Targeting the Gut-Kidney Axis in Diarrhea with Kidney-Yang Deficiency Syndrome: The Role of Sishen Pills in Regulating TMAO-Mediated Inflammatory Response.

BACKGROUND Sishen Pills (SSPs) are commonly used to treat diarrhea with kidney-yang deficiency syndrome. Trimethylamine-N-oxide (TMAO) is produced through the metabolism of gut microbiota and can participate in diarrhea in kidney-yang deficiency syndrome by mediating the "gut-kidney axis" to transmit inflammatory factors. This study combined network pharmacology with animal experiments to explore whether SSPs can treat diarrhea with kidney-yang deficiency syndrome by affecting the interaction between TMAO and gut microbiota. MATERIAL AND METHODS A mouse model of diarrhea with kidney-yang deficiency syndrome was constructed by using adenine and Folium sennae decoction, and SSP decoction was used for treatment. This study utilized network pharmacology to predict the potential mechanisms of SSPs in treating diarrhea with kidney-yang deficiency syndrome. 16S rRNA high-throughput sequencing was used to analyze gut mucosal microbial characteristics. ELISA was used to measure TMAO, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1ß (IL-1ß), and transforming growth factor-ß1 (TGF-ß1) levels. We performed Masson and immunohistochemical (Occludin, ZO-1) staining of kidney and small intestinal tissues. The fluorescein diacetate (FDA) hydrolysis spectrophotometric method was used to assess the microbial activity in contents of the small intestine. RESULTS Network pharmacology analysis revealed that SSPs can modulate 108 target points involved in the development of diarrhea, including IL-1ß and TNF. The experimental results demonstrated that SSP decoction significantly improved the general behavioral profiles of the mice, and also reduced TMAO, NLRP3, IL-1ß, and TGF-ß1 levels (P<0.05). Correlation analysis revealed significant positive correlations between TMAO concentrations and NLRP3, IL-1ß and TGF-ß1 levels (P<0.05). Pathological analysis revealed improvements in renal fibrosis and increased expression of the Occludin and ZO-1 proteins in intestinal tissue. In the SSP group, there was a significant increase in microbial activity (P<0.001). According to the sequencing results, the characteristic bacteria of the SSP and NR groups included Succinatimonas hippei, uncultured Solirubrobacter sp., and Clostridium tyrobutyricum. Furthermore, TMAO, NLRP3, IL-1ß, and TGF-ß1 were significantly positively correlated (P<0.05) with Succinatimonas hippei and Clostridium tyrobutyricum. By modulating Firmicutes, Succinatimonas hippei, and Clostridium tyrobutyricum, SSP decoction lowers TMAO levels to alleviate diarrhea with kidney-yang deficiency syndrome. CONCLUSIONS TMAO likely plays a significant role in the "gut-kidney axis" of diarrhea with kidney-yang deficiency syndrome. By adjusting gut microbiota to reduce the inflammatory response that is transmitted through the "gut-kidney axis" as a result of elevated TMAO levels, SSP decoction can alleviate diarrhea with kidney-yang deficiency syndrome.

Integrated untargeted and targeted testicular metabolomics to reveal the regulated mechanism of Gushudan on the hypothalamic-pituitary-gonadal axis of kidney-yang-deficiency-syndrome rats.

Modern studies have shown that neuroendocrine disorders caused by the dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis are one of the important pathogenetic mechanisms of kidney-yang-deficiency-syndrome (KYDS). The preventive effect of Gushudan on KYDS has been reported, but its regulatory mechanisms on the HPG axis have not been elucidated. In this study, we developed an integrated untargeted and targeted metabolomics analysis strategy to investigate the regulatory mechanism of Gushudan on the HPG axis in rats with KYDS. In untargeted metabolomics, we screened 14 potential biomarkers such as glycine, lysine, and glycerol that were significantly associated with the HPG axis. To explore the effect of changes in the levels of potential biomarkers on KYDS, all of them were quantified in targeted metabolomics. With the quantitative results, correlations between potential biomarkers and testosterone, a functional indicator of the HPG axis, were explored. The results showed that oxidative stress, inflammatory response, and energy depletion, induced by metabolic disorders in rats, were responsible for the decrease in testosterone levels. Gushudan improves metabolic disorders and restores testosterone levels, thus restoring HPG axis dysfunction. This finding elucidates the special metabolic characteristics of KYDS and the therapeutic mechanism of Gushudan from a new perspective.

Integrated UHPLC-MS untargeted metabolomics and gut microbe metabolism pathway-targeted metabolomics to reveal the prevention mechanism of Gushudan on kidney-yang-deficiency-syndrome rats.

