OBJECTIVE: To observe the effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique on the relative protein expression of the signaling pathway of nucleotide-binding oligomerization domain-containing protein 1 (NOD1)/ receptor-interacting protein 2 (RIP2)/nuclear factor kappa-B (NF-κB) and the expression of proinflammatory cytokines in the rats with primary dysmenorrhea (PD), so as to explore the underlying mechanism of this acupuncture technique for pain alleviation in PD. METHODS: Thirty female SD rats of SPF grade with normal estrous cycle were randomized into a blank group, a model group and an acupuncture group, 10 rats in each one. Using the intraperitoneal injection with estradiol benzoate combined with oxytocin, PD model was prepared in the model group and the acupuncture group. In the acupuncture group, during model preparation, the intervention with "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique was delivered simultaneously, 20 min each time, once daily for consecutive 10 days. On day 11, within 30 min after the intraperitoneal injection with oxytocin, the writhing reaction (latency, frequency and score) was recorded; the morphology of uterine tissue was observed with HE staining, the contents of prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), interleukin (IL)-1β, IL-18, cyclooxygenase-2 (COX-2), and tumor necrosis factor-α(TNF-α) in the serum were detected using ELISA method; the relative protein expression of NOD1, RIP2, NF-κB p65, phosphorylation-NF-κB p65 (p-NF-κB p65) was detected in the uterine tissue using Western blot method; and the mRNA expression of NOD1, RIP2 and NF-κB p65 was detected with the quantitative real-time PCR employed. RESULTS: Compared with the blank group, in the model group, the writhing latency was prolonged (P<0.01), the writhing frequency and score increased (P<0.01) in the rats; the endometrial epithelial cells showed massive degeneration and necrosis, with severe endometrial edema and widespread shedding, combined with neutrophil infiltration; the serum PGE₂ content was dropped (P<0.01), while those of PGF_2α, IL-1β, IL-18, COX-2, and TNF-α elevated (P<0.01); the protein expression of NOD1, RIP2, NF-κB p65 and p-NF-κB p65, and the mRNA expression of NOD1, RIP2 and NF-κB p65 in uterine tissue increased (P<0.01). In comparison with the model group, in the acupuncture group, the writhing latency was prolonged (P<0.01), the writhing frequency and score were reduced (P<0.01) in the rats; there was less degeneration and necrosis of endometrial epithelial cells, with mild endometrial edema and very little neutrophil infiltration; the serum PGE₂ content increased (P<0.01), while those of PGF_2α, IL-1β, IL-18, COX-2, and TNF-α decreased (P<0.01); the protein expression of NOD1, RIP2, NF-κB p65 and p-NF-κB p65 and the mRNA expression of NOD1, RIP2 and NF-κB p65 in uterine tissue were dropped (P<0.05, P<0.01). CONCLUSION "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique can alleviate the pain symptom of PD rats, and its action mechanism may be related to inhibiting the active expression of NOD1/RIP2/NF-κB signaling pathway in the uterine tissue, thereby reducing the inflammatory response.
Animals ; Female ; Rats, Sprague-Dawley ; Rats ; Signal Transduction ; Dysmenorrhea/metabolism* ; NF-kappa B/metabolism* ; Acupuncture Points ; Humans ; Acupuncture Analgesia ; Nod1 Signaling Adaptor Protein/metabolism* ; Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism* ; Acupuncture Therapy

