ABSTRACT
ObjectiveTo observe the effect of Liuwei Dihuangtang on depression-like behavior of diabetes mellitus combined with comorbid depression （DD） rats， so as to explore its action mechanism. MethodFifty male SD rats of SPF grade were fed with high fat diet and injected with low-dose streptozotocin （STZ） via tail vein for inducing diabetes. Afterwards， the diabetic rats were exposed to chronic unpredictable mild stress （CUMS） for 28 d. The successfully modeled DD rats were randomly divided into five groups： model group， fluoxetine （10 mg·kg-1·d-1） group， and low-， medium-， and high-dose （3.375， 6.75， 13.5 g·kg-1·d-1） Liuwei Dihuangtang groups， with 10 in each group. Another 10 rats were classified into the normal control group and treated with intragastric administration of normal saline for four weeks. The tail suspension test and open field test were conducted to evaluate the depressive-like phenotype of rats. The contents of malondialdehyde （MDA）， reactive oxygen species （ROS）， 8-hydroxy-2 deoxyguanosine （8-OHdG）， superoxide dismutase （SOD）， and glutathione （GSH） in ventral hippocampus （vHIP） were measured by enzyme-linked immunosorbent assay （ELISA）， and the myelin basic protein （MBP） expression in vHIP by immunofluorescence assay. The expression levels of MBP， myelin protein lipoprotein （PLP）， myelin oligodendrocyte glycoprotein （MOG）， phosphorylated adenosine 5'-monophosphate-activated protein kinase （p-AMPK）/AMPK， phosphorylated protein kinase B （p-Akt）/Akt， phosphorylated glycogen synthase kinase 3β （p-GSK3β）/GSK3β， and nuclear factor erythroid-2 related factor 2（Nrf2） were determined by Western blotting. ResultCompared with the normal control group， the model group exhibited significantly prolonged immobility in the tail suspension test （P<0.01） and shortened residence at the central area in the open field test （P<0.01）. The immobility time in the medium- and high-dose Liuwei Dihuangtang groups declined to different degrees as compared with that of the model group （P<0.01）， while the residence time at the central area was significantly increased （P<0.05， P<0.01）. Compared with the normal control group， the model group displayed down-regulated MBP， PLP， and MOG protein expression in vHIP （P<0.01）. Compared with the model group， Liuwei Dihuangtang at the low dose up-regulated the expression of MBP （P<0.05）， but did not obviously affect the expression of MOG and PLP. Fluoxetine and Liuwei Dihuangtang at the medium and high doses up-regulated the expression of MBP， PLP， and MOG （P<0.05， P<0.01）. Comparison with the normal control group revealed that the MBP fluorescence intensity in vHIP of the model group was significantly weakened （P<0.01）. After the intervention， the MBP fluorescence intensities in the medium- and high-dose Liuwei Dihuangtang groups and fluoxetine group were enhanced in contrast to that of the model group （P<0.05， P<0.01）. SOD and GSH in the model group were lower than those in the normal control group （P<0.01）， whereas the MDA， ROS， and 8-OHdG expression levels were higher （P<0.01）. Compared with the model group， Liuwei Dihuangtang at the medium and high doses and fluoxetine all down-regulated the expression levels of MDA， ROS， and 8-OHdG （P<0.05，P<0.01）， while up-regulated SOD and GSH expression （P<0.05，P<0.01）. The expression levels of p-AMPK， p-Akt， and Nrf2 in the model group were down-regulated as compared with those in the control group， and the expression of p-GSK3β was up-regulated （P <0.01）. As demonstrated by comparison with the model group， the protein expression of p-AMPK in the low-dose Liuwei Dihuangtang group was elevated （P<0.05）， while p-Akt and Nrf2 were slightly increased， exhibiting no statistical significant difference. However， the protein expression levels of p-AMPK， p-Akt， and Nrf2 in the medium- and high-dose Liuwei Dihuangtang groups and fluoxetine group were up-regulated， while those of p-GSK3β were down-regulated （P<0.05，P<0.01）. ConclusionLiuwei Dihuangtang improves the depressive-like behavior of DD rats， which may be related to its activation of the AMPK/Akt/GSK3β/NRF2 pathway， regulation of the oxidative stress in vHIP， and enhancement of myelin repair.