Gushudan (GSD) was a traditional Chinese prescription with the remarkable effect of kidney-tonifying and bone-strengthening. However, the potential prevention mechanisms of the GSD on kidney-yang-deficiency-syndrome (KYDS) and its regulation on gut microbe metabolism still need to be further systematically investigated. This study established untargeted urinary metabolomics based on RP/HILIC-UHPLC-Q-Orbitrap HRMS and combined with multivariate statistical analysis to discover differential metabolites and key metabolic pathways. And the gut microbe metabolism pathway-targeted metabolomic based on HILIC-UHPLC-MS/MS was developed and validated to simultaneously determine 15 gut microbe-mediated metabolites in urine samples from the control group (CON), KYDS model group (MOD), GSD-treatment group (GSD) and positive group (POS). The results showed that a total of 36 differential metabolites were discovered in untargeted metabolomics. These differential metabolites included proline, cytosine, butyric acid and nicotinic acid, which were primarily involved in the gut microbe metabolism, amino acid metabolism, energy metabolism and nucleotide metabolism. And GSD played a role in preventing KYDS by regulating these metabolic pathways. The targeted metabolomics found that the levels of 10 gut microbe-mediated metabolites had significant differences in different groups. Among them, compared with the CON group, the levels of lysine, tryptophan, phenylacetylglycine and hippuric acid were increased in the MOD group, while the levels of threonine, leucine, dimethylamine, trimethylamine, succinic acid and butyric acid were decreased, which verified the disorders of gut microbe metabolism in the KYDS rats and GSD had a significant regulatory effect on this disorder. As well as by comparing analysis, it was found that the experimental results were consistent with previous metabolomics and microbiomics of fecal samples. Therefore, this integrated strategy of untargeted and targeted metabolomics not only elucidated the potential prevention mechanism of GSD on KYDS, but also provided a scientific basis for GSD preventing KYDS via the "gut-kidney" axis.

Integrated gas chromatography-mass spectrometry and ultra-high-performance liquid chromatography-mass spectrometry renal metabolomics and lipidomics deciphered the metabolic regulation mechanism of Gushudan on kidney-yang-deficiency-syndrome rats.

Kidney-yang-deficiency-syndrome is a neuroendocrine disease caused by the dysfunction of the adrenal-pituitary-target gland axis. Gushudan is a traditional Chinese medicine prescription with the functions of tonifying the kidney and strengthening bone, and its bone-strengthening effect has been confirmed by previous anti-osteoporosis research. However, its kidney-tonifying mechanism has not been clear so far. In this study, renal metabolomics and lipidomics based on gas chromatography-mass spectrometry and ultra-high-performance liquid chromatography-high resolution mass spectrometry were integrated to find the metabolic disorders in kidney-yang-deficiency-syndrome rats. Protein precipitation and liquid-liquid extraction were used to extract metabolome and lipidome from the kidney. Gushudan regulated abnormal levels of amino acids, lipids, purines, and carbohydrates, such as L-arginine, hypoxanine, stearic acid, and phosphatidylethanolamine (P-18:1/20:4), which had effects on many metabolic pathways, such as glycerophospholipid metabolism, sphingolipid metabolism, glycine, serine and threonine metabolism and purine metabolism, and so forth. By integrating metabolomics and lipidomics, this study comprehensively revealed the abnormal metabolic activities of amino acids, lipids, and nucleotides in kidney-yang-deficiency-syndrome, and the metabolic regulation mechanism of Gushudan in preventing kidney-yang-deficiency-syndrome, as well as the improvement of Gushudan in maintaining renal cell structure, mitochondrial function, and energy supply, which also provided some new evidence and connotation for "kidney-bone" axis.

Research on the effect of Dipsaci Radix before and after salt-processed on kidney yang deficiency syndrome rats and the preliminary mechanism study through the BMP-Smad signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsaci Radix (DR) is the dry root of Dipsacus asper Wall. ex DC. AIM OF THE STUDY: The purpose of this study was to compare the effects of DR on rats before and after salt-processed with kidney yang deficiency syndrome (KYDS), and we selected the BMP-Smad signaling pathway to explore the mechanism of DR. MATERIALS AND METHODS: The model of KYDS was established by subcutaneous injection of hydrocortisone, the crude DR (CDR) and salt-processed DR (SDR) were given the corresponding dose (2 g/kg, 4 g/kg, and 6 g/kg). The organ index and the contents of adrenocorticotropic hormone (ACTH), cortistatin (CORT), thyroid hormone (T4), tumor necrosis factor-alpha (TNF-α), testosterone (T), estradiol (E2), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), Na+-K+-ATPase, and growth hormone (GH) in serum were measured to evaluate the intervention effect of DR on KYDS rats. The expression of Smad 1, Smad 4, Smad 5, Smad 8, and BMP 7 protein in kidney was determined by immunohistochemistry, quantitative PCR (qPCR) and Western blot analysis. The effects of DR on 5 expression factors in the BMP-Smad signaling pathway were studied. Constituents absorbed into blood were identified by UPLC-Q-TOF/MS. RESULTS: The results showed that compared with the model group, the thymus and kidney index, as well as the contents of ACTH, CORT, cAMP, GH, Na+-K+-ATPase, T, T4, and E2 were significantly increased in the CDR and SDR groups, and the contents of cGMP and TNF-α were significantly decreased. Compared with the CDR high dose group, ACTH, Na+-K+-ATPase, T, and T4 were significantly increased in the SDR high dose group. The results of immunohistochemistry, qPCR, and Western blot analysis showed that compared with the model group, the expression levels of Smad 1, Smad 4, Smad 5, Smad 8 and BMP 7 proteins in the kidney of DR groups were significantly increased. And SDR groups tended to be better than CDR groups. 8 constituents migrating to blood were identified. CONCLUSION: This study showed that both CDR and SDR could have a good therapeutic effect on KYDS, and SDR was better than CDR. This study chose the BMP-Smad signaling pathway to study the mechanism of DR in the treatment of KYDS and provided a scientific basis for the processing mechanism of salt-processed.