This study aimed to investigate the therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea(PD) and explore the underlying mechanism in conjunction with untargeted metabolomics. Forty adult female rats were randomly divi-ded into a normal group, a model control group, ibuprofen(0.12 g·kg~(-1)) group, and high-and low-dose Leonuri Herba aqueous decoction(5 and 2.5 g·kg~(-1)) groups, with eight rats in each group. The PD rat model was prepared using intramuscular injection of estradiol benzoate combined with intraperitoneal injection of pitocin. Drugs were administered by gavage from the 4th day of modeling for 7 d. After the last administration, pitocin was injected intraperitoneally, and the writhing latency and writhing times within 30 min were recorded. The uterine and ovarian coefficients were determined. Estradiol(E_2), progesterone(Prog), oxytocin(OT), cyclooxyge-nase 2(COX-2), prostaglandin E_2(PGE_2), prostaglandin F_(2α)(PGF_(2α)), and Ca~(2+) levels in uterine tissues were measured by ELISA and biochemical kits. Morphological changes in uterine and ovarian tissues were observed by hematoxylin-eosin(HE) staining. The protein expression of oxytocin receptor(OTR), prostaglandin E_2 receptor 3(EP3), and estrogen receptor alpha(ERα) in uterine tissues was detected by immunohistochemistry. The mRNA expression of OTR, PGE_2 receptors 1-4(EP1, EP2, EP3, and EP4), and PGF_(2α) receptor(FP) in uterine tissues was detected by quantitative real-time PCR. Untargeted metabolomics analysis was performed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-QTOF-MS) technology to screen potential biomarkers and enrich metabolic pathways. The results showed that Leonuri Herba was able to significantly reduce the writhing times in PD rats(P<0.05 or P<0.01), significantly reduce the uterine and ovarian coefficients(P<0.01), and improve their histomorphology. After treatment with Leonuri Herba, PGE_2 content was significantly increased(P<0.05), COX-2, PGF_(2α) and Ca~(2+) content, and PGF_(2α)/PGE_2 was significantly decreased(P<0.05 or P<0.01), and OT content was decreased, while E_2 and Prog content tended to further increase in uterine tissues of PD rats. Correspondingly, OTR and EP3 protein expression was significantly downregulated(P<0.05 or P<0.01) and ERα protein expression was upregulated(P<0.05) in uterine tissues. The mRNA expression of FP and EP4 in uterine tissues was significantly downregulated(P<0.01), and the mRNA expression of EP1, EP3, and OTR showed a decreasing trend. The untargeted metabolomics results showed that 10 differential metabolites were restored in the plasma of PD rats after Leonuri Herba treatment. The results indicate that Leonuri Herba is effective in the prevention and treatment of PD, and the underlying mechanism may be attributed to the regulation of PGs synthesis and corresponding receptor binding.
Humans ; Rats ; Female ; Animals ; Estrogen Receptor alpha ; Oxytocin ; Dysmenorrhea/metabolism* ; Cyclooxygenase 2 ; Dinoprostone ; RNA, Messenger/metabolism* ; Dinoprost

