A new heterometallic MOF in cyanosilylation reaction, treatment and care management value in stroke by reducing oxidative stress damage

Article abstract of DOI:10.1246/bcsj.20190260

By employment the heterometallic strategy, a new highly porous metal-organic framework {(Me₂NH₂)[Mn₃K₂(TZIA)₃-(H₂O)₃] '(DMF)₄}n (1) was prepared via corresponding K(I)

Full text of DOI:10.1246/bcsj.20190260

Document type: Article
A New Heterometallic MOF in Cyanosilylation Reaction, Treatment and
Care Management Value in Stroke by Reducing Oxidative Stress Damage
Shu-Rong Chen,1,# Yi Li,2,# Bi-Yu Wu, and Hui Xu*
1
3
1Department of Geriatrics, Quanzhou 1st Hospital Affiliated to Fujian Medical University, Quanzhou, Fujian, P. R. China
2Department of Thyroid & Breast Surgery, the Fifth Affiliated Hospital, Sun Yat-Sen University,
Zhuhai, Guangdong, P. R. China
3Department of Fundamental Nursing, The Nursing and Health College of Zhengzhou University,
Zhengzhou, Henan, P. R. China
E-mail: hui_xu12@sina.com
Received: September 18, 2019; Accepted: October 19, 2019; Web Released: October 26, 2019
Hui Xu
Hui Xu, M.M., Associate Professor. She worked in the Nursing and Health College of Zhengzhou University.
Her research fields focus on health management of stroke and cognitive impairment, health promotion
among older adults and health policy of aged-care.
Abstract
ing the everyday life of people.1,2 Every year, stroke caused
deaths of 5.7 million as well as first time illnesses of 16 million,
by 2030, those numbers could reach up to 7.8 and 23 million.
By employment the heterometallic strategy, a new highly
porous metal-organic framework {(Me NH )[Mn K (TZIA) -
3
2
2
3
2
3
(
H O) ]¢(DMF) } (1) was prepared via corresponding K(I)and
Despite the fact that over the last decade, stroke mortality has
declined due to the use of more targeted preventive drugs, such
as statins and antihypertensives, strokes remain a risk factor
2
3
4 n
Mn(II) salts and hetero-donor organic ligand 5-(1H-tetrazol-5-
yl)isophthalic acid (H TZIA) via solvothermal conditions in
water and DMF mixed solvent. X-ray study of a single crystal
shows that ionic K was immobilized on the surface of pores
via trinuclear Mn ­tetrazole coordination motif. The resulting
activated 1a has been explored in detail by BET analysis to
probe its porosity. The removal of water molecules in order
3
4
seriously threatening human health. Thus, in this research,
+
we aimed to explore new candidates with excellent protective
activity in stroke treatment and clinical care management.
Porous coordination polymers (PCPs) or metal-organic
frameworks (MOFs) are crystalline materials composed of
coordination bonds of organic ligands and metal clusters/
2
+
+
to generate 1a makes it possible for K ions to be applied as
5
­7
an efficient and size-selective multiphase catalyst for cyano-
silylation of acetaldehyde under the mild conditions without
solvent. Furthermore, ELISA assay was performed to measure
IL-18 and IL-1β level in hCMEC/D3 human brain microvas-
cular endothelial cells after compound treatment. Besides, ROS
detection and RT-PCR was performed to detect ROS produc-
tion as well as the related gene expression. Next, the protective
effect of the compound was evaluated in animal model in vivo.
ions. MOFs are well known for their beauty along with pres-
enting diverse structures and catalysis, separation, gas storage,
8­10
luminescence, sensing, as well as other advanced functions.
Metal ions in MOFs acting as skeleton connecting sites may be
used as the surface of the heterogeneous catalyst to separate
Lewis acid sites. Unfortunately, in solution, many MOFs are
unstable. Stability is essential for application to industrial
processes and recovery of heterogeneous catalysts. Therefore,
preparing MOFs with stability remains a great challenge. MOF
stability is decided largely by secondary building units (SBUs)
or metal ions. The recent literature has revealed that cluster-
based units could endow MOFs with better framework stability
compared with those based on the single metal ions.1
Keywords: Hetero-donor organic ligand j Porous framework j
Cyanosilylation reaction
1,12
1
. Introduction
Trialkylsilyl cyanide (TMSCN) reaction with carbonyl com-
spounds is an important approach to C-C bond formation in
organic synthesis. It can be used for forming derivatives of
cyanohydrin, and can also be converted into many pharma-
According to the World Health Organization, stroke ranks
No. 2 in leading preventable death worldwide and No. 4 in lost
productivity, which has been a serious social problem disturb-
Bull. Chem. Soc. Jpn. 2020, 93, 43–49 | doi:10.1246/bcsj.20190260
© 2020 The Chemical Society of Japan | 43

