Pharmacokinetics, safety, and efficacy of Fuqi Guben Gao in the treatment of kidney-yang deficiency syndrome: a randomized, double-blind phase I trial.

Introduction: Fuqi Guben Gao (FQGBG) is a botanical drug formulation composed of FuZi (FZ; Aconitum carmichaelii Debeaux [Ranunculaceae; Aconiti radix cocta]), Wolfberry (Lycium barbarum L. [Solanaceae; Lycii fructus]), and Cinnamon (Neolitsea cassia (L.) Kosterm. [Lauraceae; Cinnamomi cortex]). It has been used to clinically treat nocturia caused by kidney-yang deficiency syndrome (KYDS) for over 30 years and warms kidney yang. However, the pharmacological mechanism and the safety of FQGBG in humans require further exploration and evaluation. Methods: We investigated the efficacy of FQGBG in reducing urination and improving immune organ damage in two kinds of KYDS model rats (hydrocortisone-induced model and natural aging model), and evaluated the safety of different oral FQGBG doses through pharmacokinetic (PK) parameters, metabonomics, and occurrence of adverse reactions in healthy Chinese participants in a randomized, double-blind, placebo-controlled, single ascending dose clinical trial. Forty-two participants were allocated to six cohorts with FQGBG doses of 12.5, 25, 50, 75, 100, and 125 g. The PKs of FQGBG in plasma were determined using a fully validated LC-MS/MS method. Results: FQGBG significantly and rapidly improved the symptoms of increased urination in both two KYDS model rats and significantly resisted the adrenal atrophy in hydrocortisone-induced KYDS model rats. No apparent increase in adverse events was observed with dose escalation. Major adverse drug reactions included toothache, thirst, heat sensation, gum pain, diarrhea, abdominal distension, T-wave changes, and elevated creatinine levels. The PK results showed a higher exposure level of benzoylhypaconine (BHA) than benzoylmesaconine (BMA) and a shorter half-life of BMA than BHA. Toxic diester alkaloids, aconitine, mesaconitine, and hypaconitine were below the lower quantitative limit. Drug-induced metabolite markers primarily included lysophosphatidylcholines, fatty acids, phenylalanine, and arginine metabolites; no safety-related metabolite changes were observed. Conclusion: Under the investigated dosing regimen, FQGBG was safe. The efficacy mechanism of FQGBG in treating nocturia caused by KYDS may be related to the improvement of the hypothalamus-pituitary-adrenal axis function and increased energy metabolism. Clinical Trial Registration: https://www.chictr.org.cn/showproj.html?proj=26934, identifier ChiCTR1800015840.

Comparing the pharmacological effects of the prepared folium of Epimedium brevicornu Maxim. and Epimedium sagittatum Maxim. on kidney-Yang deficiency syndrome and liver injury complications.

Yinyanghuo, a famous herb, includes the folium of Epimedium brevicornu Maxim. and Epimedium sagittatum Maxim. It is believed that their processed products, the prepared slices of the folium of Epimedium brevicornu Maxim. (PFEB) and Epimedium sagittatum Maxim. (PFES) have greater efficacy in tonifying kidney Yang to treat kidney-Yang deficiency syndrome (KDS). However, there are few studies comparing the pharmacological effects of PFEB and PFES, and the underlying mechanisms. This study compared their effects on improving hypothalamic-pituitary-adrenal (HPA) axis, immune system and sexual characteristic, as well as repairing liver injury complications in the KDS model mice. Additionally, the mechanisms of the effects relevance to their main components were explored. It was found that PFEB was more effective than PFES in increasing cAMP/cGMP ratio, SOD activity, CRH and ACTH levels, eNOS and testosterone levels, splenic lymphocytes proliferation, while in decreasing MDA content, atrophy of spleen and thymus, splenic lymphocytes apoptosis, and PDE5 level. PFES showed stronger protection than PFEB in decreasing triglyceride and hepatic lipid. The contents of baohuoside I and epimedin A, B were much higher in PFEB, while Epimedin C, Icariin, 2-O″-rhamnosylicaridide II were higher in PFES. Consequently, PFEB exhibits superior efficacy over PFES in tonifying the kidney-Yang by improving the neuroendocrine-immune network, including HPA axis, immune systems, and corpus cavernosum. However, PFES has better recovery effect on mild hepatic lipid caused by KDS. The efficacy difference between PFEB and PFES in kidney-Yang and liver may be attributed to the content variations of baohuoside I.