This study aims to decipher the mechanism underlying the effect of Shaofu Zhuyu Decoction on endometriosis(EMT)-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis based on mitogen-and stress-activated protein kinase 1/2(MSK1/2).We employed a random number table to randomly assign SPF female non-pregnant rats into the sham group, and treated the rest rats with autologous transplantation+refrigerator freezing for the modeling of the syndrome of cold coagulation and blood stasis.The modeled rats were then randomly assigned into the control group and high-, medium-and low-dose Shaofu Zhuyu Decoction groups.The rats in the low-, medium-, and high-dose decoction groups were respectively administrated with 9, 4.5, and 2.3 g·kg~(-1) decoction through gavage once a day for 2 consecutive weeks, and those in the control group were administrated with 0.24 mg·kg~(-1) gestrinone through gavage once every 3 days for 2 weeks.After that, the size of ectopic focus in each rat was measured via laparotomy.Enzyme-linked immunosorbent assay(ELISA) was adopted to determine the expression of interleukin(IL)-6, IL-10, prostaglandin E2(PGE2), tumor necrosis factor-α(TNF-α).Western blot was employed to determine the protein levels of MSK1/2 and dual-specificity phosphatase 1(DUSP1) and real-time quantitative polymerase chain reaction(RT-PCR) to determine the mRNA levels of the two genes in rat eutopic endometrial tissue.Compared with the sham group, the model group showed increased levels of IL-6, PGE2, and TNF-α while decrease level of IL-10 in the serum(P<0.01).Compared with the model group, the high-and medium-dose decoction groups and the gestrinone group had declined levels of IL-6, PGE2, and TNF-α while risen level of IL-10 in the serum(P<0.01).The model group had lower protein levels and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the sham group(P<0.01). The high-and medium-dose decoction groups and the gestrinone group had higher protein and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the model group(P<0.01).The results indicated that Shaofu Zhuyu Decoction can regulate the abnormal expression of pro-inflammatory cytokines TNF-α, IL-6, and PGE2 and anti-inflammatory cytokines IL-10 and DUSP1 via MSK1/2 to alleviate EMT-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis.
Animals ; Female ; Rats ; Anti-Inflammatory Agents/therapeutic use* ; Cytokines ; Dinoprostone ; Drugs, Chinese Herbal/therapeutic use* ; Dual-Specificity Phosphatases ; Dysmenorrhea/genetics* ; Endometriosis/genetics* ; Gestrinone/therapeutic use* ; Interleukin-10 ; Interleukin-6 ; Mitogen-Activated Protein Kinase 8/therapeutic use* ; Mitogens/therapeutic use* ; RNA, Messenger ; Tumor Necrosis Factor-alpha/metabolism*

BACKGROUND: Nicotine is a major toxic component of tobacco smoke and has been recognized as a risk factor to induce oxidative tissue damage, which is a precursor to cardiovascular diseases, lung-related diseases, and cancers. Peaches (Prunus persica) have been used for the treatment of degenerative disorders, such as hypermenorrhea, dysmenorrhea, and infertility in Asian countries. In this study, we investigated the effects of white-fleshed peach on the excretion of nicotine metabolites and 1-hydroxypyrene in smokers and chronic nicotine-induced tissue damages in mice. METHODS: The concentrations of cotinine and 1-hydroxypyrene were measured in urine of smokers before or after intake of white-fleshed peaches. In addition, ICR mice were injected with nicotine (5 mg/kg body weight) and then orally administered with white-fleshed peach extracts (WFPE) (250 or 500 mg/kg body weight) for 36 days. The oxidative stress parameters and the activities of antioxidant enzymes were measured in liver and kidney tissues. Also, histological changes and nitrotyrosine expression were assessed. RESULTS: Intake of white-fleshed peaches increased the urinary concentration of nicotine metabolites and 1-hydroxypyrene in 91.67% and 83.33% of smokers, respectively. WFPE decreased the malondialdehyde levels and recovered the activities of antioxidant enzymes in nicotine-injected mice. In addition, WFPE inhibited nitrotyrosine expression and inflammatory responses in the liver, kidney, and lung tissues of nicotine-treated mice. CONCLUSIONS: White-fleshed peaches may increase the metabolism of toxic components in tobacco smoke in smokers and protect normal tissues against nicotine toxicity in mice. Therefore, supplementation of white-fleshed peaches might be beneficial to smokers.
Animals ; Asian Continental Ancestry Group ; Cardiovascular Diseases ; Cotinine ; Dysmenorrhea ; Female ; Humans ; Infertility ; Kidney ; Liver ; Lung ; Malondialdehyde ; Menorrhagia ; Metabolism ; Mice ; Mice, Inbred ICR ; Nicotine ; Oxidative Stress ; Prunus persica* ; Risk Factors ; Smoke ; Tobacco

