[Effect of mild moxibustion on vascular aging in senescence rats based on local MCP-1/MMP-2/TGF-ß1 pro-inflammatory signal loop].

OBJECTIVE: To observe the effect of mild moxibustion on monocyte chemotaxis protein 1 (MCP-1)/matrix metalloproteinase 2 (MMP-2)/transforming growth factor ß1 (TGF-ß1) pro-inflammatory signal loop in senile rats, so as to explore its mechanisms underlying improving vascular aging (VA). METHODS: Twenty-four male VA SD rats were randomized into senium (VA) control, medication and moxibustion groups, and other 8 young SD rats (aged 2 months) were used as the young control group. The VA model was established by intraperitoneal injection of D-galactose (300 mg·kg-1·d-1) once daily for 4 weeks, and verified by serum total testosterone (TT) and free testosterone (FT) levels. For rats of the moxibustion group, mild moxibustion was applied to bilateral "Shenshu"(BL23) and "Guanyuan"(CV4) for 20 min, once a day, 5 days a week for 8 weeks. Rats of the medication group were treated by intraperitoneal injection of testosterone propionate (7 mg·kg-1·[3 d]-1) once daily for 8 weeks except weekends, and rats of the senium control and young control groups treated by intraperitoneal injection of the same dose of normal saline, once daily for 8 weeks except weekends. The duration of exhausted swimming (DES) before and after the treatment was recorded. H.E. staining and Masson staining were used to observe histopathological changes and collagen fiber content of the thoracic aortic tissue, respectively. The contents of serum TT, FT and angiotensin 2 (Ang Ⅱ) were determined by ELISA. The immunoactivity of aortic MCP-1 was detected by immunohistochemistry, and the expression levels of aortic MCP-1, MMP-2 and TGF-ß1 were detected by Western blot. RESULTS: Compared with the young control group, the levels of DES, serum TT and FT contents were significantly decreased (P<0.01), while those of serum AngⅡ and collagen fiber contents, aortic MCP-1 immunoactivity and MMP-2, TGF-ß1 and MCP-1 protein expression considerably increased in the senium control group (P<0.01). After the interventions, the decreased levels of DES, serum TT and FT contents and the increased levels of serum AngⅡ, collagen fiber contents, aortic MCP-1 immunoactivity and MMP-2, TGF-ß1 and MCP-1 protein expression were reversed in both medication and moxibustion groups (P<0.01, P<0.05). The therapeutic effect of mild moxibustion was significantly superior to that of medication in down-regulating the aortic collagen fiber, serum AngⅡ contents and MCP-1 immunoactivity and protein expression (P<0.05). H.E. staining showed thickened endometrium and disordered arrangement of vascular smooth muscles of the aorta in the senium group, and thinner endometrium and regular and ordered arrangement of aortic vascular smooth muscles in both moxibustion and medication groups. CONCLUSION: Mild moxibustion may improve vascular aging in senescence rats, which is possibly by suppressing vascular MCP-1/MMP-2/TGF-ß1 pro-inflammatory signal loop.

[Electroacupuncture intervention increases testosterone level of aged rats by activating ERK/Nrf2/HO-1 signaling of Leydig cells].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on anti-oxidant function of Leydig cells in aged rats with low testosterone, so as to reveal its underlying mechanism of anti-aging of male reproduction. METHODS: Eighteen 20 months old SD rats were divided into aged control, medication and EA groups(n=6 in each group), and other 6 young male SD rats (2 months of age) were used as the youth control group. The rats of the youth and the aged control groups received subcutaneous injection of 0.9% normal saline (7 mg·kg-1·3 d-1) for 8 weeks, and those of the medication group received abdominal subcutaneous injection of Testosterone Propionate (7 mg·kg-1·3 d-1) for 8 weeks. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Shenshu" (BL23) and "Guanyuan" (CV4) for 15 min, once daily for 8 weeks except the weekends. The levels of serum total testosterone (TT) and free testosterone (FT) were determined by enzyme linked immune sorbent assay (ELISA), the immunoactivity of phosphorylated extracellular signal-regulated protein kinase (p-ERK) was detected by immunohistochemistry, and the expression levels of ERK, p-ERK, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins in the testis tissues were determined by Western blot. RESULTS: Before and after the treatment, the levels of serum TT and FT in the aged control group were significantly lower than those of the youth control group (P<0.01). After the treatment, the serum TT and FT contents in the EA and medication groups were obviously higher than those in the aged control group (P<0.01). Compared with the youth control group, the expression levels of ERK, p-ERK, Nrf2 and HO-1 proteins were significantly down-regulated in the aged control group (P<0.01), while in comparison with the aged control group, the expression levels of ERK, p-ERK, Nrf2 and HO-1 proteins were significantly up-regulated in the medication and EA groups (P<0.01). The therapeutic effect of EA was notably superior to that of medication in up-regulating the immunoactivity of p-ERK, and the expression levels of ERK, p-ERK, Nrf2 and HO-1 proteins (P<0.01). No significant differences were found between the EA and medication groups in the increased levels of serum TT and FT after the intervention (P>0.05). CONCLUSION: EA intervention may increase testosterone level of the aged rats, which may be related to its effects in triggering ERK /Nrf2 /HO-1 signaling (anti-oxidative stress signal pathway) in the testis.