Integrated serum pharmacochemistry, network pharmacology, and pharmacokinetics to clarify the effective components and pharmacological mechanisms of the proprietary Chinese medicine Jinkui Shenqi Pill in treating kidney yang deficiency syndrome.

The proprietary Chinese medicine Jinkui Shenqi Pill (PCM-JKSQP) is a classic compound used for the effective clinical treatment of kidney yang deficiency syndrome (KYDS), a metabolic disease accompanied by kidney injury. However, its active ingredients and therapeutic mechanisms are not clear. This study employed serum pharmacochemistry, network pharmacology, and pharmacokinetics (PK) to identify the bioactive components of PCM-JKSQP and preliminarily clarify its mechanism in treating KYDS. One hundred and forty chemical components of PCM-JKSQP, 47 (20 parent compouds and 27 metabolites) of which were absorbed into the blood, were identified by ultra-high-performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The topological parameters of network pharmacology and high concentrations in blood found six parent components as PK markers (cinnamic acid, paeonol, loganin, morroniside, apigenin, and poricoic acid A). PK analysis further identified these six compounds as active ingredients. Protein-protein interaction (PPI) analysis and molecular docking simulation predicted and verified eight core targets (TP53, ESR1, CTNNB1, EP300, EGFR, AKT1, ERBB2, and TNF). Most were concentrated in the MAPK, HIF-1, and PI3K-AKT signaling pathways, indicating that these six active ingredients may mainly exert therapeutic effects through these three pathways via their core targets. The PK results also showed these six components were absorbed quickly, although cinnamic acid and paeonol were rapidly metabolized, with a short half-life and retention time. Loganin and morroniside did not have high peak concentrations, and apigenin and poricoic acid A had long retention times. This study provides a new overall perspective for exploring the bioactive components and mechanisms underlying the effects of PCM-JKSQP in treating KYDS.

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.

The acute toxic effect of Chinese medicine Fuzi is exacerbated in kidney yang deficiency mice due to metabolic difference.

ETHNOPHARMACOLOGICAL RELEVANCE: The proper application of toxic medicines is one of the characteristics of traditional Chinese medicines, and the use of traditional Chinese medicines follows the principle of dialectical treatment. It is necessary to combine different "syndrome" or "disease" states with the toxicity of traditional Chinese medicines to form a reliable toxicity evaluation system. Fuzi, the lateral root of Aconitum carmichaelii Debx, is recognized as a panacea for kidney yang deficiency syndrome, however, its toxic effects significantly limit its clinical application. AIM OF THE STUDY: Herein, our research aimed to explore the toxic effects of Fuzi on syndrome models, and tried to reveal the underlying mechanisms. MATERIALS AND METHODS: Firstly, the mouse model of kidney yang deficiency syndrome was established through intramuscular injection of 25 mg/kg hydrocortisone per day for 10 consecutive days. Then, the acute toxicity of Fuzi in normal mice and kidney yang deficiency model mice was explored. Finally, the plasma metabolite concentrations and liver CYP3A4 enzyme activity were analyzed to reveal the possible mechanisms of the different pharmacological and toxicological effects of Fuzi in individuals with different physical constitutions. RESULTS: It was found that the treatment with Fuzi (138 g/kg) had serious toxic effects on kidney yang deficiency mice, leading to the death of 80% of the mice, whereas it showed no lethal toxicity in normal mice. This indicates that Fuzi induced greater toxicity in kidney yang deficiency mice than in normal ones. The liver CYP3A4 enzyme activity in kidney yang deficiency mice was decreased by 20% compared to the controls, resulting in slower metabolism of the toxic diester diterpenoid alkaloids in Fuzi. CONCLUSION: In conclusion, our study showed that changes of the metabolic enzyme activity in individuals with different syndromes led to different toxic effects of Chinese medicines, emphasizing the crucial importance of considering individual physical syndromes in the clinical application of traditional Chinese medicine, and the significance of conducting safety evaluations and dose predictions on animal models with specific syndromes for traditional Chinese medicines.

Dysfunction of cecal microbiota and CutC activity in mice mediating diarrhea with kidney-yang deficiency syndrome.