To clarify the active components in Guizhi Fuling capsule in treatment of intrinsic dysmenorrhea, pelvic inflammation and hysteromyoma, main components were gradually knocked out from the capsules, the effects of knockout capsules on uterine contraction, TNF-α secretion, murine splenocytes (SPL) and hysteromyoma cells proliferation were evaluated, respectively. The inhibition of capsules on uterine contraction was weakened by gradient knockout of paeoniflorin, paeonol, and amygdalin. The suppression of capsulte on TNF-α secretion was reduced by gradient knockout of gallic acid, cinnamaldehyde, pentagalloylglucose, and pachyman. The promotion of SPL cells proliferation was reversed by gradient knockout of gallic acid, paeoniflorin, cinnamaldehyde, quercetin, and pachyman. The depression of capsules on hysteromyoma cells proliferation was attenuated by gradient knockout of paeoniflorin, paeonol, pentagalloylglucose, and albiflorin. In conclusion, the compounds mentioned-above could be the key active basis of Guizhi Fuling capsule in treatment of intrinsic dysmenorrhea, pelvic inflammation and hysteromyoma.
Animals ; Capsules ; administration & dosage ; chemistry ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Dysmenorrhea ; drug therapy ; metabolism ; physiopathology ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Molecular Imprinting ; methods ; Tumor Necrosis Factor-alpha ; metabolism

In this study, the active components and potential molecular .mechanism of Guizhi Fuling formula in treatment on dysmenorrhea, pelvic inflammation, and hysteromyoma were investigated using network pharmacological methods. Sterols and pentacyclic triterpenes, with high moleculal network degree, revealed promising effects on anti-inflammatory, analgesic, anti-tumor, and immune-regulation, according to D-T network analysis. On the other hand, the targets with high degree were involved in inflammatory, coagulation, angiopoiesis, smooth muscle contraction, and cell reproduction, which showed the novel function in anti-dysmenorrhea, pelvic inflammation, and hysteromyoma. Furthermore, the formula was indicated to play a key role in smooth muscle proliferation, inhibition of new vessels, circulation improvement, reduction of hormone secretion, alleviation of smooth muscle, block of arachidonic acid metabolism, and inflammation in uterus. Thus, the main mechanism of Guizhi Fuling formula was summarized. In conclusion, Guizhi Fuling formula was proven to alleviated dysmenorrhea, pelvic inflammation, and hysteromyoma by acting on multiple targets through several bioactive compounds, regulating 21 biological pathways.
Drugs, Chinese Herbal ; therapeutic use ; Dysmenorrhea ; drug therapy ; genetics ; metabolism ; Female ; Gene Regulatory Networks ; drug effects ; Humans ; Leiomyoma ; drug therapy ; genetics ; metabolism ; Pelvic Inflammatory Disease ; drug therapy ; genetics ; metabolism

In 2012, the preparation process and quality standard for Guizhi Fuling capsule were improved. To compare the effects and differences of capsules before (2011) and after(2012-2014) the improvement, evaluation models for intrinsic dysmenorrhea, pelvic inflammation and hysteromyoma were applied in rats. Models were induced by oxytocin, liqiud bacteria mixture and estrogen loading, respectively. The capsules (12 batchs/year, 48 bathcs in all), sampled randomly in 2011-2014, the effects were assessed using the three models. In anti-dysmenorrhea models, remarked reduction of writhing frequency, ET-1 and PGF2α content in uterus could be detected, as well as extension of writhing latency. In pelvic inflammation rats, depression of TNF-α and raise of IL-2 were induced by earh batch of capsules. In hysteromyoma model, uterine weight and smooth muscle proliferation, including E2 and P level in plasma, were lowered obviously by all batchs of capsules. Secondly, Guizhi Fuling capsules produced in 2012-2014 revealed better effectiveness than the ones manufactured in 2011. Moreover, pharmacodynamics indexes of the samples made in 2011 differed significantly between groups, which could not be observed in the ones ot 2012-2014. After tne preparation process and quality standard improvement, the effectiveness and homogeneity of Guizhi Fuling capsules were enhanced.
Animals ; Capsules ; administration & dosage ; chemistry ; standards ; Depression ; drug therapy ; genetics ; metabolism ; Dinoprost ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; standards ; Dysmenorrhea ; drug therapy ; genetics ; metabolism ; Female ; Humans ; Interleukin-2 ; genetics ; metabolism ; Pelvic Inflammatory Disease ; drug therapy ; genetics ; metabolism ; Quality Improvement ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo explore the molecular mechanisms of main active ingredients of Siwu decoction analogous formulae for treating primary dysmenorrhea of gynecology blood stasis syndrome by network pharmacology study, and to investigate the correlations between multi-compounds, multi targets and multi pathways.