Twelve-component pharmacokinetic study of rat plasma after oral administration of You-Gui-Wan in osteoporosis rats with kidney-yin deficiency and kidney-yang deficiency.

You-Gui-Wan is a widely used traditional Chinese medicine preparation for the treatment of osteoporosis with kidney-yang deficiency, and is composed of both yang-invigorating and kidney-tonifying herbs, and yin-nourishing and kidney essence-replenishing herbs. Considering that the pharmacokinetics of drugs might differ in different pathological conditions, it is necessary to study the pharmacokinetic characteristics of You-Gui-Wan under different osteoporotic conditions. In this study, the pharmacokinetic behaviors of You-Gui-Wan in osteoporosis rats with kidney-yin and kidney-yang deficiency were compared. The results showed that the absorption, metabolism, and disposition of You-Gui-Wan varied widely in animals with different types of osteoporosis. The active components belonging to the yang-invigorating herbs, such as aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypacoitine, benzoylmesaconine, chlorogenic acid and pinoresinol diglucoside, had a higher uptake and slower elimination in osteoporosis rats with kidney-yang deficiency, which corresponds to the opinion that You-Gui-Wan is used to treat kidney-yang deficiency syndrome, and indicates the scientific nature of Bian-Zheng-Lun-Zhi.

Metabolomics and serum pharmacochemistry revealed the preventive mechanism of Gushudan in kidney-yang-deficiency-syndrome rats.

Kidney-yang-deficiency-syndrome (KYDS) is a metabolic disease caused by neuroendocrine disorder. Gushudan (GSD) is a traditional Chinese medicine prescription with the effect of nourishing kidney and strengthening bones. In this study, the mechanism of preventive effect of GSD on KYDS was explored by integrating metabolomics and serum pharmacochemistry. Reversed-phase/hydrophilic interaction chromatography-ultra-high-performance liquid chromatography-Quadrupole-Orbitrap high-resolution mass spectrometry (RP/HILIC-UHPLC-Q-Orbitrap HRMS)-based serum metabolomics indicated metabolic disturbances of KYDS rats, and 50 potential biomarkers including l-threonine, succinic acid and phytosphingosine were obtained, which were mainly involved in alanine, aspartate and glutamate metabolism, citrate cycle (tricarboxylic acid cycle) and glycerophospholipid metabolism, among others. Serum pharmacochemistry identified 29 prototypical ingredients and 9 metabolites of GSD after administration, such as icaritin and xanthotoxol. The combination of 10 serum migration ingredients in GSD, including icaritin and osthole, with 7 important targets, including AKT serine/threonine kinase 1 (AKT1) and MAPK14, was found to be key for GSD to prevent KYDS in the network pharmacology study. This study provided a new idea for the research of pathogenesis of diseases and the pharmacodynamic mechanism of traditional Chinese medicine.

1H NMR serum metabolomics and its endogenous network pharmacological analysis of Gushudan on kidney-yang-deficiency-syndrome rats.

The pharmacodynamics, 1H NMR metabolomics and endogenous network pharmacology strategy approaches were integrated to investigate the preventive mechanism of Gushudan (GSD) on kidney-yang-deficiency-syndrome (KYDS) rats in this study. Firstly, the KYDS rat model was achieved by hydrocortisone induction, and the efficacy of GSD on KYDS model rats was assessed by the pharmacodynamic indicators. Next, the comprehensive untargeted serum metabolic profile of rats was obtained in 1H NMR metabolomics study, 29 potential biomarkers closely associated with KYDS were identified, which were mainly involved in carbohydrate metabolism, amino acid metabolism and intestinal flora metabolism. In addition, the potential biomarkers-targets-pathways-disease metabolic network was further investigated for deeper understanding the preventive effects of GSD on KYDS rats and its mechanism, which was further obtained for the important targets related to biomarkers and diseases such as NOS3, PTGS2 and CXCL8, and important metabolic pathways such as glyoxylate and dicarboxylate metabolism, arginine and proline metabolism, and microbial metabolism in diverse environments. Finally, compared with our previous anti-osteoporosis study of GSD, it suggested that some similar metabolic pathways, which would provide some scientific reference of the existence of the kidney-bone axis under the traditional Chinese medicine (TCM) theory of "kidney dominates bone".