ABSTRACT
ObjectiveTo observe the effect of Liuwei Dihuangtang （LWDHT） on depression-like behaviors of rats with diabetes mellitus and depression （DD） and explore its mechanism. MethodThe diabetes mellitus （DM） model was induced by the high-fat diet and tail vein injection of low-dose streptozotocin （STZ） in 50 male Sprague-Dawley rats of SPF grade. Then the DD model was induced by chronic unpredictable mild stress （CUMS） for 28 days in DM rats. Fifty DD rats were randomly divided into model group， fluoxetine group （10 mg·kg-1·d-1）， and low-， medium-， and high-dose LWDHT groups （3.375， 6.75， 13.5 g·kg-1·d-1）， with 10 rats in each group. Another 10 healthy rats were assigned into a control group and received normal saline by gavage. After four weeks of drug intervention， the forced swimming assay was carried out to assess the depression-like behaviors of rats. The expression levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） in the anterior cingulate cortex （ACC） were detected by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence was used to detect the expression of myelin basic protein （MBP） in ACC and the co-localization of ionized calcium-binding adapter molecule 1 （Iba1） with intracellular microtubule-associated protein 1 light chain 3 （LC3）. The protein expression levels of MBP， myelin proteolipid protein （PLP）， myelin oligodendrocyte glycoprotein （MOG）， Beclin-1， LC3， p62， and microglia （MG） phenotypic protein-related inducible nitric oxide synthase （iNOS）， and arginase 1 （Arg1） were detected by Western blot. ResultCompared with the control group， the model group showed shortened swimming time and prolonged immobility time （P<0.01）. Compared with the model group， the medium- and high-dose LWDHT groups showed reduced immobility time （P<0.05， P<0.01）. Compared with the control group， the model group showed decreased protein expression of MBP， PLP， and MOG in the ACC region （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed up-regulated protein expression of MBP， PLP， and MOG （P<0.05， P<0.01）. Compared with the control group， the model group showed decreased MBP fluorescence intensity in the ACC region （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed increased MBP fluorescence intensity in the ACC region （P<0.05， P<0.01）. Compared with the control group， the model group showed increased expression of iNOS （P<0.01） and slightly increased Arg1 protein expression. Compared with the model group， the medium- and high-dose LWDHT groups and the fluoxetine group showed down-regulated iNOS expression and up-regulated Arg1 protein expression （P<0.05， P<0.01）， but there was no significant difference between the fluoxetine group and the medium-，high-dose LWDHT groups. Compared with the control group， the model group showed increased expression levels of proinflammatory factors IL-1β and TNF-α in the ACC region （P<0.01） and slightly increased expression levels of anti-inflammatory factors IL-4 and IL-10. Compared with the model group， the fluoxetine group， and the medium- and high-dose LWDHT groups showed down-regulated expression of IL-1β and TNF-α （P<0.05， P<0.01） and up-regulated expression of IL-4 and IL-10 （P<0.05， P<0.01）. Compared with the control group， the model group showed reduced expression levels of Beclin-1 and LC3Ⅱ （P<0.01） and increased expression level of p62 （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed up-regulated Beclin-1 and LC3Ⅱ expression （P<0.01） and down-regulated p62 expression （P<0.01）. Compared with the control group， the model group showed decreased LC3+Iba1+ cells in the ACC region （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed increased LC3+Iba1+ cells （P<0.05， P<0.01）. ConclusionLWDHT can alleviate the depression-like behaviors in DD rats presumedly by promoting MG autophagy， regulating MG phenotypic changes， and increasing MG clearance of myelin sheath fragments. Meanwhile， MG phenotypic transformation also inhibits ACC inflammation in DD rats， improves the local microenvironment of oligodendrocyte proliferation and differentiation， and ultimately promotes the repair and remyelination of damaged myelin sheath.
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Objective:To observe the effects of modified Liuwei Dihuangtang on serum fibroblast growth factor 23 （FGF23）， full-length intact parathyroid hormone （iPTH）， and 1，25-dihydroxyvitamin D<sub>3 </sub>［1，25（OH）<sub>2</sub>D<sub>3</sub>］ levels and Klotho and FGF23 protein expression in renal and bone tissues of rats exposed to high phosphorus combined with adenine， so as to explore the mechanism of modified Liuwei Dihuangtang against renal osteopathy. Method:One hundred and thirty healthy adult SD rats were randomly divided into five groups， namely normal group（<italic>n</italic>=10），high phosphorus group（<italic>n</italic>=30），model group（<italic>n</italic>=30），modified Liuwei Dihuangtang group（<italic>n</italic>=30） ， and calcitriol group（<italic>n</italic>=30），and rats in each group were further classified based on three time points， namely 8，10， and 12 weeks. Rats in the normal group were fed with normal diet， the ones in the high phosphorus group with high phosphorus diet， and those in the other groups with