Expression of Th17/Treg Cells in Peripheral Blood and Related Cytokines of Patients with Ulcerative Colitis of Different Syndrome Types and Correlation with the Disease

Objective. To explore the expression of helper T cells 17 (Th17)/regulatory T cells (Treg) in peripheral blood and related cytokines of patients with different types of ulcerative colitis (UC) and analyze their correlation with the disease. Methods. From January 2018 to December 2019, 53 patients diagnosed with UC in our hospital were selected. According to their medical syndromes, they were divided into the damp-heat internal accumulation group (n = 35) and the spleen-kidney yang deficiency group (n = 18). 21 healthy volunteers were selected as the control group. The Mayo scoring standard was used to determine the severity of the patient’s condition. The expression levels of Th17/Treg cells and related cytokines in peripheral blood were compared between the groups. Pearson correlation was used to analyze the correlation between the ratio of Th17 and Treg cells in the peripheral blood of UC patients and the ratio of TH17/Treg with Mayo score. Results. The peripheral blood Th17 cell ratio and Th17/Treg ratio of the damp-heat internal accumulation and spleen-kidney yang deficiency group were higher than those of the control group; the Treg cell ratio was lower than that of the control group; the peripheral blood Th17 cell ratio and Th17/Treg ratio of the damp-heat internal accumulation group were higher those of the spleen-kidney yang deficiency group; and the proportion of Treg cells was lower than that of the spleen-kidney yang deficiency group ( P < 0.05 ). The expression levels of serum IL-6, IL-17, IL-22, and TNF-α in the damp-heat internal accumulation and spleen-kidney yang deficiency group were higher than those of the control group; IL-10 and TGF-β were lower than those of the control group; the levels of serum IL-6, IL-17, IL-22, and TNF-α in the damp-heat internal accumulation group were higher than those of the spleen-kidney yang deficiency group; and both IL-10 and TGF-β were lower than those of the spleen-kidney yang deficiency group ( P < 0.05 ). The peripheral blood Th17 cell ratio and Th17/Treg ratio in the moderately active period group and severely active period group were higher than those of the lightly active period group; the Treg cell ratio was lower than that of the lightly active period group; the peripheral blood Th17 cell ratio and Th17/Treg ratio in the severely active period group were higher than those in the moderately active period group; and the proportion of Treg cells was lower than that of the moderately active period group. Pearson correlation analysis showed that the proportion of Th17 cells and Th17/Treg in peripheral blood of UC patients were both positively correlated with Mayo score (r = 0.762, r = 0.777, P < 0.001 ). Treg was negatively correlated with Mayo score (r = −0.790, P < 0.001 ). Conclusion. There are differences in the expression of peripheral blood Th17/Treg cells and related cytokines among UC patients with different syndromes, and the damp-heat content is the most significant. The higher the ratio of Th17 cells in peripheral blood and the degree of Th17/Treg imbalance, the lower the ratio of Treg cells, and the more severe the condition of UC patients, which can provide a preliminary quantitative basis for the TCM classification and severity of the diagnosis of UC.

Pharmacokinetics of the Couple of Radix Aconiti Lateralis Preparata and Cinnamomum Cassia in Rats with Osteoporosis with Kidney-yang Deficiency by UPLC and Microdialysis Method

Background: To confirm the metabolites of the Aconite and Cinnamon herb couple in rat, and characterize its pharmacokinetics.Methods: Microdialysis probes were inserted into the jugular vein and knee joint of Sprague Dawley rat, and dialysates of different administration groups were collected. Target analytes were separated on a hydrophilic interaction liquid chromatography column (ACQUITY UPLC BEH C8 2.1×100 mm, 1.7 μm) and analyzed with an ultra-performance liquid chromatography (UPLC) under multiple reaction monitoring modes.Results: The experiment shows that the concentrations of quality of the three metabolites in mixing extraction of the herb couple were 0.3701% for trans-cinnamic acid, 0.1249 % for mesaconitine, and 0.0469 % for hypoaconitine, and Cinnamon group was 0.1731 % for trans-cinnamic acid, Aconite group were 0.0017 % for mesaconitine, and 0.0300 % for hypoaconitine. The concentrations of quality of the metabolites in rat plasma of the herb couple group were 0.0028% for trans-cinnamic acid, 0.0947% for mesaconitine, and 0.1124% for hypoaconitine. And the concentrations of quality of Aconite group and Cinnamon group were 0.0019% for trans-cinnamic acid, 0.0307% for mesaconitine, and 0.0220% for hypoaconitine. Pharmacokinetic results showed that the mean half-lives of the microdialysis samples of blood of Cinnamon group was 492.18 min for trans-cinnamic acid, and Aconite group were 102.48 min for mesaconitine and 93.27 min for hypoaconitine, and the herb couple ware 181.36 min for trans-cinnamic acid, 103.9 min for mesaconitine and 116.01 min for hypoaconitine, and the microdialysis samples of joint of Cinnamon group was 190.85 min for trans-cinnamic acid, and Aconite group were 48.51 min for mesaconitine and 46.01 min for hypoaconitine, and the herb couple was 131.19 min for trans-cinnamic acid, 49.36 min for mesaconitine and 146.68 min for hypoaconitine.Conclusions: The mixed extraction of aconite and cinnamon can promote the dissolution of the active ingredients. The herb couple can promote the absorption of the active ingredients, improve the distribution of the active ingredients in the joint and blood, prolong the half-lives of the active ingredients. It shows that the compatibility of aconite and cinnamon can increase the bioavailability of the drug and improve the clinical efficacy.

