significantly reduced lymphadenopathy, salivary gland infiltrates and proteinuria in mrl-lpr/lpr mice treated with ultrasoluble curcumin/turmeric: increased survival with curcumin treatment

by:Petolar     2020-07-10
Curcumin for commercial use (CU)
Ginger powder from food spices (TU)
As a potential treatment for multiple tumors and inflammatory diseases, it has been widely studied.
The lack of dissoluble/biological utilization hinders the therapeutic effect of curcumin in human diseases.
We dissolve the curry in water under heating/pressure to get up to 35-
Double the solubility (
(Super)UsC)).
We assume that the University of Southern California or the ultrasonic ginger (UsT)
Will improve systemic erythema (SLE)
And schagne syndrome (SS)-
Like the disease in MRL. lpr/lpr mice.
Method 18 female MRL-lpr/lpr (6 weeks old)
18 female MRLMpJ mice (6 weeks old)were used. Female MRL-
Lpr/License Plate Recognition developed lupus mice-
Just like the disease at 10 weeks, the average death at 17 weeks. MRL-
MpJ mice developed into lupus-
Diseases like around 47 weeks usually die in 73 weeks.
Six mice of each strain only received high pressure sterilized water (lpr-water or MpJ-water group), UsC (lpr-CU or MpJ-CU group)or UsT (lpr-TU or MpJ-TU group)
In a water bottle.
Results in MRL, the UsC or UsT improved the SLE-
Lpr/lpr mice by significantly reducing lymphatic proliferation, urinary protein, and lesions (tail)
And their own antibodies. lpr-
Compared with lpr, CU group has a survival advantage of 20%. water group. However, lpr-
The average life of the TU group was 16 days shorter than that of the lpr
Water Group caused by complications unrelated to lupuslike illness.
CU/TU treatment significantly inhibited lymph node lesions compared to lprwater group (p=0. 03 and p=0.
02 respectively)
By inducing cell apoptosis
The mean weight of lymph nodes was 2606 ± 1147,742 ± 331 and 385 ± 68 mg, respectively, lpr-water, lpr-CU and lpr-TU mice.
Transfer enzyme dUTP Nick end labeling Test display lpr-CU and lpr-
Cell apoptosis occurred in TU mice.
Significantly reduced cell infiltration of the spit glands in salivary
Group TU compared to TU
In the water group, the trend of decreased renal damage was observed in lprCU and lpr-TU groups.
Conclusion these studies suggest that UsC/UsT can be shown to be therapeutic interventions.
Purpose: Commercial useCU)
Ginger powder from food spices (TU)
As a potential treatment for multiple tumors and inflammatory diseases, it has been widely studied.
The lack of dissoluble/biological utilization hinders the therapeutic effect of curcumin in human diseases.
We dissolve the curry in water under heating/pressure to get up to 35-
Double the solubility (
(Super)UsC)).
We assume that the University of Southern California or the ultrasonic ginger (UsT)
Will improve systemic erythema (SLE)
And schagne syndrome (SS)-
Like the disease in MRL. lpr/lpr mice.
Method 18 female MRL-lpr/lpr (6 weeks old)
18 female MRLMpJ mice (6 weeks old)were used. Female MRL-
Lpr/License Plate Recognition developed lupus mice-
Just like the disease at 10 weeks, the average death at 17 weeks. MRL-
MpJ mice developed into lupus-
Diseases like around 47 weeks usually die in 73 weeks.
Six mice of each strain only received high pressure sterilized water (lpr-water or MpJ-water group), UsC (lpr-CU or MpJ-CU group)or UsT (lpr-TU or MpJ-TU group)
In a water bottle.
Results in MRL, the UsC or UsT improved the SLE-
Lpr/lpr mice by significantly reducing lymphatic proliferation, urinary protein, and lesions (tail)
And their own antibodies. lpr-
Compared with lpr, CU group has a survival advantage of 20%. water group. However, lpr-
The average life of the TU group was 16 days shorter than that of the lpr
Water Group caused by complications unrelated to lupuslike illness.