[Effects of Electroacupuncture and Intracerebral Injection of VEGF on Caspase12, Caspase3, and GRP78 Genes in Rats with Cerebral Ischemia-Reperfusion Injury].

OBJECTIVE: To determine the effects of electroacupuncture (EA) and intracerebral injection of vascular endothelial growth factor (VEGF) on caspase12, caspase3, and glucose regulated protein 78 kD (GRP78) genes of rats with cerebral ischemia reperfusion injury. METHODS: 60 SD rats were randomly divided into sham-operation group, model group, EA group and EA+VEGF group with 15 rats in each group. Middle cerebral artery occlusion (MCAO) method was used to establish the model of cerebral ischemia reperfusion injury. Electro-acupuncture intervention was introduced 1 day after the injury in the EA group and EA+VEGF group: 30 minutes each session and once a day for a total of 14 d [acupoint selection: Baihui (GV 20), Quchi(Li 11), Zusanli (ST36)]. The rats in the EA+VEGF group were also injected with 10 µL of VEGF165 (0.025 µg/µL) into the lateral ventricle after the first session of EA. Five rats in each group were sacrificed after obtaining a neurological function score (mNSS) at day 0 (1 d after modeling, before EA intervention), day 7 and day 14, respectively. Nissl staining was used to observe the histomorphology of cerebral infarction areas. Immunohistochemistry was used to CM(155mm]detect GRP78 activity in the ischemic brain tissues. Real-time fluorescence quantitative PCR (real-time PCR) was used to detect the expressions of caspase12, caspase3 and GRP78 mRNA in the ischemic brain tissues. RESULTS: Compared with the sham-operation group, rats in the model group had higher mNSS scores ( P<0.05), showed signs of cerebral infarction (with reduced numbers of and disordered Nissl bodies and unclear structure), increased GRP78 immunopositive cells, increased expression of GRP78 mRNA ( P<0.05), and increased expressions of caspase12 and caspase3 mRNA ( P<0.05). Compared with the model group, EA and EA+VEGF decreased mNSS scores at day 7 and 14 ( P<0.05), showing alleviated signs of cerebral infarction, increased GRP78 immunopositive cells ( P<0.05), increased GRP78 mRNA expression ( P<0.05), and decreased caspase12 and caspase3 mRNA expressions ( P<0.05). The most obvious changes were found in the EA+VEGF group ( P<0.05). No significant changes were observed in the sham-operation group over time ( P<0.05). In comparison, mNSS scores, the signs of cerebral infarction, and the expressions of caspase12 and caspase3 decreased over time in the other groups ( P<0.05), accompanied with increased GRP78 immunopositive cells and the expression of GRP78 gene ( P<0.05). CONCLUSION: Electroacupuncture and intracerebral injection of VEGF promote tissue repair of rats with cerebral ischemic injury, possibly through down-regulating the expressions of caspase12 and caspase3 genes and up-regulating the expression of GRP78 gene. The effect of electroacupuncture in combination with intracerebral injection of VEGF is superior to that of the single use of electroacupuncture.

[Warm needle moxibustion, mild moxibustion and electroacupuncture interventions have an anti-aging effect possibly by regulating hypothalamus-pituitary-testis axis in aged male rats].