Metabolomics of Spleen-Yang deficiency syndrome and the therapeutic effect of Fuzi Lizhong pill on regulating endogenous metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Spleen-Yang deficiency (SYD) is one of the primary causes of many digestive diseases, such as ulcerative colitis (UC), and irritable bowel syndrome (IBS), but its endogenous metabolic characteristics are still unclear. Fuzi Lizhong pill (FLZP) is well-known for its powerful capacity for treating SYD; however, its mechanisms require further study. AIM OF THE STUDY: Herein, our present study aimed to investigate the essence of SYD from the perspective of metabolomics, and tried to reveal the anti-SYD action mechanisms of FLZP. MATERIALS AND METHODS: Firstly, the compound factor modeling method with the principle of "indiscipline in diet + excessive fatigue + intragastric administration of Senna water extracts" was used to establish Sprague Dawley (SD) rats as SYD model. Then, the visceral index, motilin (MTL), malonaldehyde (MDA), Interleukin 1 alpha (IL-1α), and Interleukin 6 (IL-6) levels were used to verify the anti-SYD effect of FLZP. In addition, serum samples were analyzed by UPLC-QE/MS metabolomics technique. Finally, the metabolic pathways associated with specific biomarkers were analyzed to research the possible mechanism underlying the action of FLZP. RESULTS: The expression of MTL, MDA, IL-1α, and IL-6 were regulated by FLZP, which suggested that it has relieved diarrhea and gastrointestinal motility disorder caused by SYD and had an anti-peroxidation, anti-inflammatory, and immune regulation effect. A total of 75 metabolites were found to be the potential biomarkers of SYD. Moreover, FLZP regulates 21 metabolites and 10 vital pathways including the tricarboxylic acid (TCA) cycle, sphingolipid metabolism, and histidine metabolism. CONCLUSION: SYD primarily causes disorders of amino acid metabolism, lipid metabolism, carbohydrate metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, and translation. In addition, FLZP regulated carbohydrate, lipid, and amino acid metabolisms, gastrointestinal motility, digestive juice secretion, immune regulation, as well as antioxidant effects. Hence, FLZP had a good therapeutic effect on treatment of SYD. It might be a promising therapeutic agent for the treatment of SYD-related diseases.

The cold-soaking extract of Chinese yam (Dioscorea opposita Thunb.) protects against erectile dysfunction by ameliorating testicular function in hydrocortisone-induced KDS-Yang rats and in oxidatively damaged TM3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Clinical applications and pharmacological research suggest that Dioscorea opposita Thunb. (Chinese yam), a well-known traditional Chinese medicine which has been used for more than 2000 years to nourish kidney-yang and protect the male reproductive system, might be efficacious for the treatment of erectile dysfunction (ED). AIM OF THE STUDY: This study aimed to investigate the active component extract of Chinese yam, determine its effectiveness in hydrocortisone-induced "kidney-yang deficiency syndrome" (KDS-Yang) rats and in oxidatively damaged TM3 cells and explore the underlying mechanism on restoring erectile function. MATERIALS AND METHODS: We clarified the Chinese yam cold-soaking extract (CYCSE) as the main active extract of Chinese yam by a CCK8 assay and further identified its composition. The KDS-Yang rats were induced by intragastric administration of hydrocortisone. After 10 d of CYCSE intervention, cavernous and testis morphology were stained with hematoxylin and eosin. Inducible nitric oxide synthase (iNOS), cyclic guanosine monophosphate (cGMP), testosterone, 8-hydroxy-2-deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay kits. Leydig cells were performed using immunohistochemistry. Reactive oxygen species were measured using a DCFH-DA fluorescent probe, and testicular collagenous fibers were stained with a Masson kit. Detection of testicular apoptosis was performed by a TUNEL assay. Nrf2 and NQO1 mRNA expression levels were measured by qRT-PCR. The protein expression levels of Nrf2, HO-1, TGF-ß1 and SMAD2/3 were analyzed by Western blot. RESULTS: We demonstrated in KDS-Yang rats and oxidatively damaged TM3 cells that CYCSE successfully restored erectile function through ameliorating testicular function. Our data suggested that CYCSE can stimulate the NO/cGMP pathway and restore the cavernous morphology to protect against KDS-Yang-induced ED. It also protected testis morphology, increased Leydig cell proliferation and stimulated testosterone secretion. In the damaged testes, excessive increases in 8-OHdG and inhibition of SOD activity were ameliorated, and the Nrf2/HO-1 signaling pathway was enhanced after treatment with CYCSE, indicating that the antioxidant defense system was activated. These findings were also validated in vitro. Additionally, fibrosis of the testes and TM3 cells was reversed by CYCSE through the TGF-ß1/SMAD2/3 pathway. CONCLUSION: CYCSE has a therapeutic effect on KDS-Yang-induced ED, and the mechanism includes stimulation of testosterone secretion, resistance to oxidative stress and prevention of fibrosis. These findings provide a new scientific verification for the application of Chinese yam in the treatment of KDS-Yang-induced ED.

