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Objective:To study the effect and mechanism of Zuoguiwan on the autophagy of cells in the skin during the development of congenital kidney-deficient mice， and to explore the application value of kidney tonifying and essence filling method in abnormal growth and development. Method:36 rats with a 2∶1 ratio of male to female were paired， and the pregnant rats were stimulated by combined stress method to build a model of congenital kidney-deficient rats. According to different treatment methods， the pregnant rats were divided into the control group， the kidney-deficient group， and the Zuoguiwan low and high dose groups （2， 8 g·kg<sup>-1</sup>）. The control group was given normal saline without modeling. Kidney deficiency group was modeled before intragastric administration of normal saline. Zuoguiwan small dose and high dose groups received intragastric administration of Zuoguiwan suspension. 21 days after the birth of mice， the back skin was taken to observe the skin microstructure and morphology of the mice by HE staining and detect the thickness of dermis and epidermis. The gene and protein expression levels of Wingless3a and <italic>β</italic>-catenin in the skin of neonatal rats were measured by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Double immunofluorescence assay was used to detect the skin autophagy indicators Microtubule-associated protein light chain 3 （LC3）and Sequestosome 1 （SQSTM1-p62）. Result:In the control group， the skin layers were clear and the structure was normal， in the kidney deficiency group， the collagen arrangement in the dermis was loose， the skin composition in Zuoguiwan low and high dose groups was basically normal. Compared with the control group， the epidermal layer of the kidney deficiency group was thickened and the dermis was thinned （<italic>P</italic><0.05）. Compared with the kidney deficiency group， the thickness changes of epidermal layer and dermis layer were significantly restored in Zuoguiwan low and high dose groups （<italic>P</italic><0.05）. Compared with the control group， the expression of Wnt3a and <italic>β</italic>-catenin protein and gene in the kidney deficiency group significantly decreased （<italic>P</italic><0.05）. Compared with the kidney deficiency group， Wnt3a and <italic>β</italic>-catenin protein and gene expression increased in Zuoguiwan low and high dose groups （<italic>P</italic><0.05）. At the same time， immunofluorescence assay showed that compared with the control group， the expression of LC3 was up-regulated and the expression of SQSTM1-p62 was down-regulated in the kidney deficiency group， while Zuoguiwan could reverse such abnormal expression （<italic>P</italic><0.05）. Conclusion:Zuoguiwan can down-regulate the autophagy level of skin cells and improve the abnormal skin development of congenital renal deficiency by increasing the expression of Wnt3a and <italic>β</italic>-catenin protein and gene in the skin of neonatal rats.
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Objective:To investigate the mechanism of Shugan Bushen Yulin decoction in inhibiting voltage-dependent anion-selective channel protein 2 （VDAC2） gene methylation， affecting sperm mitochondrial function， and improving sperm motility through the cyclic adenosine monophosphate/protein kinase A （cAMP/PKA） pathway. Method:Forty male SD rats were randomly divided into the blank group， model group， high- and low-dose Shugan Bushen Yulin decoction groups， and L-carnitine group， with eight rats in each group. Adenine （0.05 g·kg<sup>-1</sup>） was administered by gavage for 14 d for inducing oligospermia and asthenospermia. Rats in the Shugan Bushen Yulin decoction groups were treated with intragastric administration of 32.4， 8.1 g·kg<sup>-1 </sup>Shugan Bushen Yulin decoction， respectively， while those in the L-carnitine group received 0.27 g·kg<sup>-1</sup> L-carnitine by gavage. Following the measurement of sperm motility using an automatic sperm analyzer， the pathological changes in testicular tissue were observed by hematoxylin-eosin （HE） staining. Sperm mitochondrial membrane potential was detected by flow cytometry. The expression of VDAC2 in the testicular tissue was determined by immunofluorescence assay. Real-time polymerase chain reaction （Real-time PCR） was conducted for detecting VDAC2 mRNA expression in testicular tissue. The methylation of VDAC2 gene was examined using bisulfite sequencing. The cAMP expression in testicular tissue was detected by enzyme-linked immunosorbent assay （ELISA）， and the PKA protein expression in testicular tissue by Western blot. Result:Compared with the blank group， the model group exhibited significantly decreased sperm density and motility （<italic>P</italic><0.01）， increased mitochondrial membrane potential （<italic>P</italic><0.01）， down-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in testicular tissue （<italic>P</italic><0.01）， and elevated VDAC2 gene methylation （<italic>P</italic><0.01）. Compared with the model group， L-carnitine and Shugan Bushen Yulin decoction at the high and low doses all remarkably increased the sperm density and motility and mitochondrial membrane potential （<italic>P</italic><0.01）， up-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue （<italic>P</italic><0.01）， and lowered the methylation of VDAC2 in testicular tissue （<italic>P</italic><0.01）. The comparison with the L-carnitine group showed that the sperm density and motility and mitochondrial membrane potential in the low-dose Shugan Bushen Yulin decoction group declined significantly （<italic>P</italic><0.01）. The VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue were significantly down-regulated （<italic>P</italic><0.01）， while the methylation of VDAC2 was significantly enhanced （<italic>P</italic><0.01）. Conclusion:Shugan Bushen Yulint decoction may inhibit VDAC2 gene methylation， increase VDAC2 expression， regulate cAMP/PKA pathway， and change mitochondrial membrane potential to enhance the sperm motility.