OBJECTIVE: To observe the effect of warm needle moxibustion (WNM), mild moxibustion and electroacupuncture (EA) of "Shenshu" (BL23) and "Guanyuan" (CV4) on serum gonadal hormone levels and related gene expression in the hypothalamus and testis in aged rats, so as to explore their underlying mechanisms in anti-aging. METHODS: Forty male SD male rats were randomly divided into aged control, WNM, mild moxibustion, EA and medication groups (n ＝ 8 in each group), and other 8 male young adult SD rats were used as the youth control group. WNM (acupuncture plus moxibustion), mild moxibustion or EA (2 Hz /100 Hz, 1 mA) was separately applied to CV4 and bilateral BL23 for 15 min, once daily, 5 days a week for 8 weeks. Rats of the medication group received subcutaneous injection of testosterone propionate injection (7 mg/kg), once every 3 days, and those of the aged and youth groups received the same dose of 0.9% NaCl solution for 8 weeks. The levels of serum testoste-rone (T) and gonadotropin-releasing hormone (GnRH) were measured by ELISA. The expression of Kisspeptin (KISS-1), G protein-coupled receptors 54 (GPR54), RFamide-related peptide-3 (RFRP-3) and GPR147 mRNAs in the hypothalamus and GnRH-R, GPR54 and GPR147 mRNAs in the testis tissues were detected by quantitative real-time PCR. RESULTS: The levels of serum T and GnRH, and the expressions of KISS-1, GPR54, RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 mRNAs in the testis in the aged rats were significantly lower than those in the youth rats (P<0.05), while the expression level of GPR147 mRNA of the testis tissue was obviously higher in the aged group than that in the youth group (P<0.05). Follo-wing the treatment, the lowered serum T and GnRH contents, the down-regulated expression levels of hypothalamic KISS-1, GPR54, RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs, as well as the up-regulated testicle GPR147 mRNA expression in the aged rats were reversed in the WNM, mild moxibustion and EA groups (not the medication group except serum T) in comparison with the aged control group (P<0.05). The therapeutic effects of WNM, mild moxibustion and EA were significantly superior to those of medication in up-regulating serum GnRH content and expression levels of KISS-1, GPR54, RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R, GPR54 and GPR147 mRNAs in the testis (P<0.05). No significant differences were found among the WNM, mild moxibustion and EA groups in serum T and GnRH contents, and the expression levels of KISS-1, GPR54, RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 and GPR147 mRNAs in the testis (P>0.05)．. CONCLUSION: The WNM, mild moxibustion and EA therapies have an anti-aging effect by increasing serum T and GnRH levels in aged rats, which is possibly related to their effects in up-regulating the expression of hypothalamic KISS-1, GPR54, RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs, as well as down-regulating testicle GPR147 mRNA expression. The therapeutic effect of WNM, mild moxibustion and EA is evidently superior to that of medication.