CU/TU treatment significantly inhibited lymph node lesions compared to lprwater group (p=0. 03 and p=0.
02 respectively)
By inducing cell apoptosis
The mean weight of lymph nodes was 2606 ± 1147,742 ± 331 and 385 ± 68 mg, respectively, lpr-water, lpr-CU and lpr-TU mice.
Transfer enzyme dUTP Nick end labeling Test display lpr-CU and lpr-
Cell apoptosis occurred in TU mice.
Significantly reduced cell infiltration of the spit glands in salivary
Group TU compared to TU
In the water group, the trend of decreased renal damage was observed in lprCU and lpr-TU groups.
Conclusion these studies suggest that UsC/UsT can be shown to be therapeutic interventions.
Systemic erythema (SLE)
It is a devastating chronic inflammatory autoimmune disease that can affect almost any organ of the body.
Women occur 10 times more than men, and autoantibody is the main feature of the disease, appearing 10 years before the diagnosis of the disease.
1-5 sj gren syndrome (SS)
Like other diseases, this is a weak systemic and chronic inflammatory, autoimmune disease characterized by self-antibody.
The main feature of SS is decreased secretion of tear gland (sicca complex)
, Resulting in Kernal inflammation and dry mouth. 6–8MRL-
Animal model of Lpr/lpr SLE mice/SSMRL-
Lpr/lpr mice are similar to human SLE in many ways and are therefore a widely used animal model. 9–18 MRL-
Lpr/License Plate Recognition mouse has a singlegene mutation (lpr)
The loss of fas apoptosis gene on chromosome 19 of mice, therefore, the defect of lymphocytes apoptosis death.
Cd4-cd8-T a large accumulation of lymphocytes and develop into a disease similar to human SLE.
Although lymphatic proliferation is less prominent in most people, other features, such as nephritis, skin lesions, arthritis, and nervous system performance, also match well.
However, double lymph node diseasenegative T-
Cell proliferation may mimic the defective apoptosis mechanism and its removal of human lupus. MRL-
Saliva and tear gland lymphocytes infiltration in Salivary/lpr mice is similar to human SS, but there is no loss of function. 13–15MRL-
Although MPJ miceMRL/MpJ mice have a normal Fas gene, their immune symptoms are higher than that of MRL-lpr/lpr mice.
The longevity of MRL/MpJ melee female mice was 73 weeks, while that of male mice was 93 weeks. MRL-
Lpr/lpr female mice usually die at 17 weeks of age, while male mice die at 22 weeks of age. MRL-
MpJ mice as control of MRL-lpr/lpr mice (
To a large extent, it is not known what causes it.
There is no effective treatment other than an immune inhibitor.
The current treatment has great side effects.
Therefore, it is most important to conduct research to identify new therapeutic drugs with few side effects.
Quantitative studies by ultrasound showed a significant increase in oxidative damage in patients19-21, which led us to study an antioxidant/Antioxidant
The inflammatory substance obtained from the root-like stem of ginger is called Curry, 22-31 (turmeric). Curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione)
A naturally occurring \"nutrient component\" consisting of 4-6% yellow ginger.
Commercial combination of turmeric (77%)
A compound of noroxygen (DMC)(17%)
And ginger (BDMC)(3%).
When abbreviated as CU, Curcumin will refer to a commercially available mixture containing all three curcuminoid compounds.
Copper has been studied in prostate cancer, esophagus, lung cancer, breast cancer and oral cancer.
Recently, CU has been shown to inhibit nuclear factor-
KB and increased apoptosis of both
1 and OE33 esophageal adenocarcinoma cell lines 36, 37 CU also induce the release of pigment C, the activation of aspases and p53 and produce anti-vascular production by down-regulation of vascular endothelial growth factor.
It has been shown that oil 38, 39 TU has antibacterial, mosquito-killing, pesticide, antibacterial, antioxidant, mutation-resistant and anti-cancer activities.
40-43 most in vitro or in vivo studies were conducted with CU dissolved in organic solvents or CU powder mixed in animal feed.
We have proved heat treatment (100°C)
Increase the solubility of CU/TU 12-fold/3-
Fold separately in water.