Two-Way Impacts Between Macrophages on Vascular Endothelium and Characteristics of TCM Syndromes in Dyslipidemic Mice with the Phlegm-Dampness Retention syndrome and the Spleen and Kidney Yang Deficiency syndrome Using RNA-Seq

Background: ‘Treating the same disease with different methods’ is a Traditional Chinese Medicine (TCM) therapeutic concept. That means although patients are diagnosed with the same disease, they may have different syndromes that require distinct drug administrations. This study aimed to identify the differentially expressed genes and related biological processes in dyslipidemia with the Phlegm-Dampness Retention (PDR) syndrome and the Spleen and Kidney Yang Deficiency (SKYD) syndrome using transcriptomic analysis.Methods: Ten ApoE knockout (ApoE-/-) mice were used for the establishment of dyslipidemic disease-syndrome models via multifactor-hybrid modeling, with 5 in the the PDR group and 5 in the SKYD group. Five C57BL/6J mice were employed as normal controls (NC) group. Test model quality. Aortic endothelial macrophages in mice were screened using flow cytometry. Transcriptomic analysis was performed for macrophages using RNA-Seq.Results: ①The quality assessment of the disease-syndrome model showed that TG, TC, and LDL-C levels significantly increased in the PDR and SKYD groups versus the NC group (P < 0.05). Combined with HE staining of aorta, the disease model was successfully established. ②The quality assessment of the syndrome models showed that mice in the PDR group presented with typical manifestations of the PDR syndrome, and mice in the SKYD group had the related manifestations of the SKYD syndrome, indicating that the syndrome models were successfully constructed. ③After comparing the differentially expressed gene (DEG) expressions in macrophages in dyslipidemia mice with different syndromes, 4142 genes were identified with statistical significance (P < 0.05). The Gene Ontology (GO) analysis for the DEGs showed that biological process of difference between PDR group and SKYD group include both adverse and protective processes were included.Conclusion: The DEGs between the PDR syndrome and the SKYD syndrome indicate different biological mechanisms between the onset of the two syndromes. They have distinctive biological processes, including adverse and protective processes, corresponding to the invasion of pathogenic factors into the body and the fight of healthy qi against pathogenic factors, respectively, in the TCM theory. Our results have demonstrated the biological evidence behind ‘treating the same disease with different treatments’ in TCM.

Effects of Haima Duobian Pill in a Rat Model of Kidney Yang Deficiency Syndrome

Objective. Modern research shows that Haima Duobian pill (HDP) can relieve the kidney yang deficiency syndrome (KYDS), but the mechanism is still unclear. The aim of this work was to study the effects of HDP in a rat model of KYDS. Materials and Methods. The network pharmacology methods were used to predict the therapeutic effects of Haima Duobian pill. Adenine was used to establish the rat model of kidney yang deficiency syndrome. The general physical signs of rats were observed after different doses of Haima Duobian pill (HDP) were given. Serum cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), estradiol (E2), and gonadotropin-releasing hormone (GnRH) levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. Then, the histopathologic changes and sperm activity were detected. Results. HDP could improve the general signs of kidney yang deficiency syndrome rats. After the rats were treated with HDP, the expression of cGMP and E2 was significantly inhibited and the expression of cAMP and T was significantly increased. The pathological damage of testis, epididymis, and seminal vesicle was alleviated, and the sperm activity was improved. Conclusion. For adenine-induced kidney yang deficiency syndrome in rats, HDP had a significant therapeutic effect.

