Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by cas...Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.展开更多
The meibomian gland is a unique sebaceous gland located in the eyelid.Its main function is to secrete lipids and form the lipid layer of the tear film to delay the evaporation of waterborne tears,increase the surface ...The meibomian gland is a unique sebaceous gland located in the eyelid.Its main function is to secrete lipids and form the lipid layer of the tear film to delay the evaporation of waterborne tears,increase the surface tension of the tear film,and to lubricate the contact area of the eyelid and eyeball.Abnormal secretion of the meibomian gland is known as meibomian gland dysfunction(MGD),which has become the most important cause of evaporative dry eye disease(DED).The clinical pathophysiological process and underlying molecular mechanisms of MGD are not clear.As serious side effects may occur with the long-term use of hormonotherapy and non-steroidal anti-inflammatory drugs(NSAIDs)for the treatment of MGD,meibomian gland physiotherapy is considered the most effective and safest therapy for MGD.This review summarizes the physical therapy modalities of western medicine(WM)and traditiond Chinese medicine(TCM)for the treatment of MGD to provide optimal treatments for these patients and to further lay a foundation for mechanistic studies of MGD.展开更多
Background Comeal neovascular leakage can lead to edema and secondary scarring. Previous studies have shown that pericytes play a key role in maturation of angiogenesis. The present studies investigate the relationshi...Background Comeal neovascular leakage can lead to edema and secondary scarring. Previous studies have shown that pericytes play a key role in maturation of angiogenesis. The present studies investigate the relationship between vascular permeability and pericyte coverage of endothelial cells in rat corneal neovascular induced by alkali bums. Methods Corneal neovascular vessels induced by alkali bums was performed in Sprague-Dawley rats. Corneas were excised on 1,2, 3, 5, 7 and 10 days after cauterization. The vascular permeability rate was measured by the Evans blue method. The microvessel pericyte coverage index （MPI） was applied to quantify the pericyte coverage through double immunofluorescent staining of frozen sections of corneas with CD31 as the endothelial and α-smooth muscle actin （α-SMA） as the pericyte markers. The correlation between permeability rate and MPI was analyzed. Pericyte coverage was confirmed ultrastructually using transmission electron microscopy. Results The vascular permeability rate was （1.14±0.17）, （0.24±0.08）, （0.29±0.16）, （0.14±0.10）, （0.09±0.06） and （0.05±0.04）μg· ml^-1 · mm^-2respectively on 1, 2, 3, 5, 7 and 10 days after cauterization. The MPI was 0, 16.07%, 11.95%, 43.84%, 73.97% and 86.21% respectively at the above mentioned time points. The correlation coefficient between MPI and the permeability rate was -0.943 （P=-0.005）. Conclusions Pericyte recruitment was significantly correlated with the permeability of comeal neovascularization induced by alkali bums in rats. Therapeutic strategies aiming at anti-leakage should be most effective if they promote pericytes proliferation in the course of corneal neovascularization.展开更多
Background The treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells （MSCs） for spinal cord injury. Metho...Background The treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells （MSCs） for spinal cord injury. Methods In this study, rhesus MSCs were isolated and induced by cryptotanshinone in vitro and then a process of RT-PCR was used to detect the expression of glutamic acid decarboxylase （GAD） gene. The induced MSCs were tagged with Hoechst 33342 and injected into the injury site of rhesus spinal cord made by the modified Allen method. Following that, behavior analysis was made after 1 week, 1 month, 2 months and 3 months. After 3 months, true blue chloride retrograde tracing study was also used to evaluate the reestablishment of axons pathway and the hematoxylin-eosin （HE） staining and immunohistochemistry were performed after the animals had been killed. Results In this study, the expression of mRNA of GAD gene could be found in the induced MSCs but not in primitive MSCs and immunohistochemistry could also confirm that rhesus MSCs could be induced and differentiated into neurons. Behavior analysis showed that the experimental animals restored the function of spinal cord up to grade 2 -3 of Tarlov classification. Retrograde tracing study showed that true blue chollide could be found in the rostral thoracic spinal cords, red nucleus and sensory-motor cortex. Conclusions These results suggest that the transplantation is safe and effective.展开更多