44 then we found that heat/pressure (121°C/15u2005psi)
Treatment to improve the solubility of CU 35-fold (
Ultrasonic CU (UsC);
Unpublished data). Our antibody-
Antigen suppression studies performed with CU45 suggest possible therapeutic intervention pathways for experimental SLE and SS.
To test this, we gave the water, the University of Southern California, or the ultrasonic ginger powder (UsT)to six MRL-
Lpr/License Plate Recognition or peaceful moving mice. MRL-
Animal model of Lpr/lpr SLE mice/SSMRL-
Lpr/lpr mice are similar to human SLE in many ways and are therefore a widely used animal model. 9–18 MRL-
Lpr/License Plate Recognition mouse has a singlegene mutation (lpr)
The loss of fas apoptosis gene on chromosome 19 of mice, therefore, the defect of lymphocytes apoptosis death.
Cd4-cd8-T a large accumulation of lymphocytes and develop into a disease similar to human SLE.
Although lymphatic proliferation is less prominent in most people, other features, such as nephritis, skin lesions, arthritis, and nervous system performance, also match well.
However, double lymph node diseasenegative T-
Cell proliferation may mimic the defective apoptosis mechanism and its removal of human lupus. MRL-
Saliva and tear gland lymphocytes infiltration in Salivary/lpr mice is similar to human SS, but there is no loss of function. 13–15MRL-
Although MPJ miceMRL/MpJ mice have a normal Fas gene, their immune symptoms are higher than that of MRL-lpr/lpr mice.
The longevity of MRL/MpJ melee female mice was 73 weeks, while that of male mice was 93 weeks. MRL-
Lpr/lpr female mice usually die at 17 weeks of age, while male mice die at 22 weeks of age. MRL-
MpJ mice as control of MRL-lpr/lpr mice (
To a large extent, it is not known what causes it.
There is no effective treatment other than an immune inhibitor.
The current treatment has great side effects.
Therefore, it is most important to conduct research to identify new therapeutic drugs with few side effects. MRL-
Although MPJ miceMRL/MpJ mice have a normal Fas gene, their immune symptoms are higher than that of MRL-lpr/lpr mice.
The longevity of MRL/MpJ melee female mice was 73 weeks, while that of male mice was 93 weeks. MRL-
Lpr/lpr female mice usually die at 17 weeks of age, while male mice die at 22 weeks of age. MRL-
MpJ mice as control of MRL-lpr/lpr mice (
To a large extent, it is not known what causes it.
There is no effective treatment other than an immune inhibitor.
The current treatment has great side effects.
Therefore, it is most important to conduct research to identify new therapeutic drugs with few side effects.
Quantitative studies by ultrasound showed a significant increase in oxidative damage in patients19-21, which led us to study an antioxidant/Antioxidant
The inflammatory substance obtained from the root-like stem of ginger is called Curry, 22-31 (turmeric). Curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione)
A naturally occurring \"nutrient component\" consisting of 4-6% yellow ginger.
Commercial combination of turmeric (77%)
A compound of noroxygen (DMC)(17%)
And ginger (BDMC)(3%).
When abbreviated as CU, Curcumin will refer to a commercially available mixture containing all three curcuminoid compounds.
Copper has been studied in prostate cancer, esophagus, lung cancer, breast cancer and oral cancer.
Recently, CU has been shown to inhibit nuclear factor-
KB and increased apoptosis of both
1 and OE33 esophageal adenocarcinoma cell lines 36, 37 CU also induce the release of pigment C, the activation of aspases and p53 and produce anti-vascular production by down-regulation of vascular endothelial growth factor.
It has been shown that oil 38, 39 TU has antibacterial, mosquito-killing, pesticide, antibacterial, antioxidant, mutation-resistant and anti-cancer activities.
40-43 most in vitro or in vivo studies were conducted with CU dissolved in organic solvents or CU powder mixed in animal feed.
We have proved heat treatment (100°C)
Increase the solubility of CU/TU 12-fold/3-
Fold separately in water.