high crystallinity by solvothermal synthesis. On the basis of
the crystal data collected under room temperature, the results
of refinement along with structural decomposition showed that
complex 1 can crystallize in the square P6 /m space group
3
and has a three-dimensional framework which has nanotube
2
+
channels. There is one crystallographically independent Mn
+
ion, one K ion, one ¯2-bridging water molecule as well as a
3
¹
2+
half TZIA ligand in the asymmetric unit. Mn ion among
molecular units have tetrahedral coordination geometry, which
3
¹
is defined via two O atoms from four independent TZIA
ligands as well as two N atoms from the tetrazole groups,
which forms a distorted tetrahedral coordination environment.
2
+
Three Mn ions were linked by the groups of tetrazolium to
form the trinuclear molecular building block (MBB) with
triangular shape. N atoms of the group of tetrazolium bind two
+
Figure 1. (a) View of asymmetric unit for complex 1.
b) View of the Mn-K MBB. (c) View of the formation
of the 1D channels; (d) The spacing filling mode for the
D framework of 1.
K
ions to trinuclear MBB via coordination and distribute
+
(
symmetrically on the triangle. K ions are partially linked via
isophthalate along with water acid on two independent TZIA
ligands generating a new MBB. MBBs are bridged via sharing
the motifs of Mn ­tetrazole to provide the building unit with
rod-like Co-K. The rod-like structure unit is connected with the
coordination of K and Mn centers through TZIA ligand
carboxyl group to provide the porous skeleton which is 1-D
hexagonal channels with nanotube-shape (considering Van der
3¹
3
2+
ceuticals along with chemicals, for example, β-hydroxy amino
alcohols, alpha-hydroxy ketones, alpha-hydroxy acids, etc.
1
3
+
2+
3¹
Hence, it is an attractive target for chemists to find catalysts
with high efficacy for carbonyl compound cyanosilication cata-
lyzed by TMSCN. In recent decades, many of the homoge-
neous catalysts, such as N-heterocyclic carbenes, inorganic or
organic salts, Lewis bases and Lewis acids, can catalyze reac-
tion, and they have been widely investigated.1 Nevertheless,
the reactions of homogeneous catalyists usually require pains-
taking purification, so in recent years, research in the field has
been mainly focused on the use of the heterogeneous catalysts,
particularly MOFs, with the aim of overcoming the problem of
+
Waals radius, pore size = 12 ¡). Ignoring the [(CH ) NH ]
ions within channels as well as solvent molecules with disorder
state, PLATON estimates an achievable volume of 53.2%.
3
2
2
4,15
PXRD, TGA and BET Analysis.
With the aim of
checking the as-prepared complex 1 phase purity, the PXRD
pattern of complex 1 was collected at room temperature by
using freshly prepared samples, and the results are shown in
Figure 2a. The observed along with the calculated diffraction
peaks well reflect sample purity. The discrepancy of reflective
strength for observed along with simulated results is because of
distinct crystal orientation within powder samples. Complex 1
thermogravimetric analysis was carried out and the thermo-
dynamic stability of complex 1 was discussed. According to
Figure 2b, the weightloss of complex 1 is 23.8% continuously
from 25 °C to 212 °C, which is related to removal of four lattice
DMF units along with three coordinated water molecules, the
framework could be thermally stable up to 340 °C. Inspired
via its latent porosity along with appropriate size of pore, we
were encouraged to explore the microporous properties of the
material. Complex 1 was assessed for 5 hours at 313 k under
the condition of high vacuum after full exchange of acetone,
resulting in solvent removal or activation for 1a. Obviously,
complex 1a maintains its microporous properties, because the
characteristic peaks are fundamentally consistent with the pat-
1
6­18
product catalyst separation under solvent-free conditions.
In this study, by employment the heterometallic strategy, a new
highly porous metal-organic framework {(Me NH )[Mn K -
2
2
3
2
(
TZIA) (H O) ]¢(DMF) } (1) (Figure 1) was triumphantly
3 2 3 4 n
prepared via corresponding K(I) and Mn(II) salts and hetero-
donor organic ligand 5-(1H-tetrazol-5-yl)isophthalic acid
H3TZIA) under solvothermal conditions in water and DMF
(
mixed solvent. X-ray diffraction of a single crystal showed that
+
K
ions were immobilized on pore surfaces via a trinuclear
2
+
Mn ­tetrazole coordination motif. The resulting activated 1a
has been explored in detail by BET analysis to probe its
porosity. The removal of water molecules in order to generate
+
1
a makes it possible for K ions to be applied as an efficient
and size-selective multiphase catalyst for solvent-free cyano-
silylation of acetaldehyde under mild conditions. In biological
research, the protective effect of this new compound was
evaluated both in vitro and in vivo. The ELISA assay of IL-18
and IL-1β indicated inhibition activity of the compound in the
inflammatory level in hCMEC/D3 endothelial cells. Besides,
the ROS detection and RT-PCR results revealed the compound
could also inhibit ROS production via reducing the related gene
expression. Finally, in vivo study suggested the compound has
excellent prevention activity in vivo.
1
9
tern of simulated XRD (Figure 2a). After removing solvent
molecules from the newly established framework, some dis-
placements are expected to be discovered between 1 and 1a. In
addition, the TGA analysis of 1a confirmed that there was no
lattice or coordinated solvents in the framework of 1a, which
is evidenced by the absence of obvious weight loss between
the temperatures of 25 and 367 °C. According to Figure 2c, at
7
7 K, the adsorption isotherm of N shows that compound 1a
2
2
. Results and Discussion
Crystal Structure of Complex 1. In water and DMF
mixed solvent, the target complex 1 can be easily acquired with
has a type I adsorption isotherm and there is a mild lag between
desorption along with adsorption, it can use the dynamics
characteristics of frame and multi-channel explanation. N2
44 | Bull. Chem. Soc. Jpn. 2020, 93, 43–49 | doi:10.1246/bcsj.20190260
© 2020 The Chemical Society of Japan