The Effect of Shen Qi Wan Medicated Serum on NRK-52E Cells Proliferation and Migration by Targeting Aquaporin 1 (AQP1).

BACKGROUND Shen Qi Wan (SQW) as a well-known formula for the amelioration of kidney yang deficiency syndrome (KYDS), and it has been widely employed in traditional Chinese medicine (TCM). This study aimed to investigate the effect and underlying mechanism of SQW medicated serum on proliferation and migration in NRK-52E cells. MATERIAL AND METHODS We employed the real-time cell analysis (RTCA) system to investigate the effect of SQW medicated serum on proliferation and migration in NRK-52E cells. In addition, the migration was further investigated by using a wound-healing assay. The mRNA and protein expression level of aquaporin 1 (AQP1) of NRK-52E cells with SQW medicated serum-treated were quantified by real-time quantitative polymerase chain reaction (q-PCR) and western blot assay, respectively. Furthermore, NRK-52E cells were transfected with lentivirus AQP1-RNAi to assess migratory cell abilities in vitro. RESULTS The migratory abilities of NRK-52E cells were significantly increased after SQW medicated serum treatment (P<0.05), and no significant difference in cell proliferation. In addition, SQW medicated serum was significantly upregulated the mRNA and protein expression level of AQP1 in NRK-52E cells (P<0.05). Additionally, the in vitro metastasis test proved that knockdown of AQP1 suppressed migratory abilities according to RTCA and wound healing test while was reversed by SQW medicated serum (P<0.05). CONCLUSIONS Our study demonstrates that SQW medicated serum effectively promotes the migration of NRK-52E cells by increasing AQP1 expression, and AQP1 may be as a therapeutic target of SQW for renal injury treatment under KYDS.

A Novel Strategy Facilitates Reference Gene Selection by RT-qPCR Analysis in Kidney Yang Deficiency Syndrome Mice Infected with the Influenza A (H1N1) Virus.

In reverse transcription-quantitative polymerase chain reaction (RT-qPCR) studies, endogenous reference genes are routinely used to normalize the expression of target gene studies. In order to precisely evaluate the relative expression of genes in the cells of mice suffering from Kidney Yang Deficiency Syndrome (KYDS) in response to influenza A virus (IAV) H1N1 using RT-qPCR, it is crucial to identify reliable reference genes. In the present study, 15 candidate reference genes (Actb, ß2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhae, 18S rRNA, Rpl13, Ubc, Rpl32, and Ppia) were investigated in lung cells from KYDS mice infected with IAV H1N1. NormFinder, BestKeeper, and GeNorm were used to assess the stability of reference genes. The results were authenticated over extended experimental settings by a group of 10 samples. In the present study, we explored a novel method using dual-gene combinations; the difference in gene expression between the model and normal control groups was statistically analyzed by an independent-samples t-test, and the difference in the mean value between the two groups was compared. A P value > 0.05 and the lowest absolute value of the difference indicated the optimal reference two-gene combination. Four additional host innate immune system-related genes (TLR3, TLR4, TLR7, and RIG-I) were analyzed together with the two treatment datasets to confirm the selected reference genes. Our results indicated that none of these 15 candidate reference genes can be used as reference gene individually for relative quantitative fluorescence PCR analysis; however, the combination of Grcc10 and Ppia, based on the process of calculating the higher P value and lower difference values between groups, was the best choice as a reference gene for the lung tissue samples in KYDS mice infected with IAV. This technique may be applied to promote the selection process of the optimal reference gene in other experiments.

Integrated adrenal and testicular metabolomics revealed the protective effects of Guilingji on the Kidney-Yang deficiency syndrome rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Guilingji (GLJ) is a well-known traditional Chinese medicine (TCM) prescription for the treatment of Kidney-Yang deficiency syndrome (KYDS). AIM OF THE STUDY: This study aimed to address the protective effects of GLJ against KYDS in rats with pharmacodynamic indicators and target tissues (adrenal gland and testis) metabolomics. MATERIALS AND METHODS: The rats were injected intraperitoneally (i.p) hydrocortisone to simulate KYDS and administered orally of GLJ for 30 days. Traditional pharmacodynamic indicators (body weight, behavioral indicators, biochemical parameters and histological examination) were performed to evaluate the efficacy of GLJ. Furthermore, adrenal gland and testis metabolic profiles obtained by UHPLC-Q Exactive Orbitrap-MS coupled with multivariate analysis were conducted to explore the metabolic regulation mechanism of GLJ. RESULTS: After administration of GLJ, the weight, levels of behavioral indicators and biochemical parameters of rats were increased compared with those of the model group, and the abnormalities of morphology in adrenal and testicular tissues were improved. Furthermore, GLJ had recovering effects via the adjustment of vitamins metabolism, which was accompanied by lipids metabolism, amino acid metabolism and nucleotides metabolism. CONCLUSIONS: The study firstly integrated the target tissues metabolic profiles, which were complementary, and GLJ had protective effects on KYDS rats via the regulation of steroid hormone biosynthesis, oxidant-antioxidant balance and energy acquisition.

Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a metabolic disease caused by a neuro-endocrine disorder. The You-gui pill (YGP) is a classic traditional Chinese medicine (TCM) formula for the treatment of KYDS and has been widely used to warm and recuperate KYDS clinically for hundreds of years in China. However, it is unknown whetherthe corresponding targets and metabolic pathways can also be found via using metabonomics based on one platform (e.g., 1H NMR) to study different biological samples of KYDS. At the same time, relevant reports on further molecular verification (e.g., RT-qPCR analysis) of these targets associated with biomarkers and metabolic pathways have not yet, to our knowledge, been seen in KYDS's research. In the present study, a comprehensive strategy integrating systems pharmacology and 1H NMR-based urinary metabonomics analysis was proposed to identify the target proteins and metabolic pathways that YGP acts on KYDS. Thereafter, further validation of target proteins in kidney tissue was performed through quantitative real-time PCR analysis (RT-qPCR). Furthermore, biochemical parameters and histopathological analysis were studied. As a result, seven target proteins (L-serine dehydratase; phosphoenolpyruvate carboxykinase; spermidine synthase; tyrosyl-tRNA synthetase, glutamine synthetase; 3-hydroxyacyl-CoA dehydrogenase; glycine amidinotransferase) in YGP were discovered to play a therapeutic role in KYDS via affecting eight metabolic pathways (glycine, serine and threonine metabolism; butanoate metabolism; TCA cycle, etc.). Importantly, three target proteins (i.e., 3-hydroxyacyl-CoA dehydrogenase; glutamine synthetase; and glycine amidinotransferase) and two metabolic pathways (butanoate metabolism and dicarboxylate metabolism) related to KYDS, to our knowledge, had been newly discovered in our study. The mechanism of action mainly involved energy metabolism, oxidative stress, ammonia metabolism, amino acid metabolism, and fatty acid metabolism. In short, our study demonstrated that targets and metabolic pathways for the treatment of KYDS by YGP can be effectively found via combining with systems pharmacology and urinary metabonomics. In addition to this, common and specific targets and metabolic pathways of KYDS treated by YGP can be found effectively by integration with the analysis of different biological samples (e.g., serum, urine, feces, and tissue). It is; therefore, important that this laid the foundation for deeper mechanism research and drug-targeted therapy of KYDS in future.

Optimization of Extraction Technology of Majun Mupakhi Ela and its Effect on Hydrocortisone-induced Kidney Yang Deficiency in Mice.

We used Box-Behnken design-based (BBD) response surface methodology (RSM) in this research to optimize the extraction process of Traditional medicine Majun Mupakhi Ela (MME) and evaluate its effect on hydrocortisone-induced kidney yang deficiency. Three independent parameters were applied to evaluate the maximum phosphodiesterase type 5 (PDE5) inhibition activity of MME extracts in vitro. The optimal processing conditions (extraction time 2 h, solid-liquid ratio 1:16, extraction once) gave a maximum PDE5 inhibition rate of 84.10%, flavonoid content of 0.49 mg/ml, icariin content of 0.028 mg/ml and targeted extraction yield of 26.50%. In animal experiments, MME extracts significantly increased the adrenal mass index, semen weight index, preputial gland weight index, and penis weight index in mice; in the middle and high dose group, the level of serum testosterone increased by 7664.29% and 14207.14% respectively, compared with the model group, and the level of PDE5 decreased by 67.22% and 74.69% respectively compared with the control group. These results indicate that MME has a significant positive effect on the hypothalamus-pituitary-gonadal axis, improve mating ability and not only has inhibits PDE5 activity but also significantly inhibits the expression of PDE5 in penile tissues, potential to become erectile dysfunction (ED) therapies for the clinical management of patients with kidney yang deficiency.

Er Shen Wan extract alleviates polyuria and regulates AQP 2 and AVPR 2 in a rat model of spleen-kidney Yang deficiency-induced diarrhea.