Objective: Previous studies have indicated that diarrhea with kidney-yang deficiency syndrome leads to a disorder of small intestine contents and mucosal microbiota. However, the relationship of TMA-lyase (CutC) activity and TMAO with diarrhea with kidney-yang deficiency syndrome remains unexplored. Therefore, this study explores the relationship between cecal microbiota and choline TMA-lyase (CutC) activity, as well as the correlation between trimethylamine oxide (TMAO), inflammatory index, and CutC activity. Method: Twenty SPF-grade male KM mice were randomly divided into the normal group (CN) and the diarrhea model group (CD). Diarrhea mouse models were established by adenine combined with Folium sennae administration. CutC activity, TMAO, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were detected, and the cecal content microbiota was sequenced. Result: After 14 days, diarrhea occurred in the CD group. Compared with the CN group, there was no significant change in the activity of CutC in the small intestine of the CD group, while the activity of CutC in the cecum was significantly increased, and the levels of TMAO, IL-6, and TNF-α showed a significant increase. The Chao1 index, Observed_species index, Shannon index, and Simpson index all exhibited a decreasing trend. The main changes at the bacterial genus level were Alistipes, Enterorhabdus, Desulfovibrio, Bacteroides, Candidatus_Saccharimonas, and [Ruminococcus]_torques_group. The results of LEfSe analysis, random forest analysis and ROC curve analysis revealed Paludicola, Blautia, Negativibacillus, Paraprevotella, Harryflintia, Candidatus_Soleaferrea, Anaerotruncus, Oscillibacter, Colidextribacter, [Ruminococcus]_torques_group, and Bacteroides as characteristic bacteria in the CD group. Correlation analysis showed a significant negative correlation between cecal CutC activity and Ligilactobacillus, and a significant positive correlation with Negativibacillus and Paludicola. The level of TMAO was significantly positively correlated with CutC activity and IL-6. Conclusion: Diarrhea with kidney-yang deficiency syndrome significantly affects the physiological status, digestive enzyme activity, CutC activity, TMAO levels, and inflammatory response in mice. Additionally, there are changes in the composition and function of cecal microbiota, indicating an important impact of diarrhea with kidney-yang deficiency syndrome on the host intestinal microbiota balance. The occurrence of diarrhea with kidney-yang deficiency syndrome may be associated with dysbiosis of intestinal microbiota, increased CutC activity, elevated TMAO levels, and heightened inflammatory factor levels.

Adenine's impact on mice's gut and kidney varies with the dosage administered and relates to intestinal microorganisms and enzyme activities.

This study aimed to investigate the effects of different dosages of adenine on intestinal microorganisms and enzyme activities, laying the experimental groundwork for subsequent exploration of the microbial mechanisms underlying diarrhea with kidney yang deficiency syndrome. Twenty-four mice were assigned to the following four groups: the control (NC) group, low-dosage adenine (NML) group, middle-dosage adenine (NMM) group, and high-dosage adenine (NMH) group. Mice in the NML, NMM, and NMH groups received 25 mg/(kg·d), 50 mg/(kg·d), and 100 mg/(kg·d) of adenine, respectively, 0.4 mL/each, once a day for 14 days. The NC group received 0.4 mL sterile water. Parameters including body weight, rectal temperature, intestinal microorganisms, enzyme activities, and microbial activity were measured. Results indicated that mice in the experimental group displayed signs of a poor mental state, curled up with their backs arched, and felt sleepy and lazy, with sparse fur that was easily shed, and damp bedding. Some mice showed fecal adhesion contamination in the perianal and tail areas. Dosage-dependent effects were observed, with decreased food intake, body weight, rectal temperature, and microbial activity and increased water intake and fecal water content. Enzyme activity analyses revealed significantly higher activities of protease, sucrase, amylase, and cellulase in intestinal contents and lactase, sucrase, amylase, and cellulase in the mucosa of the NMM group compared to those of other groups. Ultimately, the higher adenine dosage was associated with more pronounced symptoms of kidney yang deficiency syndrome, with 50 mg/kg adenine exhibiting the most substantial impact on the number of intestinal microbial colonies and enzyme activities.