44 then we found that heat/pressure (121°C/15u2005psi)
Treatment to improve the solubility of CU 35-fold (
Ultrasonic CU (UsC);
Unpublished data). Our antibody-
Antigen suppression studies performed with CU45 suggest possible therapeutic intervention pathways for experimental SLE and SS.
To test this, we gave the water, the University of Southern California, or the ultrasonic ginger powder (UsT)to six MRL-
Lpr/License Plate Recognition or peaceful moving mice.
Materials and Methods purchased scurcumin from Sigma Chemical.
St. Louis, Missouri.
Bought from a local grocery store.
Self-antigen Sm and rna protein (RNP)
It\'s AK of immune vision. HEp-
2 Anti-nuclear antibody (ANA)
Test kit and anti-hepatitis Bdouble-stranded DNA (anti-dsDNA)
The test kit comes from INOVA Diagnostics in San Diego, California, USA.
The 10 urine reagent strips of Accustrip are from Jant Pharmacal Corporation in Encino, California, USA.
The secondary antibody binding is from the Jackson Laboratory in Bar Harbor, Maine.
All of the other chemicals are grade 5. AnimalsMRL-Lpr/License Plate Recognition and mri-
Buy MpJ mouse from Jackson Lab (
Balang, Massachusetts, United States of America).
Animals are kept at the OMRF Laboratory Animal Resource Center of the oklaoma medical research foundation with a light/dark cycle of 12 hours.
Standard gamma-feeding in mice-
Irradiation week (
Picolab Rodent animal diet 20, label diet, St.
Louis, Missouri, USA)
And water ads.
For mice selected for curry or ginger huanggen treatment, a heated/pressure-dissolved curry or ginger huanggen is provided in a water bottle.
According to the established guidelines, all experimental designs are approved by the Animal Care and Use Committee of the OMRF institution.
Curry and ginger dissolved cu and TU are hot
Dissolution/pressure-
Dissolve in water of 5 mg/ml (UsC and UsT)
And provide mice in the water bottle.
Heat CU in ultra-pure water of 5 ml and boil in aluminum foil-
Cover the cone bottle on the hot plate for 1 hour and sterilize for 30 minutes under high pressure. The heat-
Dissolution/pressure-
Dissolved CU is filtered with Whatman 1 filter after cooling. This filtrate (UsC)
The mouse in the water bottle.
TU in ultra pure water of 5 mg/ml, heated on the hot plate to boil for 1 hour, and centrifuge at mg/ml for 20 minutes after cooling.
The supernatant (UsT)
The mouse in the water bottle was provided.
We do not have the TU heated by high pressure sterilization, because there is no significant difference in the dissolution of TU during high pressure sterilization after heating on the hot plate.
Ultra-pure water for high pressure sterilization and cooling will be supplied to control mice in water bottles.
For the UsC uptake study, we used the fact that the emission spectrum of CU fluorescence at 458 nm was 505-575 nm.
46. For this experiment, the Epstein-Barr virus (EBV)
Transforming human peripheral blood single nuclear cells (PBMCs)(
200000 000 cells/mL)
Added to a room in the lab. Tek 8-
Tissue culture slide in chamber.
The chamber slide was placed on the stage plug-in of Zeiss 710 co-focal microscope.
You can view the cells and arrange the settings before you add UsC. UsC (350u2005µL)
Into eb transformed cells.
After joining the UsC, cells are observed and photographed every five seconds.
Control cells heat treated with 50 μ l
The treated water does not show any fluorescence (data not shown).
Experimental treatmentweek-old female MRL-Lpr/License Plate Recognition and mri-
MpJ mice were used in the experiment. MRL-
Lpr/license plate recognition to establish lupus-like in female mice
Just like the disease at 10 weeks, the average death at 17 weeks. Female MRL-
MpJ mouse lupus-
Like a disease later in life47th week)
Usually died after 73 weeks. MRL-Lpr/License Plate Recognition and mri-
MpJ mice were divided into 6 groups, with 6 mice in each group (
3 Animals/cages)as follows.
Provide the animals in the drinking water bottle with high pressure sterilized water, UsC or UsT. Group 1 (lpr-water)—
Two groups were given high pressure sterilization water (lpr-CU)—
Group 3 (lpr-TU)—
In view of the UsT Group month (MpJ-water)—
5 groups were given high pressure sterilization water (MpJ-CU)—
Group 6 (MpJ-TU)—
Give UsTWater, replenish the UsC or UsT in the beverage bottle every three days, and calculate the consumption every week in each group.