Background Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 （MEN1）gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage...Background Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 （MEN1）gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage leukemia （MLL） protein, a histone H3 lysine 4 methyltransferase, and plays a critical role in hematopoiesis and leukemogenesis.Previous studies have shown that menin promotes transforming growth factor beta （TGF-β） signaling in endocrine cells.However, little is known regarding the impact of TGF-β pathway on menin in hematopoietic system. Here, with leukemia cell lines generated from conditional MEN1 or TGF-p receptor （TβRII） knockout mouse models, we investigated the possible cross-talk of these two pathways in leukemia cells.Methods MEN1 or TβRII conditional knockout mice were bred and the bone marrow cells were transduced with retroviruses expressing oncogeneic MLL-AF9 （a mixed lineage leukemia fusion protein） to generate two leukemia cell lines. Cell proliferation assays were performed to investigate the effect of TGF-β treatment on MLL-AF9 transformed leukemia cells with/without MEN1 or TβRII excision. Menin protein was detected with Western blotting and mRNA levels of cell proliferation-related genes Cyclin A2 and Cyclin E2 were examined with real-time RT-PCR for each treated sample.In vivo effect of TGF-p signal on menin expression was also investigated in mouse liver tissue after TβRII excision.Results TGF-β not only inhibited the proliferation of wild type MLL-AF9 transformed mouse bone marrow cells, but also up-regulated menin expression in these cells. Moreover, TGF-P failed to further inhibit the proliferation of Men1-null cells as compared to Men1-expressing control cells. Furthermore, excision of TβRII, a vital component in TGF-β signaling pathway, down-regulated menin expression in MLL-AF9 transformed mouse bone marrow cells. In vivo data also confirmed that menin expression was decreased in liver samples of conditional TβRII knockout mice after TβRII excision.Conclusion These results provided the first piece of evidence of cross-talk between menin and TGF-β signaling pathways in regulating proliferation of leukemia cells, suggesting that manipulating the cross-talk of the two pathways may lead to a novel therapy for leukemia.展开更多
基金We thank for the funding support from the National Natural Science Foundation of China(No.81260550)Key Laboratory Construction Project of Traditional Chinese Medicine for Prevention and Treatment of Five Sense Organ Diseases in Hunan Province(No.2017TP1018)Key Subject Construction Project of Traditional Chinese Medicine Ophthalmology of the State Administration of Traditional Chinese Medicine(No.ZK1801YK015).
文摘Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.
基金We thank for the funding support from the Research and Innovation Project of Graduate Students in Hunan Province(No.CX20190538)the First-class Open Fund for Integrated Chinese and Western Medicine(No.2018ZXYJH05)+1 种基金the Traditional Chinese Medicine First-Class Discipline Open Fund(No.2018ZYX57)the Construction Project of Hunan Engineering Technology Research Center for the Prevention and Treatment of Otorhinolaryngologic Diseases and Protection of Visual Function with Chinese Medicine(No.2018YGC02).
文摘The meibomian gland is a unique sebaceous gland located in the eyelid.Its main function is to secrete lipids and form the lipid layer of the tear film to delay the evaporation of waterborne tears,increase the surface tension of the tear film,and to lubricate the contact area of the eyelid and eyeball.Abnormal secretion of the meibomian gland is known as meibomian gland dysfunction(MGD),which has become the most important cause of evaporative dry eye disease(DED).The clinical pathophysiological process and underlying molecular mechanisms of MGD are not clear.As serious side effects may occur with the long-term use of hormonotherapy and non-steroidal anti-inflammatory drugs(NSAIDs)for the treatment of MGD,meibomian gland physiotherapy is considered the most effective and safest therapy for MGD.This review summarizes the physical therapy modalities of western medicine(WM)and traditiond Chinese medicine(TCM)for the treatment of MGD to provide optimal treatments for these patients and to further lay a foundation for mechanistic studies of MGD.
基金National Science Fund for Distinguished Young Scholars(No.30225044)Fund for Innovative Research Groups of China (No. 30321004)Natural Science Foundation of Guangdong Province of China (No. 36652).