ETHNOPHARMACOLOGICAL RELEVANCE: Er Shen Wan (ESW), has been empirically used for treating spleen-kidney Yang deficiency (SKYD) syndrome in Traditional Chinese medicine (TCM) for centuries and shows a variety of activities. The medicinal formula is a mixture of two component herbs, Psoraleae Fructus (PF, Bu-Gu-Zhi in Chinese) and Myristicae Semen (MS, Rou-Dou-Kou in Chinese). The current study was designed to evaluate ESWP antidiuretic treatment of polyuria and to explore potential mechanisms of renal water metabolism in the rat model of SKYD-induced diarrhea. MATERIALS AND METHODS: An animal model of 'SKYD-induced diarrhea syndrome' has been established to evaluate the therapeutic effect and action mechanism according to the clinical syndrome and symptoms. The optimal dose (3.5 g/kg) of ESWP was given to rats by gavage for two weeks. Urinary volumes after 24 h were recorded. After the end of the trial, macroscopic morphological and histological examination of the kidney were conducted. Serum levels of Arginine vasopressin (AVP) and aldosterone (ALD) were also measured. Additionally, quantitative real-time RT-PCR (RT-qPCR) and immunohistochemistry (IHC) analyses were performed to clarify the regulation of aquaporin 2 (AQP 2) and arginine vasopressin type 2 receptor (AVPR 2) in the kidney at the gene and tissue expression levels respectively. RESULTS: After the administration of ESWP, urinary output volume after 24 h was found to be significantly decreased in rats. Elevated plasma levels of AVP and ALD were detected. Histological kidney damage appeared to be impeded, and histological disease scores were reduced. In addition, the expression levels of AQP 2 and AVPR 2 were significantly increased. CONCLUSION: This study suggests that ESWP may elicit significant effects on the treatment of polyuria. Potential mechanisms at least partially involve hormone regulation, and alleviating renal pathological damage. Simultaneously, ESWP may alter renal water absorption by increasing AQP 2 and AVPR 2 expression levels. Thus, the in vivo experimental evidence indicates that ESWP has a therapeutic effect on the SKYD syndrome, which is consistent with its traditional usage.

miRNA Expression Profile of Saliva in Subjects of Yang Deficiency Constitution and Yin Deficiency Constitution.

BACKGROUND/AIMS: Based on the theory of constitution in Traditional Chinese Medicine (TCM), the Chinese Han population has been classified into nine constitutions. Of these, Yang deficiency constitution mainly exhibit cold intolerance while Yin deficiency constitution mainly exhibit heat intolerance. Some studies have been carried out to explore the modern genetic and biological basis of such constitution classification, but more remains to be done. MicroRNA (miRNA) serves as post-transcriptional regulators of gene expression and may play a role in the classification process. Here, we examined miRNA expression profile of saliva to further improve the comprehensiveness of constitution classification. METHODS: Saliva was collected from Chinese Han individuals with Yang deficiency, Yin deficiency and Balanced constitutions (n=5 each), and miRNA expression profile was determined using the Human miRNA OneArray®v7. Based on 1.5 Fold change, means log2|Ratio|≥0.585 and P-value< 0.05, differentially expressed miRNA was screened. Target genes were predicted using DIANA-TarBasev7.0 and analysis of KEGG pathway was carried out using DIANA-mirPathv.3. RESULTS: We found that 81 and 98 differentially expressed miRNAs were screened in Yang deficiency and Yin deficiency constitution, respectively. Among them, 16 miRNAs were identical and the others were unique. In addition, the target genes that are regulated by the unique miRNAs were significantly enriched in 27 and 20 signaling pathways in Yang deficiency and Yin deficiency constitution, respectively. Thyroid hormone signaling pathway is present in both constitutions. These unique miRNAs that regulated target genes of thyroid hormone signaling pathway may be associated with cold intolerance or heat intolerance. CONCLUSION: The results of our study show that Yang deficiency and Yin deficiency constitutions exhibit systematic differences in miRNA expression profile. Moreover, the distinct characteristics of TCM constitution may be explained, in part, by differentially expressed miRNAs.

[Expression of G-protein coupled estrogen receptor in the testis of the male mouse with kidney yin or kidney yang deficiency and its impact on the reproductive function of the mouse].