Study on Neuroendocrine-Immune Function of Cistanche deserticola and Its Rice Wine Steaming Products in Glucocorticoid-Induced Rat Model

The desert-dwelling Cistanche herb was first recorded in the “Shen Nong Herbal Classic” and is listed as the top-grade herbal medicine in this publication. The Chinese Pharmacopoeia records that pieces of Cistanche deserticola (CD) and rice wine-steamed Cistanche deserticola (WCD) can be used in the clinic as the main types of decoctions. After being steamed with rice wine, the antiaging and tonifying kidney-yang effects are enhanced. In this study, we detected the chemical content of CD and WCD and the pharmacological mechanism of invigorating kidney-yang deficiency in model rats. Aim. The purpose of this study was to examine the effects of CD and WCD on the neuroendocrine-immune function of kidney-yang deficiency in glucocorticoid-overdosed model rats. Materials and methods. Sprague Dawley (SD) rats were selected. The rats were subcutaneously injected with corticosterone water suspension for the glucocorticoid-overdosed model rats. The positive control rats were gavaged with Jinkuishenqi pills and high-, medium-, and low-dose CD/WCD suspensions (1.646 g/(kg day), 5.48 g/(kg day), 2.74 g/(kg day), and 1.37 g/(kg day), respectively); the blank control (BC) and model control (MC) groups were given the same volume of distilled water as those in the drug group for 40 consecutive days at a dose of 1 mL/100 g. After the last administration, the blood was collected from the abdominal aorta, and serum levels of T, CRH, ACTH, CORT, cortisol, IL-10, IL-6, IL-2, TNF-α, and IFN-γ were measured. Organ indexes of the thymus gland and the spleen were calculated. The expression of Bax, Bcl-2, caspase-3, Fas, and FasL in the adrenal gland was measured by immunohistochemistry. The pathological changes in the thymus gland and the adrenal gland were observed by HE staining (×200). T lymphocyte subsets in peripheral blood were detected by flow cytometry, and the expression of CaM mRNA in the hypothalamus and hypophysis tissues was also measured by RT-PCR. Results. Compared with the MC group, the CD and WCD groups exhibited increases in activity, the organ index of the thymus and the spleen, the serum levels of T, CRH, ACTH, CORT, cortisol, IL-2, and IL-10, the ratio of CD4+/CD8+, and the expression of Bcl-2, caspase-3, Fas, FasL, and CaM in the hypophysis tissue. The CD and WCD groups also exhibited reductions in the IL-6, TNF-α, and IFN-γ levels in serum and the expression of CaM mRNA in the hypothalamus. Conclusions. Each dose of CD and WCD could counteract the dysregulated sex hormone and immune factors in glucocorticoid-overdosed model rats, enhancing and restoring the effect of the hypothalamic nerve cells and improving immune function.

Establishment and Comparison of an Experimental Kidney Yang Deficiency Syndrome Rat Model Caused by Hydrocortisone, Adenine, and Dexamethasone Combined with Adriamycin in Rats

Objective: The paper is aimed to establish a method for renal injury in rats with kidney yang deficiency syndrome (KYDS) of the stable by comparing different model establishment methods and optimize the final modeling method.Method: In order to mimic water metabolism disorder of KYDS- a diagnostic pattern in traditional Chinese medicine (TCM), we made a comparison of hydrocortisone-induced, adenine-induced, dexamethasone combined with adriamycin-induced KYDS rat model according to testing general behavior, detecting Hypothalamus-pituitary-adrenal (HPA) axis function and renal function also making use of drug action. Additionally, the effect of adenine at a dose of 100 mg/kg, 150 mg/kg, and 200 mg/kg, respectively, on establishment of KYDS model was investigated in rats. Finally, Shenqi Wan (SQW, 3 g/kg) was used to estimate the deterioration of adenine that is associated with KYDS. Result: Three KYDS model rats all exhibited weight loss, anal temperature drop, reduced food intake and dysfunction of the Hypothalamus-pituitary-adrenal (HPA) axis, which could be alleviated by SQW. However, different from other two methods, adenine-induced rats were prominently manifested as water and fluid metabolism disorders (sharply increased water intake and urine volume). Renal injury in adenine-induced rats was more obvious than it in hydrocortisone-induced rats and dexamethasone combined with adriamycin-induced rats. And renal toxicity caused by adenine was reversible by SQW treatment. Water metabolism disorder in adenine-induced rats was more closer to clinical symptoms of patients with KYDS. Subsequently, we found different dose of adenine gavage (100, 150, 200 mg/kg) for 14 days result in general behavior change, dysfunction of HPA axis and renal function damage. But only medium-dose of adenine induced toxicity could be reversed by SQW treatment for 21 days.Conclusion: The result of the comparison between the three modeling methods showed that adenine-induced was more effective to establish the KYDS model, and only a medium-dose of adenine for inducing renal poison could be recovered by SQW treatment. Therefore, a medium-dose of adenine was optimal for the establishment of the KYDS rat model.Trial registration : This is an animal experiment, trial registration is not necessary.