Clinical Study of Modified Shenqi Pill （肾气丸） Plus Tongdu Tiaoshen Acupuncture （通督调神针刺） for Neurogenic Bladder After Spinal Cord Injury of Kidney-Yang Deficiency Syndrome / 中医杂志

ObjectiveTo observe the clinical efficacy of modified Shenqi Pill （肾气丸） plus Tongdu Tiaoshen Acupuncture （通督调神针刺） in the treatment of neurogenic bladder after spinal cord injury of kidney-yang deficiency syndrome. MethodsForty-six patients were randomly divided into 23 cases each in the control group and the treatment group. Both groups were given conventional treatment， i.e. oral methylcobalamin tablets （0.5 mg each time， 3 times a day） and paraplegic conventional acupuncture （once a day， 6 consecutive days a week）. The control group was given simple bladder function rehabilitation training on the basis of the conventional treatment； and the treatment group was given modified Shenqi Pill orally （1 dose a day， 150 ml each time， taken warmly in morning and evening） and Tongdu Tiaoshen Acupuncture （once a day， 6 consecutive days per week） in addition to what were given to the control group. The treatment course lasted for 4 weeks. The 24 h urination frequency， 24 h urine leakage frequency， 24 h single urine volume， bladder residual urine volume， international lower urinary tract symptom （LUTS） score， traditional Chinese medicine （TCM） syndrome score were compared between the two groups， and clinical effectiveness and TCM syndrome effectiveness were compared between the two groups after treatment. ResultsTwenty patients in each group were finally analyzed in this study. The number of 24 h urination， the number of 24 h urine leakage， bladder residual urine volume， LUTS score， and the TCM syndrome scores decreased after treatment in both groups， and the 24 h single urine volume increased （P＜0.01）； and much more improvement was found of each index in the treatment group than in the control group （P＜0.05 or P＜0.01）. The total clinical effectiveness and TCM syndrome effectiveness in the treatment group was 85.00% （17/20） respectively， which were statistically significantly higher than 45.00% （the total clinical effectiveness， 9/20） and 60.00% （TCM syndrome effectiveness， 12/20） in the control group （P＜0.01）. ConclusionModified Shenqi Pill plus Tongdu Tiaoshen Acupuncture can signi-ficantly improve the clinical symptoms of neurogenic bladder patients after spinal cord injury of kidney-yang deficiency syndrome， having better effectiveness than simple bladder function rehabilitation training， and its mechanism may be related to the improvement of the injured nerve function innervating the bladder.

A serum metabolomics study of vascular cognitive impairment patients based on Traditional Chinese medicine syndrome differentiation.

Objective: Vascular cognitive impairment (VCI) accounts for approximately 50%-70% of all dementia cases and poses a significant burden on existing medical systems. Identifying an optimal strategy for preventing VCI and developing efficient symptomatic treatments remains a significant challenge. Syndrome differentiation represents a fundamental approach for personalized diagnosis and treatment in Traditional Chinese Medicine (TCM) and aligns with the principles of precision medicine. The objective of this study was to elucidate the metabolic characteristics of VCI based on TCM syndrome differentiation, thus providing novel insights into the diagnosis and treatment of VCI. Methods: A 2-year cross-sectional cognitive survey was conducted in four communities in Beijing between September 2020 and November 2022. The syndrome differentiation of participants was based on the Kidney-Yang Deficiency Syndrome Scale (KYDSS), which was originally developed by Delphi expert consultation. The identification of serum metabolites was performed by Ultra performance liquid chromatography (UPLC) analysis coupled with an electrospray ionization quadruple time-of-flight mass spectrometer (ESI-QTOF MS). Multivariate, univariate, and pathway analyses were used to investigate metabolic changes. Logistic regression models were also used to construct metabolite panels that were capable of discerning distinct groups. Phospholipase A2 (PLA2) levels were measured by a commercial ELISA kit. Results: A total of 2,337 residents completed the survey, and the prevalence of VCI was 9.84%. Of the patients with VCI, those with Kidney-Yang deficiency syndrome (VCIS) accounted for 70.87% of cases and exhibited more severe cognitive impairments. A total of 80 participants were included in metabolomics study, including 30 with VCIS, 20 without Kidney-Yang deficiency syndrome (VCINS), and 30 healthy control participants (C). Ultimately, 45 differential metabolites were identified when comparing the VCIS group with group C, 65 differential metabolites between the VCINS group and group C, and 27 differential metabolites between the VCIS group and the VCINS group. The downregulation of phosphatidylethanolamine (PE), and phosphatidylcholine (PC) along with the upregulation of lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), phosphatidic acid (PA) and phospholipase A2 (PLA2) can be considered as the general metabolic characteristics associated with VCI. Dysfunction of glycerophospholipids, particularly LPEs and PCs, was identified as a key metabolic characteristic of VCIS. In particular Glycerophospho-N-Arachidonoyl Ethanolamine (GP-NArE) was discovered for the first time in VCI patients and is considered to represent a potential biomarker for VCIS. The upregulation of PLA2 expression was implicated in the induction of alterations in glycerophospholipid metabolism in both VCIS and VCINS. Moreover, robust diagnostic models were established based on these metabolites, achieving high AUC values of 0.9322, 0.9550, and 0.9450, respectively. Conclusion: These findings contribute valuable information relating to the intricate relationship between metabolic disorders in VCI, neurodegeneration and vascular/neuroinflammation. Our findings also provide a TCM perspective for the precise diagnosis and treatment of VCI in the context of precision medicine.

[Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway].

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

[Zuogui Pills and Yougui Pills in differential treatment of diminished ovarian reserve based on metabolomics].

Based on the metabolomics, this paper systematically analyzed the metabolic substance basis of Zuogui Pills and Yougui Pills in syndrome differentiation and treatment of diminished ovarian reserve(DOR), so as to provide a scientific basis for the traditional Chinese medicine(TCM) syndrome differentiation and treatment of DOR. Patients with DOR of kidney-Yin deficiency syndrome were collected from outpatient department of hospitals and treated with Zuogui Pills for 12 weeks. And kidney-Yang deficiency syndrome were treated with Yougui Pills for 12 weeks. Based on the non-targeted metabolomic research techniques, the potential biomarkers of Zuogui Pills and Yougui Pills in the treatment of DOR with kidney-Yin deficiency and kidney-Yang deficiency, respectively, were screened out, and metabolic pathways of biomarkers were analyzed. The pregnancy rate, basic serum hormone levels [basal follicle-stimulating hormone(bFSH), basal-luteinizing hormone(bLH), basal-estradiol(bE_2), and anti-Müllerian hormone(AMH)], TCM syndrome type score, and Kupperman score were recorded and statistically analyzed after treatment. The results showed that 23 patients with DOR of kidney-Yin deficiency syndrome and 25 patients of kidney-Yang deficiency syndrome were collected. Twenty-six differential metabolites, including L-carnitine, acetyl-CoA, coenzyme A, and coenzyme Q_(10)(CoQ10), were mapped to 12 metabolic pathways in patients with kidney-Yin deficiency treated with Zuogui Pills. Twenty-two differential metabolites, such as adipoyl-CoA, L-lysine, lysine arginine, and α-tocopherol, were mapped to 11 metabolic pathways in patients with kidney-Yang deficiency. After treatment, bFSH and bLH of patients with DOR were significantly lower than those before treatment(P<0.05). Although the comparison of bE_2 and AMH had no significant differences, there was a improvement trend. The TCM syndrome type score and Kupperman score of patients with DOR after TCM treatment were significantly lower than those before treatment(P<0.05).

Kidney Yang Deficiency Syndrome Exacerbates Aß25-35-Induced Pathological Changes, and Ginsenoside Re Ameliorates Synapse Lesions in Aß25-35- Injected Rats with Kidney Yang Deficiency Syndrome.

BACKGROUND: Traditional Chinese medicine (TCM) indicates that Alzheimer's disease (AD) is considered the consequence produced by Kidney Yang Deficiency Syndrome (KDS-Yang), which has similar clinical characteristics to glucocorticoid withdrawal syndrome. Ginsenoside Re (G-Re) has been found to ameliorate the symptoms and pathological impairments of AD. However, it's not clear whether G-Re could protect memory and synapse lesions against kidney deficiency dementia. METHODS: Subcutaneous injection of hydrocortisone for 14 days was used to produce KDS-Yang. On the 15th day, Aß25-35 peptide was injected into the intracerebroventricular (icv) of KDS-Yang rats. Spine density was analyzed by Golgi staining and the ultrastructural morphology of the synapse was detected using Transmission Electron Microscopy (TEM). Western blot was used to examine the expression of pS396, pS404, Tau-5, tGSK-3ß, pS9GSK-3ß, Syt, Syn I, GluA1, GluN2B, PSD93, PSD95, ß2-AR and pS346-b2-AR. RESULTS: Hyperphosphorylation of tau in Aß25-35-injected rats with KDS-Yang was stronger than in Aß25-35-injected rats at the sites of Ser396 and Ser404. G-Re improved spatial memory damage detected by Morris water-maze (MWM), enhanced spines density, the thickness of postsynaptic density (PSD) and increased the expression of Syt, Syn I, GluA1, GluN2B, PSD93 and PSD95. Moreover, GRe decreased the hyperphosphorylation of ß2-AR at serine 346 in Aß25-35-injected rats with KDS-Yang. CONCLUSION: KDS-Yang might exacerbate AD pathological lesions. Importantly, G-Re is a potential ingredient for protecting against memory and synapse deficits in kidney deficiency dementia.