METHODMajor active compounds from Siwu decoction analogous formulae, including ligustilide, butylidene phthalide, senkyunolide, ferulic acid, gallic acid, peoniflorin, jioglutin A, catalpol, transanethole, zingiberone, commiphoric acid, eugenol, isorhamnetin-3-O-neohesperidoside, wulingzhic acid, alpha-cyperone, cyperene, costunolide, costic acid, tetrahydropalmatine, protopine, amygdalin, 24-methylene cycloartanol, oleic acid, linoleic acid, 3-p-coumaroylquinic acid, hydroxysafflor yellow A, coptisine, berberine, jatrorrhizine, baicalein, baicalin, wogonin were collected to build component-protein networks based on PharmMapper database. The targets information access was used to construct and visualize components-targets-pathways network model using the kyoto encyclopedia of genes and genomes (KEGG) pathway database and Cytoscape software.

RESULT AND CONCLUSIONSerine threonine protein kinases play an important role in the process of cells. They were potential targets in the effect of Siwu decoction analogous formulae. The effect of main active ingredients involved 51 the pathway. Besides the same ones, Shaofu Zhuyu decoction had more effect on lipid metabolism, Xiangfu Siwu decoction on amino acid metabolism pathways, Taohong Siwu decoction on carbohydrate metabolism, while, Qinlian Siwu decoction on ErbB, VEGF signal transduction pathway. Siwu decoction and its derived formulae not only had common targets and pathways, but also had their own emphasis. This reflected the formulae effect mode of multi-ingredients, multi-targets and multi-pathways. It may provide clues to deeper study of molecular mechanism of Siwu decoction analogous formulae action.

Drugs, Chinese Herbal ; chemistry ; pharmacology ; Dysmenorrhea ; drug therapy ; genetics ; metabolism ; Female ; Gynecology ; Humans ; Medicine, Chinese Traditional ; methods ; Signal Transduction ; drug effects ; genetics

OBJECTIVETo explore the efficacy and the reproductive endocrinal mechanism of herbal-partitioned moxibustion in the treatment of primary dysmenorrhea.

METHODSOne hundred and seventy-one cases of primary dysmenorrhea were randomized into an herbal-partitioned moxibustion group (group A), an starch-partitioned moxibustion group (group B) and an acupuncture group (group C), 57 cases in each one. In the group A, moxibustion isolated with herbal medicine was applied to Shenque (CV 8). In the group B, moxibustion isolated with starch was used at Shenque (CV 8). In the group C, acupuncture was given at Sanyinjiao (SP 6). The changes of estradiol (E2), progesterone (P) and prostaglandin levels (PGF2alpha) were observed before and after treatment, and the therapeutic effects were compared among the 3 groups.

RESULTSThe therapeutic effect in the group A was better than those in the other two groups [compared the cured rate: 89.8% (44/49) vs 60.0% (30/50), 60.4% (32/53), both P < 0.05]. In the group A, E2 level [(110.99 +/- 12.90) pg/mL vs (83.94 +/- 8.91) pg/mL, P < 0.05] and PGF2alpha level [(24.58 +/- 3.01) pg/mL vs (14.34 +/- 1.48) pg/mL, P < 0.01] were decreased and P level was increased [(4.65 +/- 0.68) ng/mL vs (6.68 +/- 0.95) pg/mL, P < 0.05]. In the group B and C, PGF2alpha level were reduced. Concerning to the regulating of E2 and PGF2alpha levels, the results in the group A were better than those in the group B and C [(-30.16 +/- 10.20) pg/mL vs (10.79 +/- 15.01) pg/mL, (22.81 +/- 12.22) pg/mL; (-13.10 +/- 2.40) pg/mL vs (-6.52 +/- 1.88) pg/mL, (-3.14 +/- 1.19) pg/mL, (see text) P < 0.05]. Concerning to the regulation of P level, the results in the group A and B were better than that in the group C (all P < 0.05).

