ABSTRACT
ObjectiveTo investigate the effects of spleen-Yin deficiency on gastrointestinal absorption， water metabolism and intestinal flora in rats with spleen-Yin deficiency syndrome. MethodA rat model of spleen-Yin deficiency syndrome was established by using the composite factors， including irregular meat and vegetable diet， weight-bearing fatigue swimming and gavage with warm-heat injury-Yin drugs. The changes of body weight， food intake， water intake and duration of swimming in the blank and model groups were observed. Hematoxylin-eosin（HE） staining was used to observe the histopathological damage of the stomach and colon. Urinary excretion rate of D-xylose was determined by phloroglucinol method. The content of gastrin（GAS） in serum was determined by enzyme-linked immunosorbent assay（ELISA）. The relative expression levels of vasoactive intestinal peptide（VIP）， aquaporin 3（AQP3） and AQP4 in gastric tissues were detected by Western blot. The relative mRNA expression levels of VIP， AQP3 and AQP4 in gastric tissues were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the changes of intestinal flora were analyzed by 16S rDNA sequencing. ResultCompared with the blank group， the results of general physical signs showed that the body weight and food intake of rats in the model group were significantly decreased， the water intake was significantly increased（P<0.05， P<0.01）， and the duration of swimming was significantly decreased（P<0.01）. Pathological examination results showed that in the mucosa of gastric tissues of rats in the model group appeared to be misaligned， the mucosa of colonic tissues could be seen to be obviously thinned or mutilated， and the epithelial cells appeared to be necrotic or even exfoliated. Compared with the blank group， the urinary D-xylose excretion rate of rats in the model group was significantly decreased（P<0.01）， and the serum GAS content was significantly decreased（P<0.05）. Compared with the blank group， Western blot results showed that the relative expression level of VIP protein in gastric tissues of rats in the model group was significantly decreased， while the relative expression levels of AQP4 and AQP3 proteins were significantly increased（P<0.01）. Compared with the blank group， Real-time PCR results showed that the relative expression level of VIP mRNA in gastric tissues of rats in the model group was significantly decreased（P<0.01）， and the relative mRNA expression levels of AQP3 and AQP4 were significantly increased（P<0.05， P<0.01）. Compared with the blank group， the results of intestinal flora analysis showed that the number of operational taxonomic units（OTUs） and α-diversity increased and β-diversity decreased significantly in the model group， the abundance of Porphyromonadaceae was increased significantly， and the abundance of Oscillibacter_ruminantium was decreased significantly（P<0.05）. Spearman correlation analysis showed that Porphyromonadaceae was significantly positively correlated with AQP4 protein level， while Oscillibacter_ruminantium was significantly positively correlated with VIP protein level， and negatively correlated with AQP3 and AQP4 protein levels（P<0.05）. Linear discriminant analysis effect size（LEfSe） analysis results showed that there were significant differences in a variety of intestinal bacteria between groups， and the intestinal bacteria of the model group were significantly enriched in the phylum/order/family/genus of Elusimicrobia， Betaproteobacteria， Burkholderiales， Sutterellaceae and Parasutterella（P<0.05）. ConclusionSpleen-Yin deficiency syndrome can weaken the digestion and absorption capacity of gastrointestinal tract， and cause the disturbance of water metabolism and intestinal flora. AQP4， AQP3 and VIP protein levels of gastric mucosa are closely related to Porphyromonadaceae and Oscillibacter_ruminantium. And AQP4， AQP3 and VIP may be involved in the regulation of intestinal flora in order to affect the physiological function of spleen governing transportation and transformation.