Blood and urine were collected every two weeks and every week.
47, 48 protein semi-test
Quantitative analysis by oil ruler (
URS Accustrip month)from week 7. MRL-
MpJ mice were killed at the end of 36 weeks. MRL-
Lpr/lpr mice died for 32 weeks due to disease.
It was also evaluated by the duodenum sulfuric acid ester-polypropylene gel swimming apparatus in the urine of mice.
After Cell and tube type were removed, an equal sample of mouse urine was analyzed on a 4-20% gradient gel and stained with Komas bright blue.
The presence of antibodies against Sm or RNP self-antigen in 49, 50 Sm and RNP ELISASera with continuous bleeding from different experimental groups was analyzed.
For this purpose, Sm or RNP self-antigen is used as a direct ELISA for solids-
Phase antigen is performed as described.
Basically, cattle Sm or RNP are applied to 96-
In the coating buffer, overnight at 4 °c, 5 g/mL.
The plate was 3% non-
Fat dry milk in phosphate buffer salt water (PBS)
For 2 hours at room temperature.
The primary serum was added and incubated for 2 h at room temperature.
After washing 4 times with PBS containing 0. 05% Tween-20 (PBST)
, Add appropriate alkaline phosphate dressed-in at room temperature and incubate for 2 u2005 h.
After washing four times with PBST, add the substrate and read the absorbance at 405 nm.
Indirect immune fluorescence-
Using 2 slides according to the manufacturer\'s instructions, ANA was determined by indirect immune fluorescence (IIF)assay.
Dilute the serum with PBS (
1: 40, 1: 80, 1: 160 or 1: 320).
The diluted samples were added to the slide, incubated for about 40 min and washed with PBS.
The slide was then incubated with a mouse-collagen conjugate diluted with PBS at 1:100.
Clean and observe slides with a fluorescence microscope. C.
Luciliae assayC.
The Luciliae slide is used according to the manufacturer\'s instructions for determining the resistance
DsDNA antibody was detected by IIF.
To put it simply, dilute the serum with PBS (
1:10, 1:100, 1:200, 1:300 or 1:500).
The diluted samples were added to the slide, incubated for about 40 min and washed with PBS.
The slide was then incubated with a mouse-collagen conjugate diluted with PBS at 1:100.
Clean and observe slides with a fluorescence microscope.
At room temperature, fresh mouse urine rotates for 5 min at 200g and applies 5 μ l particles to the microscope slide, cover the coverslip and check with an optical microscope.
Tissue pathology studies in the 10% neutral collection of the animal sacrifice, kidney, spit gland and spleen
Buffer formaldehyde, stored at room temperature.
The fixed tissue is embedded in paraffin, sliced at 5 μm and stained with H & E or periodic acid Shifu reagent (PAS).
PAS staining was performed on animals in all treatment groups to assess the severity of nephritis.
Optimum cutting temperature for some tissues at 50: 50: Tissue freezing medium (
Durham Triangle Biomedical Center, North Carolina, United States)
Freeze-save at-80 °c for immune fluorescence staining.
The stained slides were evaluated and scored blindly.
As shown in the figure, the statistical analysis values for each group are given at mean ± sd or SE.
Statistical analysis was performed by Student t test or Mann-Whitney U test and p-Whitney test
In order to evaluate the statistical significance of the observed changes, the values were obtained. p Extended service life 20%.
Two Mice belonging to the lpr
At 15 and 16 weeks, the water group died due to the disease, and the LNs and urine protein increased.
The first two dead in Lpr
The CU group appeared at 19 weeks and 20 weeks, but there was no obvious lymph node lesion in animals (figure 2bB).