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Objective:To study the effect of Zuoguiwan on the differentiation of CD4-CD8-T cells in thymus during the development of kidney-deficient fetal rats and the application value of kidney and essence-tonifying method, in order to explore the theoretical connotation of "kidney being the origin of congenital constitution". Method:Male and female rats were paired at a ratio of 2∶1, and three stress methods were combined to construct the kidney-deficient model. Pregnant rats were divided into control group, model group, thymosin group (0.003 g·kg-1), and low-dose Zuoguiwan group and high-dose Zuoguiwan group (2,8 g·kg-1). Normal saline was given to the control group， model group was used for modeling and given normal saline by gavage, thymosin group was given thymosin capsule solution by gavage, and low-dose Zuoguiwan group and high-dose Zuoguiwan group were given Zuoguiwan suspension by gavage. Embryos that were 16 days, 19 days and born on the first day (E16, E19, P1) were selected to calculate fetal rat thymus index. Electron microscope was used to observe thymic epithelial cell ultrastructure of fetal rats. Enzyme-linked immunosorbent assay (ELISA) method was used to detect thymus thymosinβ4 (TMSβ4) and thymosinα1 (Tα1) content, fluid cytology detection of fetal rats on the surface of CD4, CD8, CD25, CD44 expression for identifying T cell type. Result:The morphology of thymus epithelial cells in the model group was irregular, the nucleus was deformed, and the chromatin of the nucleus was significantly increased. The results of ELISA showed that the content of TMSβ4 and Tα1 in thymus increased in the model group and the low-dose and high-dose Zuoguiwan groups. Flow cytometry showed that, compared with the control group, CD4-CD8-T cells were significantly increased in the model group at E16, E19 and P1 stages (P<0.05), suggesting that T cells were blocked during the development from DN to DP. CD4-CD8-CD25-CD44-T (DN4) was significantly increased (P<0.05), suggesting that T cell development was inhibited during DN4-DP transition. Compared with the model group, CD4-CD8-T cells were significantly reduced in low and high-dose groups (P<0.05) at E16, E19 and P1 stages. At the same time, DN4 was significantly reduced (P<0.05), suggesting that Zuoguiwan could promote the development of T cells from DN4 stage to DP stage as thymosin. Conclusion:Zuoguiwan can improve the structure of thymus epithelial cells in rats with kidney deficiency, and induce the differentiation of T cells in thymus from DN4 stage to DP stage by increasing the secretion of TMSβ4 and Tα1 in thymus of embryonic rats, indicating that Zuoguiwan can promote the development and maturation of T cells and regulate the immune status of fetal rats with kidney deficiency by tonifying kidney and spermatogenesis.