adenine and high phosphorus diet for inducing renal osteopathy. Rats in the normal group，high phosphorus group， and model group were intragastrically administered with distilled water （10 mL·kg<sup>-1</sup>·d<sup>-1</sup>），the ones in the modified Liuwei Dihuangtang group with modified Liuwei Dihuangtang （2.556 g·kg<sup>-1</sup>·d<sup>-1</sup>） ， and those in the calcitriol group with calcitriol （0.09 μg·kg<sup>-1</sup>·d<sup>-1</sup>）. Result:Compared with the normal group and high phosphorus group at the weeks of 8，10 and 12，the model group displayed significantly elevated blood urea nitrogen（BUN），serum creatinine（SCr），serum phosphorus，iPTH，FGF23，renal interstitial fibrosis score， and FGF23 expression in renal and bone tissues， but lowered serum calcium and 1，25（OH）<sub>2</sub>D<sub>3</sub> and Klotho protein expression in renal and bone tissues（<italic>P</italic><0.05 ，<italic>P</italic><0.01）. Compared with the model group at the weeks of 8，10 and 12， the modified Liuwei Dihuangtang and calcitriol both significantly decreased the serum BUN，SCr，serum phosphorus，iPTH， FGF23， tubulointerstitial semi-quantitative score， and FGF23 expression in renal and bone tissues， while increased the serum calcium，1，25（OH）<sub>2</sub>D<sub>3</sub>， and Klotho protein expression in renal and bone tissues （<italic>P</italic><0.05，<italic>P</italic><0.01）. There was no significant difference in the above-mentioned indexes between the modified Liuwei Dihuangtang group and the calcitriol group at the same time point. Conclusion:Klotho-FGF23 axis is probably involved in renal osteopathy. The modified Liuwei Dihuangtang effectively improves renal function，alleviates pathological changes in renal and bone tissues，and regulates calcium and phosphorus metabolism to protect the bone， which is related to its regulation of Klotho-FGF23 axis.
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Objective:To study the effect of the kidney-tonifying recipes on mouse syndrome and adrenal function induced by low-dose hydrocortisone reduction and discontinuation. Method:The 48 male ICR mice were randomly divided into normal control group, hydrocortisone group, Liuwei Dihuangtang group and Guifu Dihuangtang group, with 12 rats in each group. Each group was given 1.32 mg·kg-1·d-1 hydrocortisone for 28 days, and half a dose for 7 days, and the administration was discontinued 14 days before modeling. In the reduction and discontinuation phase, 12.5 g·kg-1·d-1 Liuwei Dihuangtang and 13.5 g·kg-1·d-1 Guifu Dihuangtang were simultaneously administered to the corresponding group for 21 d. On the 28th and 49th day of the experiment, the characteristic information of mice was detected by the experimental methodology of syndrome differentiation and treatment in mice (holding power, body surface infrared temperature). The mice were sacrificed on the 50th day of the experiment, the spleen and thymus were separated, and the spleen and thymus index were calculated, serum corticosterone content is measured by enzyme-linked immunosorbent assay (ELISA), the total RNA of the adrenal gland was extracted, and the expressions of Star, cholesterol-side-chain cleavage enzyme (Cyp11a1), Cyp21a1, Cyp11b1, low-density lipoprotein receptor (Ldlr), scavenger receptor class B type I (Scarb1/SRB1), Hmgcr, acyl-CoA-cholesterol acyltransferase-1 (Acat1), hormone sensitive lipase (Lipe/HSL), insulin-inducible gene 1 (Insig1) and sterol regulatory element binding transcription factor 2 (Srebf2) were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Western blot was used to detect the expressions of LDLR, SRB1, CYP11A1 and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) in adrenal gland. Result:Compared with the normal control group, the body weight and the body mean infrared temperature of the model group were significantly decreased after 28 days of continuous administration (P<0.01). After the administration of the drug, the mice in the model group showed a significant decrease in the grip strength (P<0.05), a significant increase in the thymus index (P<0.01), a significant decrease in the serum corticosterone content (P<0.05). Expressions of adrenal Cyp21a1 and Hmgcr genes decreased (P<0.05, P<0.01), Lipe and Acat1 gene expressions increased significantly (P<0.01), adrenal CYP11A1, SRBI protein expressions decreased significantly, and LDLR protein expression increased. Compared with the model group, the body weight of Guifu Dihuangtang group decreased significantly after 21 days of treatment with traditional Chinese medicine (P<0.01), the spleen index of Liuwei Dihuangtang group decreased significantly (P<0.01), expressions of Cyp11a1, Cyp21a1, Acat1 and Lipe genes in Guifu Dihuangtang group were significantly increased (P<0.05, P<0.01), expressions of Ldlr and Scarb1 genes were significantly decreased (P<0.05), and expressions of Ldlr, Acat1 and Lipe genes in Liuwei Dihuangtang group were significantly decreased (P<0.05, P<0.01), and expressions of CYP11A1 and SRBI proteins in Liuwei Dihuangtang and Guifu Dihuangtang increased. Conclusion:Guifu Dihuangtang can promote the recovery of adrenal function in mice with drug-induced deficiency syndrome by correcting expression of adrenal cortex synthase.