ABSTRACT
Books on Chinese herbal medicines have shown that Dendrobium has the effect of nourishing Yin and reinforcing Yin,usually used for constipation induced by spleen Yin deficiency in clinical application. D. huoshanense,as an independent species among many species of Dendrobium,has no experimental studies about its effects on spleen Yin deficiency-type constipation. The purpose of this experiment was to illustrate the therapeutic effect of D. huoshanense on the constipation of spleen Yin deficiency type in rats,investigate its preliminary mechanism,and compare it with the D. officinale and D. nobile contained in the Chinese Pharmacopoeia to clarify its characteristics. The spleen Yin deficiency model was replicated in 70 rats by the composite factor method,and then the model rats were randomly divided into 7 groups: model group,Liuwei Dihuang Pills group( LWDHP),D. huoshanense high( DHS-H),medium( DHS-M),low( DHS-L) dose groups,D. nobile group( DNS),and D. officinale group( DOS),and another 10 rats were used as normal group( Normal). After 7 continuous days of administration,the fecal water content and intestine propulsion rate of each group were detected. HE staining was used to observe the pathological damage of ileum and colon in each group. Immunohistochemistry and Western blot were used to detect aquaporin 3( AQP3) expressions,while the expression levels of the somatostatin( SS) and motilin( MTL) in the ileum of each group were detected by enzyme-linked immunosorbent assay. The results showed that as compared with the model group,the rats in each drug-administered group had increased number of fecal pellets,increased fecal water content,and the increased intestinal propulsion rate( P<0. 01),while the pathological damage of the ileum and colon was significantly reduced; the expression of AQP3 protein was significantly decreased( P<0. 01); the level of MTL was significantly increased and the level of SS was decreased( P<0. 01). All DHS groups showed a good dose-effect relationship,and the same dose treatment effect was equivalent to that of DOS,but it was superior to DNS. Therefore,DHS has a significant therapeutic effect on constipation of spleen Yin deficiency type,and its mechanism may be related to intestinal motility and water-liquid metabolism,with a good therapeutic effect.
Subject(s)
Animals , Rats , Constipation , Drug Therapy , Dendrobium , Chemistry , Drugs, Chinese Herbal , Pharmacology , Intestines , Plants, Medicinal , Chemistry , Random Allocation , Spleen , Yin Deficiency , Drug TherapyABSTRACT
This study was aimed to observe the changes of dendritic spine density in different regions of brain among spleen-yindeficiency dementia (SYDD) model rats, in order to investigate the effects ofZi-Bu Pi-Yin Recipe (ZBPYR) on dendritic spines. Spleen-yindeficiency (SYD) rats were modeled by classical method. And incubatedβ-Amyloid 1-40 (Aβ1-40) was injected into the hippocampus of each rat to make SYDD model, which received the administration of ZBPYR. Golgi staining was used to stain dendritic spine in different regions of brain in rat model for the observation of the amount and shape. The results showed that dendritic spine density in different regions of hippocampus and cortex in SYDD group was reduced than that of the SYD group. Compared with the dementia group and the SYDD group, the dendritic spine density in different regions of hippocampus and cortex of the SYDD + ZBPYR group was increased. Compared with the blank control group, the dendritic spine density in different regions of hippocampus and cortex in rats from the dementia group was reduced. It was concluded that there were different degrees of reducing in the dendritic spine density of different brain regions in SYDD group. ZBPYR improved the learning and memory impairment, which might be related to the maintenance of dendritic spine density in different brain regions.
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This study was aimed to explore the mechanism ofZi-Bu Pi-Yin Recipe (ZBPYR) on autophagy and endoplasmic reticulum stress (ERS) to improve spleen-yin deficiency diabetes-associated cognitive decline (DACD). Rats were randomly divided into the control (cont) group, the diabetes (DM) group, the spleen-yin deficiency (pi) group, the spleen-yin deficiency diabetes (piDM) group, and the spleen-yin deficiency diabetes + ZBPYR treatment (ZBPYR) group. The expression of microtubule-associated protein 1A/1B-light chain 3 (LC3Ⅱ), inositol-requiring enzymeα (IRE1α), c-Jun N-terminal kinase (JNK) were observed by western blot. The results showed that the expression of LC3Ⅱ in the DM group, pi group and piDM group decreased compared with the cont group (P < 0.05); and the expression of LC3Ⅱ of the ZBPYR group increased compared with the DM group and piDM group (P < 0.05). Compared with the cont group, the p-IRE1α of the DM group and piDM group, as well as p-JNK1 in the pi group and piDM group were increased (P < 0.05). The p-IRE1α and p-JNK1 of the ZBPYR group were decreased compared with the DM group and piDM group (P < 0.05). It was concluded that ZBPYR improved spleen-yin deficiency DACD by regulating autophagy and ERS.
