ratios were increased in M and H groups but not in the L group. p-AMPKα (Thr172)/AMPKα and p-ULK1 (Ser555)/ULK1 ratios were decreased in M, H and L groups. CONCLUSIONS: Sijunzi decoction improved HP, possibly by inhibiting mitophagy via suppression of AMPK/ULK1 signaling. This restored mitochondrial morphology and improved oxidative phosphorylation, which contributed to recovery of limb weakness in SQD model rats.

[Effects of acupoint area and non-acupoint area of eye-acupuncture on expressions of VIP and AQP 8 in colonic tissues in rats with D-IBS].

OBJECTIVE: To explore the point specificity of eye-acupuncture and the mechanism of eye-acupuncture on diarrhea-predominant irritable bowel syndrome (D-IBS). METHODS: Forty male Wistar rats of SPF grade were randomly divided into a normal group, a model group, a eye-acupuncture point (AA) group and a non-point (NA) group. The D-IBS rat model was established with the combination methods of the chronic stress and binding limbs. The AA group was treated by acupuncture at "low energizer area", "large intestine area", "liver area" and "spleen area", and the NA group by acupuncture at 3 mm apart from the same points area mentioned above, and the normal group and the model group with no intervention. The rate of feces moisture content was detected. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA of aquaporin 8 (AQP 8) in colon. Protein expressions of the vasoactive intestinal peptide (VIP) and AQP 8 in colon were detected by SABC immunohistochemistry method. RESULTS: Compared with normal group, the rate of feces moisture content at the 18th and 25th days, VIP protein in colon mucosa, myenteric nerve plexus and hypo-mucosa nerve plexus increased significantly (all P < 0.01), and AQP 8 mRNA in colon mucosa decreased significantly in model, AA and NA group (P < 0.05, P < 0.01); AQP 8 protein in colon mucosa decreased significantly in model group and NA group (both P < 0.01). Compared with model group, the rate of feces moisture content at the 25th day and VIP protein in colon mucosa decreased significantly (P < 0.01, P < 0.05), and AQP 8 mRNA and protein increased significantly (P < 0.05, P < 0.01) in AA group. Compared with AA group, the rate of feces moisture content at the 25th day and VIP protein in colon mucosa increased significantly (both P < 0.01), and AQP 8 mRNA and protein decreased significantly (both P < 0.01) in NA group. CONCLUSION: Eye-acupuncture has a good therapeutic effect on D-IBS. It is suggested that one of the mechanism is relate to increase AQP 8 in colon tissue and restrain the expression of VIP. Non-point area of eye-acupuncture has no obviously therapeutic effect and so to illustrate the point specificity of eye-acupuncture.

[Effects of oculo-acupuncture therapy on colonic serotonin reuptake transporter expression in rats with irritable bowel syndrome].

OBJECTIVE: To investigate the effect of oculo-acupuncture therapy (OAT) on the alteration of colonic serotonin reuptake transporter (SERT) expression in rats with irritable bowel syndrome (IBS) so as to explore its underlying mechanism in relieving IBS. METHODS: Thirty male Wistar rats were randomized into control, IBS model and OAT groups (n = 10/group). IBS model was established by repeated stress stimulation [water-intake inhibition (24 h), warm box keeping (40 degrees C,5 min), electric shock stimulation (30 V, 120 s), forced cool water swimming (14 t, 5 min), 24 h fasting, and tail clamping (180 s)] for 18 days. Oculo-acupuncture stimulation was given to "Xiajiao" (Lower Energizer), "Dachang" (Large Intestine), "Gan" (Liver) and "Pi" (Spleen) areas for 20 min by using filiform acupuncture needles (manipulated 1 min once every 10 min). The treatment was conducted once every 12 h for 7 days. Colonic SERT mRNA and protein expression levels were examined by RT-PCR and Western Blot. RESULTS: In comparison with the control group, the expression levels of both colonic SERT mRNA and protein in the model group were down-regulated remarkably (P < 0.05). Compared with the model group, the expression levels of SERT gene and protein in the OAT group were increased remarkably (P < 0.05). CONCLUSION: OAT can increase colonic SERT protein and gene expression in IBS rats, which may play a role in relieving IBS in OAT clinic.

HPLC determination of five polyphenols in rat plasma after intravenous administration of hawthorn leaves extract and its application to pharmacokinetic study.

A simple and specific HPLC-UV method was developed to simultaneously determine five active compounds including vitexin-4"-O-glucoside (VG), vitexin-2"-O-rhamnoside (VR), vitexin (VIT), rutin (RUT) and hyperoside (HP) in rat plasma after intravenous administrating the hawthorn leaves extract (HLE). With baicalin as internal standard (I.S.), sample pretreatment involved a one-step extraction with methanol of 0.2 ml plasma. The HPLC assay was carried out using a Phenomsil C18 analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6:1.5:18.5:74, v/v/v/v). The calibration curves were liner over the range of 2.030-500.5, 0.1513-75.64, 0.2507-12.54, 0.5128-25.64 and 0.4032-20.16 µg/ml for VG, VR, VIT, RUT and HP, respectively. The relative standard deviations (RSD) of the intra- and inter-day precisions for the analysis of the five analytes were between 1.0 and 8.9% with accuracies (relative error) below 8.2% for the analysis of the five analytes. The average extraction recoveries of five analytes were more than 82.67 ± 4.74%. The HPLC method herein described was fully validated and successfully applied to the pharmacokinetic studies after intravenous administration of HLE solution to rats over three doses.

[Effects of eye-acupuncture therapy on serum and colonic SP and VIP contents in rats with irritable bowel syndrome].

OBJECTIVE: To observe the effect of the eye-acupuncture therapy on serum and colonic substance P (SP) and vasoactive intestinal peptide (VIP) contents in rats with irritable bowel syndrome (IBS) so as to explore its underlying mechanism. METHODS: Forty male Wistar rats were equally randomized into control group, IBS model group, eye-acupuncture group and medication (Pinaverium bromide, 7.5 mg/kg, twice daily, intragastric administration) group. IBS model was established by giving the rat with chronic stress stimulation (cold-water swimming, tail clamping, electrical shock, etc.) for 18 days. Eye-acupuncture of Xiajiao (Low Energizer) Area, Pi (Spleen) Area, Gan (Liver) Area and Dachang (Large Intestine) Area was given to the rat 20 min, twice daily for 7 d. Histopathological changes of the colon tissue were displayed by HE staining; and serum and colonic SP and VIP contents were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: No significant difference was found among 4 groups in the histopathological changes of the colon. In comparison with normal control group, both serum and colonic SP and VIP contents in model group increased significantly (P < 0.01), while compared with model group, those in eye-acupuncture and medication groups lowered considerably (P < 0.01). CONCLUSION: Eye-acupuncture can reduce serum and coIonic SP and VIP contents in IBS rats, which may play a role in relieving IBS in eye-acupuncture clinic.