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Objective::To investigate the effect and possible mechanism of Liuwei Dihuangtang on memory impairment in rats with chronic depression. Method::Female Wistar rats were randomly divided into normal group (normal saline), chronic unpredictable mild stress (CUMS) model group (normal saline), and low, medium and high-dose Liuwei Dihuangtang groups (2.60, 7.81, 23.50 g·kg-1·d-1). Except for the normal group, all of the other groups were included in the chronic unpredictable mild stress model. Weight were measured every week, changes in their behavioral indicators were observed. The mRNA expressions of G protein-coupled estrogen receptor (GPR30), phosphatidylinositol 3-kinase(PI3K), cyclic adenosine monophosphate response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the rat hippocampus were determined by real-time fluorescence quantification polymerase chain reaction (Real-time PCR). The concentration of serum estrogen was detected by enzyme-linked immunosorbent assay (ELISA) method. Result::Compared with normal group, model group showed decreases in weight, activity and interest. Liuwei Dihuangtang (2.60, 7.81, 23.50 g·kg-1) significantly increased the sucrose preference of CUMS rats (P<0.01) and standing times in the open field test (P<0.01), doses of 7.81, 23.50 g·kg-1 significantly increased the total distance of the open field test (P<0.05, P<0.01), doses of 2.60, 7.81 g·kg-1 shortened the latency of water maze experiment (P<0.01), and dose of 7.81 g·kg-1 increased serum estrogen concentration (P<0.05). The mRNA expressions of GPR30, PI3K, CREB and BDNF in hippocampus of CUMS model group decreased significantly (P<0.05, P<0.01), but the mRNA expression levels of GPR30, CREB in hippocampus of 2.60 g·kg-1 dose Liuwei Dihuangtang group increased significantly (P<0.05, P<0.01), and the mRNA expression levels of GPR30, PI3K, CREB, BDNF in hippocampus of 7.81 g·kg-1 dose group increased significantly as well (P<0.05, P<0.01). Conclusion::Liuwei Dihuangtang has effect in resisting depression, and reversing depression-like behavior and learning and memory impairment in CUMS rats, with the best effect in the medium-dose Liuwei Dihuangtang group. Its mechanism may be related to increase of serum estrogen and mRNA expressions of GPR30, PI3K, CREB and BDNF in rat hippocampus.
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Objective:To observe the effect of Liuwei Dihuangtang on the mitochondrial apoptotic pathway in rats with chronic renal failure. Method:The 45 male SD rats were randomly divided into normal group （n=10）， sham group （n=10） and operation group （n=25）. The operation group received 5/6 nephrectomy， and the rats in operation group were randomly divided into the model group and the treatment group after successful modeling. The treatment group received Liuwei Dihuangtang by gavage administration， while the rest of the three groups received equal volume of distilled water by gavage administration， with a treatment course of 8 weeks. The quantitative levels of serum creatinine （SCr）， blood urea nitrogen （BUN） and urine protein in each group were determined. Hematoxylin eosin （HE） staining and Masson staining were used to observe the histopathological changes of rat kidney. The structural changes of mitochondria in renal tubular epithelial cells were observed under electron microscope. The apoptotic rate was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling（TUNEL）. The expression of B cell lymphoma -2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cytochrome C （Cyt C） and cysteine aspartic acid protease -3 （Caspase-3） were analyzed by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:As compared with the normal group， the quantitative levels of SCr， BUN and urinary protein in the model group were significantly increased （P<0.01）， and the above indicators were improved in the treatment group as compared with the model group （P<0.05）. No significant pathological changes and damage of mitochondrial structure were observed in the kidney tissues of the control group and the sham group. In the model group， there were different degrees of renal tubular atrophy and dilatation， interstitial inflammatory cell infiltration and collagen deposition， and mitochondria swelling and breaking， but such lesions were significantly alleviated in the treatment group. As compared with the normal group， apoptotic rate was increased significantly in model group （P<0.01）， the expression of Bax and Caspase-3 was significantly increased （P<0.01）， and the expression of Bcl-2 was significantly decreased （P<0.01）. As compared with the model group， apoptotic rate was significantly reduced in the treatment group （P<0.01）， the expression of Bax and Caspase-3 was significantly decreased （P<0.01）， and the expression of Bcl-2 was significantly increased （P<0.01）. In the normal group and sham group， Cyt C was mainly located in mitochondria. The expression of Cyt C in the cytoplasm of the model group was increased significantly （P<0.01）， while that of the treatment group was decreased （P<0.01）. Conclusion:Liuwei Dihuangtang can alleviate renal fibrosis and delay the progression of renal function. The mechanism of reducing apoptosis may be associated with protecting mitochondrial structure and regulating the expression of proteins related to mitochondrial apoptotic pathway.