Table 1. Catalyst 1a conditions optimization and 1 com-
parative test.
a
Entry
Cat. (mol%)
TMSCN
Temp. (°C)
Conv. /%
1
2
3
4
5
6
0
1
1
0.5
0.1
0.1 (1)
2eq
2eq
2eq
2eq
2eq
2eq
40
40
rt
rt
rt
36.6
65.2
76.6
96.5
98.5
38.2
rt
aDetermined via GC on the basis of carbonyl substrate.
ture. It shows that the 1a skeleton structure remains after
solvent removal.
Catalytic Properties of 1a. In considering the complex 1a
has coordinated unsaturated metal centers (UMCs) along with a
large channel, it can be utilized as a latent Lewis acid catalyst
for the cyanation of carbonyl compounds and tested for catal-
ytic performance. Model experiments were carried out using
benzaldehyde as a typical carbonyl under solvent-free con-
ditions by changing the amount and temperature of catalyst. As
seen in Table 1, complex 1a indicated good activity, provid-
ing 98% benzaldehyde conversion at room temperature in one
day, and the lowest catalyst loading of 0.1 mol% K, which is
the highest activity of catalysis in cyanosilylation in a quantity
(a)
2
0
of only 1/15 of Cr-MIL-101 (1.5 mol% Cr-active site). In
addition, 1a as the catalyst does not require dichloromethane,
but catalyst Cr-MIL-101 does. Only two hours later, 1a was
removed through filtration and reaction was completely
stopped, and only 45% conversion was provided by standing
for about 15 hours. This indicated that there was no homoge-
neous catalyst within the solution of reaction, and 1a was a real
+
(b)
heterogeneous catalyst. The K ion concentration in the filtrate
was determined via inductive coupled plasma emission spec-
trometry (ICP), which shows a value of 12 ¯M, indicating little
+
K
ion leached into the reaction solution.
Under the conditions of optimization (2 equivalent TMSCN,
catalyst 0.1 mol% and at room temperature), nine carbonyl sub-
strates such as cyclic ketones, and aliphatic as well as aromatic
aldehydes were cyanated. Results are gathered in Table 2,
which revealed that the reaction has extensive applicability to a
variety of substrates. Under the standard conditions, aromatic
aldehydes bearing electron-effect (electron-withdrawing NO2
and electron-donating CH ) or distinct substituents were used.
3
After about 9­12 hours of reaction, the yield of the product was
over 90%. However, under the standard conditions, aliphatic
aldehydes along with ketones usually have lower efficiency of
catalysis than the aromatic aldehydes because of their inherent
decreased reactivity. After a long reaction time (two days),
cyclobutanone conversion was only up to 83%. In order to
(
c)
Figure 2. (a) 1 PXRD mode. (b) 1a and 1 TGA curves.
+
study and explain the open K site fine activity of the catalysis,
(c) 1a data of N2 adsorption.
1
a and 1 activities were detected under the same conditions of
reaction. Cyanation reaction conversion rates of 40.3% along
with 38.2% were acquired, which is similar to the conversion
rate with no catalyst, which may be because of the saturation
3
adsorption capacity was up to 342 cm /g at 1 atm along with
7 K (Figure 2c). The estimated apparent Brunauer­Emmett­
7
+
Teller (BET) surface area as well as the Langmuir surface area
are 1100 along with 1477 m /g, respectively. The distribution
ranges of pore size were between 10 and 12 ¡. These results are
the same as those of thermal stability along with crystal struc-
geometry of the whole K ions within 1. The results also show
2
+
that the unsaturated K coordination within 1a has an impor-
2
1­23
tant function in the process of catalysis.
In addition, the
PXRD pattern of complex 1a after the cyanosilylation reaction
Bull. Chem. Soc. Jpn. 2020, 93, 43–49 | doi:10.1246/bcsj.20190260
© 2020 The Chemical Society of Japan | 45