文摘Background Comeal neovascular leakage can lead to edema and secondary scarring. Previous studies have shown that pericytes play a key role in maturation of angiogenesis. The present studies investigate the relationship between vascular permeability and pericyte coverage of endothelial cells in rat corneal neovascular induced by alkali bums. Methods Corneal neovascular vessels induced by alkali bums was performed in Sprague-Dawley rats. Corneas were excised on 1,2, 3, 5, 7 and 10 days after cauterization. The vascular permeability rate was measured by the Evans blue method. The microvessel pericyte coverage index （MPI） was applied to quantify the pericyte coverage through double immunofluorescent staining of frozen sections of corneas with CD31 as the endothelial and α-smooth muscle actin （α-SMA） as the pericyte markers. The correlation between permeability rate and MPI was analyzed. Pericyte coverage was confirmed ultrastructually using transmission electron microscopy. Results The vascular permeability rate was （1.14±0.17）, （0.24±0.08）, （0.29±0.16）, （0.14±0.10）, （0.09±0.06） and （0.05±0.04）μg· ml^-1 · mm^-2respectively on 1, 2, 3, 5, 7 and 10 days after cauterization. The MPI was 0, 16.07%, 11.95%, 43.84%, 73.97% and 86.21% respectively at the above mentioned time points. The correlation coefficient between MPI and the permeability rate was -0.943 （P=-0.005）. Conclusions Pericyte recruitment was significantly correlated with the permeability of comeal neovascularization induced by alkali bums in rats. Therapeutic strategies aiming at anti-leakage should be most effective if they promote pericytes proliferation in the course of corneal neovascularization.
文摘Background The treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells （MSCs） for spinal cord injury. Methods In this study, rhesus MSCs were isolated and induced by cryptotanshinone in vitro and then a process of RT-PCR was used to detect the expression of glutamic acid decarboxylase （GAD） gene. The induced MSCs were tagged with Hoechst 33342 and injected into the injury site of rhesus spinal cord made by the modified Allen method. Following that, behavior analysis was made after 1 week, 1 month, 2 months and 3 months. After 3 months, true blue chloride retrograde tracing study was also used to evaluate the reestablishment of axons pathway and the hematoxylin-eosin （HE） staining and immunohistochemistry were performed after the animals had been killed. Results In this study, the expression of mRNA of GAD gene could be found in the induced MSCs but not in primitive MSCs and immunohistochemistry could also confirm that rhesus MSCs could be induced and differentiated into neurons. Behavior analysis showed that the experimental animals restored the function of spinal cord up to grade 2 -3 of Tarlov classification. Retrograde tracing study showed that true blue chollide could be found in the rostral thoracic spinal cords, red nucleus and sensory-motor cortex. Conclusions These results suggest that the transplantation is safe and effective.
基金This work was supported by grants from the National Institutes of Health in USA （R01-CA-100912 and R01-CA-113962）, a Fellowship from China Scholarship Council and National Natural Science Foundation of China （No. 30600697）.Acknowledgement： We thank Dr. Harold Moses at Vanderbilt University for providing the mice with the floxed TβRII alleles.
文摘Background Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 （MEN1）gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage leukemia （MLL） protein, a histone H3 lysine 4 methyltransferase, and plays a critical role in hematopoiesis and leukemogenesis.Previous studies have shown that menin promotes transforming growth factor beta （TGF-β） signaling in endocrine cells.However, little is known regarding the impact of TGF-β pathway on menin in hematopoietic system. Here, with leukemia cell lines generated from conditional MEN1 or TGF-p receptor （TβRII） knockout mouse models, we investigated the possible cross-talk of these two pathways in leukemia cells.Methods MEN1 or TβRII conditional knockout mice were bred and the bone marrow cells were transduced with retroviruses expressing oncogeneic MLL-AF9 （a mixed lineage leukemia fusion protein） to generate two leukemia cell lines. Cell proliferation assays were performed to investigate the effect of TGF-β treatment on MLL-AF9 transformed leukemia cells with/without MEN1 or TβRII excision. Menin protein was detected with Western blotting and mRNA levels of cell proliferation-related genes Cyclin A2 and Cyclin E2 were examined with real-time RT-PCR for each treated sample.In vivo effect of TGF-p signal on menin expression was also investigated in mouse liver tissue after TβRII excision.Results TGF-β not only inhibited the proliferation of wild type MLL-AF9 transformed mouse bone marrow cells, but also up-regulated menin expression in these cells. Moreover, TGF-P failed to further inhibit the proliferation of Men1-null cells as compared to Men1-expressing control cells. Furthermore, excision of TβRII, a vital component in TGF-β signaling pathway, down-regulated menin expression in MLL-AF9 transformed mouse bone marrow cells. In vivo data also confirmed that menin expression was decreased in liver samples of conditional TβRII knockout mice after TβRII excision.Conclusion These results provided the first piece of evidence of cross-talk between menin and TGF-β signaling pathways in regulating proliferation of leukemia cells, suggesting that manipulating the cross-talk of the two pathways may lead to a novel therapy for leukemia.