CONCLUSIONThe herbal-partitioned moxibustion achieves the significant efficacy on primary dysmenorrhea, which could be related to regulating the reproductive endocrinal level. It decreases E2 and PGF2alpha levels and increases P level.

Acupuncture Points ; Adult ; Dinoprost ; metabolism ; Dysmenorrhea ; metabolism ; therapy ; Estradiol ; metabolism ; Female ; Humans ; Moxibustion ; Progesterone ; metabolism ; Young Adult

OBJECTIVETo compare the effects of instant electroacupuncture (EA) at the different acupoints on IP3 in the uterus tissue of dysmenorrhea model rats so as to investigate the specificity of acupoints.

METHODSFifty female SD rats were randomly divided into a saline group, a model group, a Sanyinjiao (SF 6) group, a Xuehai (SP 10) group and a Hegu (LI 4) group, 10 rats in each group. The rats were given subcutaneous injection of Estradiol Beozoate injection for 10 consecutive days except those in the saline group, and intraperitoneal injection of 2U Oxytocin at 1 h after the last administration to create the dysmenorrhea rats model, and the saline group was given the same dose of saline every day. On the 10th day the rats in each EA group were given EA 20 min, and the rats in the saline group and model group were bound 20 min, and the writhing response was observed at the same time. The uterine IP3 contents were detected with enzyme-linked immunosorbent assay method.

RESULTS(1) Compared with (0.311+/- 0.253) in the saline group, the writhing scores per minute of (5.867 +/- 3.442) in the model group and (2.311 +/- 0.957) in the Xuehai (SP 10) group were both increased significantly (P < 0.01, P < 0.05), and (1.833 +/- 1.355) in the Sanyinjiao (SP 6) group and (0.743 +/- 0.306) in the Hegu (LI 4) group showed no significant differences (P > 0.05). Compared with that in the model group, the writhing scores per minute decreased significantly (all P < 0.01) in all the EA groups, with no significant differences among all the EA groups (all P > 0.05). (2) Compared with (2.698 +/- 1.491) ng/mg in the saline group, IP3 contents of the uterus of (0.813 +/- 0.899) ng/mg in the model group, (1.740 +/- 0.375) ng/mg in the Sanyinjiao (SP 6) group and (0.692 +/- 0.212) ng/mg in the Hegu (LI 4) group were all lower significantly (P < 0.05, P < 0.01), and (0.743+/- 0.306) ng/mg in the Xuehai (SP 10) group showed no significant differences (P > 0.05). Compared with that in the model group, IP3 content of the uterus in the Hegu (LI 4) group showed no significant difference (P > 0.05), and those in the Sanyinjiao (SP 6) group and in the Xuehai (SP 10) group increased significantly (both P < 0 05), which were significantly higher than that in the Hegu (II 4) group (P < 0.05, P < 0.01).

CONCLUSIONThere are no significant differences among the instant EA groups in improving the dysmenorrhea symptoms, but there is obvious specificity of acupoint effects in the regulation of IP3. Electroacupuncture at "Sanyinjiao (SP 6) " Xuehai (SP 10)" has more marked effect in dysmenorrhea model rats.

Acupuncture Points ; Animals ; Disease Models, Animal ; Dysmenorrhea ; metabolism ; therapy ; Electroacupuncture ; Female ; Humans ; Inositol 1,4,5-Trisphosphate ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Uterus ; metabolism