Effect of Boschniakia Rossica Extract on BMP-5 and ActRIIA in Mice Model of Kidney-Yang Deficient

Background Boschniakia rossica (Cham. & Schltdl.) B. Fedtsch. (Bu-Lao-Cao in Chinese name) is a famous Chinese traditional drug and mainly distributed in Korea, Japan, Russia, and China. The dried whole grass of Boschniakia rossica is commonly used in traditional Chinese medicine to treat kidney deficiency, yang deficiency, waist and knee aches and constipation. In order to quantify the active ingredients in B. rossica chinensis and body weight, clinical features and serum SOD and MDA activities, cAMP, cGMP, and testosterone (T), follicle-stimulating growth hormone (FSH), mouse bone morphogenetic protein-5 (BMP-5), mouse activin receptor IIA (ActRIIA) were measured to elucidate the mechanism underlying its treatment of kidney yang deficiency potential. Methods Extracts of B. rossica were evaluated by HPLC-MS analyses. Kidney-yang deficiency syndrome was induced in mouse with hydrocortisone (10 mg/Kg, 15d) and treated with B. rossica extract. The animals were treated orally with 3 doses of B. rossica extract: 1000, 500 and 250 mg/Kg, for 15 days. Body weight and clinical characteristics were recorded daily. For biochemical analyses, the following tests were performed: superoxide dismutase (SOD) activity, malondialdehyde (MDA) activity, testosterone (T) content, follicle growth hormone (FSH) content, cyclic adenosine monophosphate (cAMP) content, mouse bone morphogenetic protein-5 (BMP-5) content and activin receptor IIA (ActRIIA) content. Results Our study found that all the mice except the blank group showed significant systemic depletion caused by hydrocortisone. Low T content, low cAMP activity and high cGMP content were consistent with the physiological and biochemical indexes of kidney-yang deficiency syndrome. After oral gavage, the pathological conditions were improved in both the Positive group and the B. rossica groups.Conclusions The high-dose extract of B. rossica showed similar therapeutic effects as Jingui Shenqi Pill. The extract of B. rossica can be used to treat with hydrocortisone-induced kidney yang deficiency syndrome by increasing serum testosterone content and decreasing FSH content. More importantly, the experiment found that activin A and BMP-5 also have effects on kidney yang deficiency syndrome.

Jingui Shenqi Pills Regulate Bone-Fat Balance in Murine Ovariectomy-Induced Osteoporosis with Kidney Yang Deficiency

Jingui Shenqi Pills (JGSQP) have been a staple of traditional Chinese medicine for thousands of years, used primarily as a treatment for kidney yang deficiency (KYD). In vitro analyses of JGSQP revealed strong induction of osteogenic differentiation and inhibition of adipogenic differentiation in bone-marrow-derived mesenchymal stem/stromal cells. However, the mechanisms by which JGSQP regulate the bone-fat balance in murine ovariectomy-induced osteoporosis with KYD have not been reported. Materials and Methods. Two-month-old female C57BL/6 mice were divided randomly into three groups: those receiving a sham operation (Sham); those undergoing bilateral ovariectomy and selection of KYD syndrome (Model); and those subjected to both bilateral ovariectomy and KYD syndrome selection for 8 weeks, followed by JGSQP treatment for 4 weeks (JGSQP). In the Sham and Model groups, mice were given the same dose of distilled water orally for 4 weeks. Animals from all three groups were euthanised at the 12th week. Vertebral microarchitecture and histomorphology were examined by micro-CT and H&E staining, respectively. In addition, we examined the mRNA expression of Akt, Wnt10b, Osterix (Osx), Fndc5, PPARγ, and Fabp4, as well as the protein of AKT, phosphorylation-AKT (p-AKT), BMP2, COL1A1, and FNDC5. Results. JGSQP treatment improved bone microarchitecture and mitigated histomorphological damage relative to the Model group. The osteoblast number (Ob.N/BS) and area (Ob.S/BS) were increased, whereas adipocyte number (adipocyte/tissue area) and area (adipocyte area/tissue area) were decreased in the JGSQP group. JGSQP treatment reduced the mRNA expression of Akt and adipogenesis-related genes (Fndc5, PPARγ, and Fabp4) while promoting osteogenesis-related genes (Wnt10b and Osx) mRNA expression. Additionally, the expression of p-AKT, BMP2, and COL1A1 proteins was increased and FNDC5 protein expression was decreased after JGSQP treatment. Conclusions. JGSQP treatment reversed murine ovariectomy-induced osteoporosis with KYD by controlling bone-fat balance via AKT pathway.