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This study was aimed to observe the effect ofZi-Bu Pi-Yin Recipe (ZBPYR) on the mRNA expressions of NMDA receptor subunits NR1, NR2A, NR2B in different brain regions of spleen-yin deficiency Alzheimer's Disease (AD) model rats. The levels of NR1, NR2A, NR2B mRNA expressions were detected by using RT-PCR method. The results showed that the levels of NR1, NR2A, NR2B mRNA expressions of AD group and spleen-yin deficiency AD group decreased significantly (P < 0.05). The levels of NR1, NR2A, NR2B mRNA expressions of ZBPYR treatment group increased significantly (P < 0.05). It was concluded that the expression levels of NMDAR mRNA in different brain regions of the ZBPYR treatment group increased significantly, which indicated that ZBPYR may up-regulate the protein expressions of NMDAR by increasing the expression levels of NMDAR mRNA, thereby to play the anti-dementia effect.
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This study was aimed to observe changes of key molecular in insulin signaling pathway in the hypothala-mus of rats to explore the mechanism of spleen yin deficiency diabetes-associated cognitive decline (DACD) and Zibu Piyin Recipe (ZBPYR) in order to provide new ideas and new clues for treatment of DACD. Rats were randomly divided into five groups, which were the control (Cont) group, diabetes (DM) group, spleen yin deficiency (pi) group, spleen yin deficiency diabetes (piDM) group and the ZBPYR group. Insulin receptor substrate 1 (IRS-1) serine phos-phorylation levels and protein kinase B (PKB/Akt) serine phosphorylation levels which were involved in the insulin signaling were observed by western blotting in the hypothalamus to determine whether there were insulin signaling obstacles in the hypothalamus of rats. The results showed that the expression of p-IRS-1ser in the DM group, pi group and piDM group was increased compared with the Cont group (P< 0.05); while the p-Akt expression of the DM group and piDM group was decreased (P< 0.05). The expression of p-IRS-1ser in the ZBPYR group decreased compared with the DM group and piDM group (P< 0.05); while the level of p-Akt increased compared with the DM group and piDM group (P < 0.05). It was concluded that insulin signaling was not transduced normally in the hy-pothalamus of the DM group, pi group and piDM group. Insulin resistance may occur in the hypothalamus. And ZBPYR can correct insulin signaling transduction disorder.
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Objective To explore the mechanism of Zibu Piyin Recipe (ZBPYR) on spleen yin deficiency diabetes-associated cognitive disorder (DACD). Methods The rats were randomly divided into control group, diabetes mellitus (DM) group, spleen yin deficiency group, spleen yin deficiency DM group and spleen yin deficiency DM+ZBPYR group (treatment group). Type 2 DM models were established by high-fat food feeding and low dose STZ intraperitoneal injection for 4 weeks. Then the classical compound method was used to construct spleen yin deficiency rat models by improper diet, over exertion and yin fluids exhaustion. The treatment group was given ZBPYR by gavage for 15 days, and the other groups were given the same amount of normal saline. Then cerebral cortex, hippocampus, stomach and liver were obtained and the changes of protein expression of PDHE1α in them were observed by Western Blot. Results The protein expression of PDHE1αin cortex of DM group and spleen yin deficiency DM group were lower than control group (P<0.05). PDHE1α expression of treatment group in cortex and stomach increased more significantly than spleen yin deficiency DM group (P<0.05). The expression of PDHE1α protein showed no significant difference among all groups in hippocampus and liver. Conclusion ZBPYR improved spleen yin deficiency DACD by regulating PDHE1αin cortex and stomach.