OBJECTIVE: To investigate the expression of the G-protein coupled estrogen receptor (GPER) in the testis of the male mouse with kidney yin or kidney yang deficiency and its influence on the reproductive function of the mouse. METHODS: We randomized 30 six-week-old male Kunming mice into three groups of equal number: kidney yang deficiency, kidney yin deficiency, and normal control, and established the models of kidney yang deficiency and kidney yin deficiency by peritoneal injection of hydrocortisone at 50 mg/kg for 5 days and 25 mg/kg for 10 days, respectively. We observed the behavioral changes of the mice using the elevated plus-maze, exhaustive swimming and field experiment, examined the semen quality with the automatic sperm quality analyzer, calculated the average number of the offspring, measured the serum testosterone (T) and estradiol (E2) levels and T/E2 ratio by Roche electrochemiluminescence assay, and determined the localization and expression of GPER in the testis by immunohistochemistry and immunofluorescence staining. RESULTS: Compared with the mice with kidney yin deficiency, those with kidney yang deficiency showed remarkably fewer entries into the open arm and central area (P <0.05) and shorter time of exhaustive swimming (P <0.05), but no statistically significant difference in the time spent in the open arm or the central area (P >0.05); the latter group also exhibited significant decreases in the epididymal sperm count (ï¼»7.27 ± 1.30ï¼½ vs ï¼»3.05 ± 1.06ï¼½ ×108/g, P <0.01), sperm motility (ï¼»54.15 ± 13.52ï¼½ vs ï¼»51.57 ± 8.75ï¼½ %, P <0.01) and average number of the offspring (6.46 vs 4.33, P <0.05), a slight increase in the rate of morphologically abnormal sperm (ï¼»13.42 ± 2.32ï¼½ vs ï¼»15.39 ± 2.48ï¼½ %, P >0.05), and markedly reduced serum T (ï¼»24.96 ± 6.18ï¼½ vs ï¼»16.72 ± 5.92ï¼½ ng/dl,P <0.05), E2 (ï¼»19.81 ± 4.01ï¼½ vs ï¼»15.24 ± 1.11ï¼½ pg/ml,P <0.05) and T/E2 ratio (1.41 vs 1.25, P <0.05). The expression of GPER was found in the cytoplasm of the Leydig cells, negative in the nuclei and cell membrane, significantly higher in the kidney yang than in the kidney yin deficiency group (P <0.05). CONCLUSIONS: The numbers of sperm and offspring decreased while the percentage of morphologically abnormal sperm increased in both the kidney yang and kidney yin deficiency mice, even more significantly in the former, which might be associated with the up-regulated expression of GPER in the testis of the mouse with kidney yang deficiency and consequently the reduced serum T level and T/E2 ratio.

Integrative metabolic and microbial profiling on patients with Spleen-yang-deficiency syndrome.

Gut microbiota is recognized as an indispensable "metabolic organ" that plays crucial roles in maintaining human health or initiating diseases. Spleen-yang-deficiency syndrome (SYDS) is a common syndrome of Traditional Chinese Medicine (TCM) clinic. It is a complex phenotype reflecting the overall changes of metabolism which are mainly caused by digestive disorders. However, little is known about the changes of gut microbiota and metabolism in patients with SYDS, as well as the crosstalk between gut microbiota and host metabolism. In the current study, an integrative metabolic and microbial profiling was performed on plasma, urine and feces from recruited SYDS and healthy individuals by using a LC-QTOFMS-based metabolomic and 16 s rRNA sequencing approaches. Our results showed a potentially significant contribution of gut dysbiosis to the metabolic disorders in SYDS. By integrating the differential gut bacteria with the metabolites, the results revealed some active bacterium of norank_f_CFT112H7, f_lachnospiraceae and bacteroides were closely involved in host mucosal integrity, bile acid metabolism and polysaccharides decomposition. Therefore, our results indicated the probable involvement of gut microbiota in mediating the metabolic changes, which warrants a further investigation on the role of gut microbiota in modulating the pathogenesis of SYDS.

Regulatory effects of Poria on substance and energy metabolism in cold-deficiency syndrome compared with heat-deficiency syndrome in rats.

Recent studies have revealed that the property of drug is mainly associated with the body's substance and energy metabolism. The present study aimed to evaluate the drug property of Poria, called Fuling (FL) in traditional Chinese medicine (TCM), in terms of its effects on the substance and energy metabolism in rat models of cold-deficiency and heat-deficiency syndromes, compared with Aconiti Lateralis Radix Praeparaia, called Fuzi (FZ) in TCM, with hot property, and Anemarrhenae Rhizoma, called Zhimu (ZM) in TCM, with cold property, as reference drugs, respectively. The appearance score, toe and rectal temperatures of the animals treated were assessed at different time points. Several indices in vivo correlated with substance and energy metabolism (glucokinas, phosphoglycerate kinase, cytochrome c reductase, cytochrome c oxydase, and Na+-K+-ATPase), endocrine system (triiodothyronine, thyroxine, and 17-hydroxycorticosteroid), nervous system (acetylcholin esterase), and cyclic nucleotide system were determined. The changes in appearance score and indices in vivo suggested the successful establishment of cold-deficiency and heat-deficiency syndrome models. FZ reversed the decreased levels of indices (substance and energy metabolism and endocrine system) and alleviated the syndrome of cold-deficiency model, and ZM showed obviously therapeutic effect on heat-deficiency syndrome (appearance score, substance and energy metabolism, and endocrine system). FL could alleviate cold-deficiency syndrome and raise the decreased levels of glucokinas, phosphoglycerate kinase, cytochrome c reductase and triiodothyronine in cold-deficiency model, but had no significant effect on heat-deficiency syndrome. Drug property of FL was inferred as trending to "flat and warm", which still need further study. It was advisable to adopt both cold-deficiency and heat-deficiency models to study the drugs with "flat" property.