Intestinal mucosal flora of the intestine-kidney remediation process of diarrhea with deficiency kidney-yang syndrome in Sishen Pill treatment: Association with interactions between Lactobacillus johnsonii, Ca2+-Mg2+-ATP-ase, and Na+-K+-ATP-ase.

This study aims to investigate the effect of Sishen Pill on the characteristics of gut mucosal microbiota in diarrhea mice with deficiency kidney-yang syndrome. Fifteen Kunming male mice were randomly divided into Normal control group (C), Model self-healing group (X) and Sishen Pill group (S), with 5 mice/cages. Hematoxylin eosin (HE) staining was used to observe the kidney structure. Serum Na+-K+-ATP-ase and Ca2+-Mg2+-ATP-ase were detected by enzyme-linked immunosorbent assay (ELISA), Analysis of intestinal mucosal flora using third-generation high-throughput sequencing. The relative abundance results in the three groups revealed that the dominant bacterial genera: Lactobacillus, Muribaculum and Candidatus-Arthromitus; bacterial species: Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus murinus, and Lactobacillus intestinalis, and differences in the presence of major microbiota between the X and S groups. A positive correlation between Lactobacillus johnsonii and both Ca2+-Mg2+-ATP-ase and Na+-K+-ATP-ase was found via correlation analysis. Sishen Pill also changed the manufacture of other secondary metabolites, as well as the metabolism of carbohydrates, glycans, energy, lipids, and other amino acids, and xenobiotics biodegradation and metabolism. In conclusion, Sishen Pill improved kidney structure, energy metabolism and the diversity and structure of intestinal mucosal flora. In addition, Lactobacillus johnsonii may be a characteristic species of Sishen Pill in treating diarrhea with kidney-yang deficiency syndrome.

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.

Integrating strategies of metabolomics, network pharmacology, and experiment validation to investigate the processing mechanism of Epimedium fried with suet oil to warm kidney and enhance yang.

Introduction: Epimedium, a traditional Chinese medicine (TCM) commonly used in ancient and modern China, is one of the traditional Chinese medicines clinically used to treat kidney yang deficiency syndrome (KYDS). There are differences in the efficacy of Epimedium before and after processing, and the effect of warming the kidney and enhancing yang is significantly enhanced after heating with suet oil. However, the active compounds, corresponding targets, metabolic pathways, and synergistic mechanism of frying Epimedium in suet oil to promote yang, remain unclear. Methods: Herein, a strategy based on comprehensive GC-TOF/MS metabolomics and network pharmacology analysis was used to construct an "active compounds-targets-metabolic pathways" network to identify the active compounds, targets and metabolic pathways involved. Subsequently, the targets in kidney tissue were further validated by real-time quantitative polymerase chain reaction (RT-qPCR). Histopathological analysis with physical and biochemical parameters were performed. Results: Fifteen biomarkers from urine and plasma, involving five known metabolic pathways related to kidney yang deficiency were screened. The network pharmacology results showed 37 active compounds (13 from Epimedium and 24 from suet oil), 159 targets, and 267 pathways with significant correlation. Importantly, integrated metabolomics and network pharmacologic analysis revealed 13 active compounds (nine from Epimedium and four from suet oil), 7 corresponding targets (ALDH2, ARG2, GSTA3, GSTM1, GSTM2, HPGDS, and NOS2), two metabolic pathways (glutathione metabolism, arginine and proline metabolism), and two biomarkers (Ornithine and 5-Oxoproline) associated with improved kidney yang deficiency by Epimedium fried with suet oil. Discussion: These finds may elucidate the underlying mechanism of yang enhancement via kidney warming effects. Our study indicated that the mechanism of action mainly involved oxidative stress and amino acid metabolism. Here, we demonstrated the novel strategies of integrating metabolomics and network pharmacology in exploring of the mechanisms of traditional Chinese medicines.