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Objective To clarify the pathogenesis of spleen yin deficiency diabetes-associated cognitive decline (DACD) and mechanism of Zinu Piyin Recipe (ZBPYR) by observing the expression of phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2α subunit (eIF2α), p-eIF2α, glucose-regulated protein 78 (GRP78) in cerebral cortex of spleen yin deficiency diabetes mellitus rats. Methods The rats were randomly divided into control group, diabetes mellitus group, spleen yin deficiency group, spleen yin deficiency diabetes mellitus group (disease-syndrome group) and spleen yin deficiency diabetes mellitus+ZBPYR group (treatment group). Type 2 diabetes mellitus models were established by high-fat food feeding for 4 weeks and low dose STZ intraperitoneal injection. Then the classical compound method was used to construct spleen yin deficiency rats model by improper diet, over exertion and yin fluids exhaustion. The reatment group was given ZBPYR and other groups were given equal volume of normal saline for 15 days, then cerebral cortex was obtained. The expression of p-PERK, p-eIF2α, eIF2α and GRP78 were observed by Western Blot and RT-PCR. Results The protein expression of p-PERK, p-eIF2α, and mRNA expression of GRP78 of diabetes mellitus group, spleen yin deficiency group and disease-syndrome group was enhanced compared with control group (P<0.05). GRP78 protein expression of diabetes mellitus group, disease-syndrome group was increased compared with control group (P<0.05). The protein expression of p-PERK, p-eIF2α, GRP78 and mRNA expression of GRP78 of treatment group was decreased compared with diabetes mellitus group and disease-syndrome group (P<0.05). Conclusion Endoplasmic reticulum stress is involved in spleen yin deficiency DACD. ZBPYR improves learning and memory ability by reducing endoplasmic reticulum stress.
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This study was aimed to observe different forms of β-amyloid peptide (Aβ) and insulin degrading enzyme (IDE) in the hippocampus and cortex in order to further explore the role of Aβ and IDE on spleen yin deficiency di-abetes-associated cognitive decline (DACD), and the effect of Zi-Bu Pi-Y in method. The rats were randomly divided into five groups, which were the blank control (Cont) group, diabetes (DM) group, spleen yin deficiency (pi) group, spleen yin deficiency diabetes (piDM) group and Zi-Bu Pi-Y in recipe (ZBPYR) group. Soluble and insoluble Aβ in the hippocampus and cortex of rats were extracted by gradient centrifugation, and then measured by ELISA. The ex-pression of IDE was observed by western blot. The results showed that the content of soluble and insoluble Aβ1-42 in the hippocampus and cortex of the DM group and piDM group were higher than the Cont group. The soluble and in-soluble Aβ1-42 content in the hippocampus and cortex of the ZBPYR group were reduced compared with the DM group and the piDM group. The soluble Aβ1-40 in the cortex of the DM group, pi group and piDM group were in-creased compared with the Cont group (P < 0.05). The soluble Aβ1-40 content of the ZBPYR group was decreased compared with the DM group and the piDM group (P < 0.05). The expression of IDE protein was decreased in the hippocampus of the DM group and the piDM group compared with the Cont group (P< 0.05), and the IDE protein level in the hippocampus of the ZBPYR group was increased compared with the DM group and the piDM group (P<0.05). The expression of IDE protein in the cortex of the DM group, pi group and piDM group was lower than the Cont group (P< 0.05). The IDE protein level in the cortex of the ZBPYR group was reduced compared to the DM group (P< 0.05). It was concluded that the increased Aβ1-42 in brain may be a major pathological change of DACD and spleen yin deficiency DACD. The decreased IDE expression may be one of the reasons to induce increasing of Aβ1-42 level. The Zi-Bu Pi-Y in method may decrease the Aβ1-42 content by upregulating IDE protein expression.