[Electroacupuncture Combined with Intracerebral Injection of VEGF Improves Neurological Dysfunction Possibly by Down-regulating Expression of Endoplasmic Reticulum Stress Related Proteins ATF 6, etc. in Cerebral Ischemia-reperfusion Injury Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) and EA combined with intracerebral injection of vascular endothelial growth factor (VEGF) on endoplasmic reticulum stress (ERS) related proteins and genes as activating transcription factor (ATF 6), inositol requiring enzyme-1 (IRE 1), CCAAT/enhancer binding protein homologous protein (CHOP), X box-binding protein-1 (XBP 1) of cerebral ischemia reperfusion injury (CIRI) rats, so as to study its repair effect for CIRI. METHODS: Forty male SD rats were equally and randomly divided into 5 groups: sham operation, model, EA, VEGF and EA+VEGF groups (n=8). The CIRI model was established by occlusion of the middle cerebral artery (MCAO) with thread embolism method. For rats of the sham operation group, the right common carotid artery was isolated without MCAO. EA (2 Hz/100 Hz, 1-3 mA) was applied to "Baihui" (GV 20), left "Quchi" (LI 11) and left "Zusanli" (ST 36) for 30 min, once a day for 14 days. For rats of the VEGF and EA+VEGF groups, 10 µL VEGF (0.025 µg/µL) was injected into the lateral ventricle 24 h after successful modeling. The rats' neurological function was assessed by using the modified neurological severity score (mNSS), and the histopathological changes of cerebral tissue were observed by Nissl staining method. The expression levels of ERS related proteins and genes ATF 6, IRE 1, XBP 1 and CHOP were determined by western blot (WB) and fluorescent quantitative PCR, separately. RESULTS: After modeling, the level of mNSS was significantly higher in the model group than in the sham operation group (P<0.05), and the number of Nissl bodies was markedly lower in the model group than in the sham operation group (P<0.05). Following the treatment, the mNSS was significantly lower in the EA, VEGF and EA+VEGF groups than in the model group (P<0.05), and the numbers of Nissl bodies were obviously higher in the EA, VEGF and EA+VEGF groups than in the model group (P<0.05), suggesting an improvement of neurological dysfunction and a repair of the injured cerebral tissue after the treatment. The levels of CIRI-induced increase of mNSS and CIRI-induced decrease of the number of Nissl bodies in the EA+VEGF group were respectively remarkably lower or higher than those of the simple EA and simple VEGF groups (P<0.05). WB and PCR showed that the expression levels of ATF 6, IRE 1, XBP1 and CHOP proteins and genes were notably higher in the model group than in the sham operation group (P<0.05), and considerably lower in the EA, VEGF and EA+VEGF groups than in the model group (P<0.05). Comparison among the three treatment groups showed that after the treatment, the expression levels of ATF 6, IRE 1, XBP1 and CHOP proteins and genes were obviously lower in the EA+VEGF group than in the EA and VEGF groups (P<0.05). CONCLUSION: EA and EA plus intracerebral microinjection of VEGF can improve neurological function and promote cerebral tissue repair in CIRI rats, which is associated with their effects in down-regulating the expression of ERS related proteins ATF 6, IRE 1, XBP1 and CHOP. The effect of EA+VEGF is superior to that of simple EA and simple VEGF.

[Electroacupuncture Intervention Improves Physical Power of Aged Rats with Low Testosterone by Reducing Chronic Inflammatory Response of Leydig Cells].

OBJECTIVE: To assess the effect of electroacupuncture (EA) on chronic inflammatory response of Leydig cells in aged rats with low testosterone, so as to investigate its underlying mechanism of anti-male reproductive aging. METHODS: Twenty-four 20 months old SD rats were randomly divided into EA, medication and aged control groups (n＝ 8 in each), and other 8 young SD rats (2 months of age) were used as the youth control group. EA (2 Hz/100 Hz, 1 mA) was applied to "Guanyuan"(CV 4) and bilate-ral "Shenshu"(BL 23) for 15 min, once daily for 8 weeks except the weekends. The medication group received abdominal subcutaneous injection of testosterone propionate (7 mg• kg－1• 3 d－1) for 8 weeks. The aged control group and the youth control group received subcutaneous injection of 0.9% normal saline, with the same dose and same treatment frequency as those of the medication group. The rats' physical power was assessed according to the exhausted swimming duration, and the levels of serum total testosterone (TT) and free testosterone(FT) were determined by ELISA. The pathological changes of the testis tissue were detected by using H．E.staining, and the immunoactivity of cyclooxygenase-2 (COX-2) in Leydig cells was detected by immunohisto-chemistry. The expression levels of nuclear factor-κB p 65 (NF-κB p 65), COX-2, interleukin-1 ß (IL-1 ß) and tumor necrosis factor-α (TNF-α) proteins in the testis tissues were determined by Western blot. RESULTS: Before and after treatment, the exhaustive swimming duration and the levels of serum TT and FT in the aged control group were significantly lower than those of the youth control group (P < 0.01). After the treatment, the exhaustive swimming duration and serum TT and FT in the EA and medication groups were notably higher than those in the aged control group (P<0.01). HE staining showed that the incompleteness of basement membrane of spermatogenic tubules, reduction of spermatogenic cells and supporting cells and irregularity of Leydig cells in the testis tissue of the aged rats were relatively milder after EA intervention. Compared with the youth control group, the expression levels of NF-κB p 65 and COX-2, IL-1 ß and TNF-α in the testicular tissue were significantly higher in the aged control group (P<0.01),while in compared with the aged control group, the expression levels of NF-κB p 65, COX-2, IL-1 ß and TNF-α proteins were significantly down-regulated in the EA group (P<0.01). CONCLUSION: EA intervention can improve the physical power of the aged rats with low testosterone, which may be related to its effects in up-regulating TT and FT levels, and in reducing chronic inflammatory response in the testis tissue.