16S rDNA sequencing combined with metabolomics profiling with multi-index scoring method reveals the mechanism of salt-processed Semen Cuscuta in Bushen Antai mixture on kidney yang deficiency syndrome.

Kidney yang deficiency syndrome (KYDS) is a classic syndrome of traditional Chinese medicine (TCM). The salt-processed product of Semen Cuscuta (YP) is the monarch drug in Bushen Antai Mixture (BAM), can improve the reproductive dysfunction caused by KYDS, and the effect is better than that of raw products of Semen Cuscuta (SP). However, its mechanism is not completely clear yet. In this study, an integrated strategy combining untargeted metabolomics with microbiology was used to explore the mechanism of YP in the BAM improving KYDS. 16S rDNA gene sequencing showed that BAM containing YP (Y-BAM) had a significantly better regulatory effect on Desulfobacterota and Desulfovibrionaceae_unclassified than BAM containing SP (S-BAM). Untargeted metabolomics studies showed that Y-BAM significantly regulated 4 metabolites and 4 metabolic pathways. In addition, multi-index analysis showed that the effect of Y-BAM on arachidonic acid metabolism, tyrosine metabolism, purine metabolism, fructose and mannose metabolism and total metabolism was closer to that of the control group compared to S-BAM. The analysis of serum biochemical indexes showed that Y-BAM had more significant regulating effect on the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) and superoxide dismutase (SOD) in serum of KYDS rats compared to S-BAM. Spearman correlation analysis showed that there was a significant correlation between intestinal microorganisms and metabolites and serum biochemical indexes. For example, Desulfovibrionaceae_unclassified was positively correlated with arachidonic acid, and negatively correlated with SOD and LH. This study suggests that YP may enhance the regulation of intestinal flora and endogenous metabolism of KYDS, so that BAM shows a better therapeutic effect on KYDS, which also reasonably explains why BAM uses Semen Cuscuta stir-baked with salt solution.

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.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway / 中国中药杂志

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Zuogui Pills and Yougui Pills in differential treatment of diminished ovarian reserve based on metabolomics / 中国中药杂志

Based on the metabolomics, this paper systematically analyzed the metabolic substance basis of Zuogui Pills and Yougui Pills in syndrome differentiation and treatment of diminished ovarian reserve(DOR), so as to provide a scientific basis for the traditional Chinese medicine(TCM) syndrome differentiation and treatment of DOR. Patients with DOR of kidney-Yin deficiency syndrome were collected from outpatient department of hospitals and treated with Zuogui Pills for 12 weeks. And kidney-Yang deficiency syndrome were treated with Yougui Pills for 12 weeks. Based on the non-targeted metabolomic research techniques, the potential biomarkers of Zuogui Pills and Yougui Pills in the treatment of DOR with kidney-Yin deficiency and kidney-Yang deficiency, respectively, were screened out, and metabolic pathways of biomarkers were analyzed. The pregnancy rate, basic serum hormone levels [basal follicle-stimulating hormone(bFSH), basal-luteinizing hormone(bLH), basal-estradiol(bE_2), and anti-Müllerian hormone(AMH)], TCM syndrome type score, and Kupperman score were recorded and statistically analyzed after treatment. The results showed that 23 patients with DOR of kidney-Yin deficiency syndrome and 25 patients of kidney-Yang deficiency syndrome were collected. Twenty-six differential metabolites, including L-carnitine, acetyl-CoA, coenzyme A, and coenzyme Q_(10)(CoQ10), were mapped to 12 metabolic pathways in patients with kidney-Yin deficiency treated with Zuogui Pills. Twenty-two differential metabolites, such as adipoyl-CoA, L-lysine, lysine arginine, and α-tocopherol, were mapped to 11 metabolic pathways in patients with kidney-Yang deficiency. After treatment, bFSH and bLH of patients with DOR were significantly lower than those before treatment(P<0.05). Although the comparison of bE_2 and AMH had no significant differences, there was a improvement trend. The TCM syndrome type score and Kupperman score of patients with DOR after TCM treatment were significantly lower than those before treatment(P<0.05).