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<p><b>OBJECTIVE</b>To observe the effect of Qianliean Pill (QP) on inflammatory factors such as IL-1β, IL-10, and tumor necrosis factor α (TNF-α) in chronic nonbacterial prostatitis (CNP) model rats, and to explore its therapeutic mechanism.</p><p><b>METHODS</b>CNP rat model was established by castration and estradiol benzoate injection. Totally 50 rats were randomly divided into 5 groups, i.e., the model group, the positive medicine group, the high dose QP group, the medium dose QP group, and the low dose QP group, 10 in each group. Besides, 10 normal rats were recruited as a normal control group. Since the 8th day of castration, Pulean Tablet (PT) at 10. 80 g/kg was administered to rats in the positive medicine group by gastrogavage. QP at 11.00, 5.50, and 2.75 g/kg was administered to rats in high, medium, and low dose QP groups by gastrogavage. Distilled water at 2 mL/100 g was administered to rats in the model group and the normal control group by gastrogavage, once daily for 30 successive days. After 30 days of medication all rats were sacrificed and their prostate tissues were extracted. The prostatic index was calculated. Pathological changes of rat prostate were observed under light microscope. Meanwhile, levels of IL-1β, IL-10, and TNF-α were detected using enzyme linked immunosorbent assay.</p><p><b>RESULTS</b>Compared with the normal control group, the prostate index obviously decreased, levels of IL-1β, TNF-α, and IL-10 in the prostate tissue significantly increased in the model group (P < 0.01). Compared with the model group, the prostate index obviously decreased in high and medium dose QP groups, and the positive medicine group (P < 0.01); levels of IL-1β, TNF-α, and IL-10 obviously decreased in each QP group and the positive medicine group (P < 0.01). Compared with the positive medicine group, the TNF-α level decreased more obviously in the high dose QP group (P < 0.05). Compared with the normal control group, inflammatory reactions occurred obviously in rats' prostate of the model group. Compared with the model group, inflammatory reactions were milder in rats' prostate of each QP group and the positive medicine group, and their degrees were improved to some extent.</p><p><b>CONCLUSION</b>QP could treat CNP, which might be achieved by regulating local immune state of the prostate, relieving inflammatory reactions of the prostate, and lowering levels of IL-β, TNF-α, and IL-10 in the prostate tissue.</p>
Subject(s)
Animals , Humans , Male , Rats , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Interleukin-10 , Metabolism , Interleukin-1beta , Metabolism , Prostatitis , Drug Therapy , Metabolism , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Qianlie Huichun capsule on the microstructure and ultranstructure of prostate glandular tissue in the model rat.</p><p><b>METHOD</b>Hynertophy of prostate model rat was established by injecting testosterone to gelding male rats. After having been fed with Qianlie Huichun capsule for 30 days, the rats were killed and prostate tissues were resected for pathomorphological studies with microscope and electromicroscope, and the diameter of glandular lumer and the height of glandular epithelial cells were measured under the microspcope for different groups of rats.</p><p><b>RESULT</b>In the model groups, the glandular epithelial cells mutiplycated notably, showing stratified and pseudostratified cells that made the glandular lumer cramped. Under the electromicroscope, the glandular epithelial cells became high columnor and the rough endoreticulum extremely expanded. But in treatment groups, the change of the diameter of the glandular lumer and the height of the glandular epithelial cells were less remarkable than those in model groups. So the differerence between the model group and the treatment groups was remarkable (P < 0.01).</p><p><b>CONCLUSION</b>Qianlie Huichun capsule can depress the glandular epithelialceu multiplication of prostate gland in model rats.</p>
Subject(s)
Animals , Male , Rats , Cell Proliferation , Dose-Response Relationship, Drug , Drugs, Chinese Herbal , Pharmacology , Epithelial Cells , Pathology , Materia Medica , Pharmacology , Plants, Medicinal , Chemistry , Prostate , Pathology , Prostatic Hyperplasia , Pathology , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of experimental varicocele on the apoptosis of spermatogenic cells in rats.</p><p><b>METHODS</b>Thirty-two Sprague-Dawley(SD) male rats were randomly divided into four groups: pseudo-operation group, 45 d experimental group, 60 d experimental group and 90 d experimental group. Experimental varicocele was created by partial ligation of left renal vein. The apoptosis number of spermatogenic cells was measured by flow cytometry.</p><p><b>RESULTS</b>No marked apoptosis peak was observed in the control group, but it was observed in all the other groups, and the height of the peak increased with the duration of experiment.</p><p><b>CONCLUSIONS</b>Varicocele can induce apoptosis of large numbers of spermatogenic cells, and the decrease of spermatogenic cells. This may be the mechanism of varicocele causing male sterility.</p>
Subject(s)
Animals , Male , Rats , Apoptosis , Flow Cytometry , Random Allocation , Spermatocytes , Cell Biology , Testis , Pathology , Varicocele , PathologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Qianlie Huichun capsular on Fas expression and cell apoptosis of prostate gland tissue in the model rat.</p><p><b>METHOD</b>Hypertrophy of prostate model rat was established by injecting testosterone to gelding male rats. After being treated with Qianlie Huichun ig 30 days, the rats were killed and prostate glands were resected for examination. The Fas expression was examined by immunobistochemical SABC. The cell apoptosis and the peak of cell apoptosis in the prostate gland of the rats were examined by flow cytometry. Compared with model group, the weight of prostage gland tissue in groups treated with Qianlie Huichun capsular was light (P < 0.01).</p><p><b>RESULT</b>Compared with model group, the Fas expression in all treatment groups increase(P < 0.05, P < 0.01). Compared with normal and model groups, cell apoptosis in all treatment groups increase at different level(P < 0.01).</p><p><b>CONCLUSION</b>The Qianlie Huchun capsular increases the Fas expression and cell apoptosis of model rats, and shows a definite treatment effect on the hypertrophy of prostate by promoting the apoptosis of prostate cell.</p>