There are no mice in Lpr-CU or lpr-
The TU group did not have urinary cell tube type until at least 15 weeks. The lpr-
Shortened life of TU group (
Average 132 days for Lpr vs 148 days-water group)
Due to complications that may not be associated with lupuslike illness (table 1A).
Five of the six mice in Lpr
There are intestinal problems in TU group (
Narrow intestinal cavity, atrophy of the stomach, blood stomach or blood intestine).
Two Mice in Lpr
The TU group died between 16 weeks and 17 cycles, but there was no evidence of erythema but intestinal complications.
No lpr-despite these deaths-
Group TU animals have lupus-
It\'s like a disease with few lymph nodes.
We assume that the lack of lymphatic proliferation may be due to the passage of non-fas-
The channel of mediation.
To verify this hypothesis, we studied CU-treated or TU-
The animals were analyzed and treated using transfer enzyme dUTP notch end labeling.
We detected apoptosis in lymphocytes (figure 5c)
Of the 3/6 mice in lpr
3/6 mice in TU group and lpr-
CU group, no significant apoptosis in lprwater group (data not shown).
These results explain a possible mechanism for treating a significant reduction in lymph node lesions in mice.
The UsC enters the cultured cells within 30 minutes and enters the B cells at room temperature and within 30 minutes (
5% CO2 in microscope chamber at 37 °c)(figure 1a, c;
Watch online supplementary video).
Cells emit fluorescence only when CU is absorbed.
The medium containing UsC does not emit fluorescence.
MCF7 cells account for CU (
Dissolved in px (DMSO))slowly (
It takes 30 min to enter the cell, 4 h for saturated cells).
Figure 1 lymph node disease in the new tabDownload powerpoint (at week 14)in the lpr-water, lpr-CU and lpr-
Mice and ultrasound groups in TU group (UsC)
Being infected with the Epstein virus (EBV)
Convert people to PBMCs. (a, A–C)EBV-
Incubation of transformed human PBMCs with UsC for 1 min; (a, D–F)EBV-
Incubation of transformed human PBMCs with UsC for 2 min; (a, G–I)EBV-
Incubation of human PBMCs with UsC for 4 min; (A, D, G)fluorescence; (B, E, H)
Superposition of transmitted light; (C, F, I)merged image (
Fluorescent transmission light superposition); (b)
Lpr of lymph node enlargement-water group (A–D), lpr-CU group (E–H)and lpr-TU group (I–L).
The arrow points to lymph node enlargement; (c)
Eb-ingest enlarged images of UsC
Convert people to PBMCs. (Left panel)fluorescence; (middle panel)
Superposition of transmitted light; (right panel)merged image (
Fluorescent transmission light superposition). CU, curcumin; TU, turmeric.
The efficient absorption and absorption of copper by cells ensures its Pharmacology.
Based on this data, we-lpr/lpr or MRL-
MpJ mice with UsC or UsT were used to test its efficacy in improving asthmalike or SS-like condition.
We allow MRL.
Lpr/lpr mice reach the prescribed end point, that is, disease death or sacrifice when death seems imminent.
There was no significant difference in weight and water, curry or ginger powder consumption in the weight or consumption of water, copper or TU in each group of mice (table 1A).
View this table: View inline View popuptable 1 body weight, self-antibody, lymph node size, kidney area, volume (
Water, ginger)
Daily consumption and pathology (FS)
In MRL-of under/parotid glandLpr/License Plate Recognition and mri-
MpJ provides water, CU, or TUTreatment treatment with UsC or UsT, significantly improving MRL-
Lpr/lpr miceBy 14 lpr-in male and female 5/6 mice-
The water group has obvious palpable lymph node lesions, which is a major feature of MRL. lpr/lpr mice (figure 1b).
A sharp decrease in lymphatic proliferation in lprCU and lpr-
Group TU compared to TUwater group (table 1A). The lpr-
There are serious lymph node lesions in the water group (
Mean lymph nodes (LN)
Weight 2606 mg).
However, lpr-
At least lymph nodes in TU group (
Average LN weight of LN mg)(p
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