Table 2. The cyanosilylation of various carbonyl com-
pounds with TMSCN.
O
1a (0.1mol%)
NC OSiMe3
+
TMSCN
R1
R2
2
solvent-free, N /r.t.
R1
R2
A
B
1 2
R = Arkyl, Alkye; R
= H
Entrya
R1
Ph
R2
t/h
Conv. /%
b
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
12
12
12
9
30
34
40
40
48
98
93
94
99
90
95
81
76
0
Figure 3. Reduced level of IL-18 and IL-1β in human brain
microvascular endothelial cells after compound treatment.
OGD was established in the hCMEC/D3 cells, followed
by compound treatment. Then, we collected cell super-
natant and level of pro-inflammatory IL-18 along with IL-
1β was measured via ELISA detection.
2-CH3C6H4
4-CH3C6H4
4-NO2C6H4
n-Propyl
2-Furyl
(CH2)4
1-Naphthyl
9-Anthryl
Compound Reduced the Expression of IL-1β and IL-18.
The pro-inflammatory cytokine expression level of IL-18 and
IL-1β reflect inflammatory response in vivo. Usually, the
excessive increase of pro-inflammatory cytokines IL-18 and
IL-1β promote mucosa inflammation, which then accelerates
the occurrence of chronic inflammation. Thus, in this experi-
ment, the pro-inflammatory cytokines IL-18 and IL-1β were
measured via ELISA assay one day after compound treatment.
According to the results of Figure 3, in the OGD group, there
was significant increased level of IL-18 and IL-1β compared
with the control group, while, in the compound treatment
group, the increased level of pro-inflammatory cytokines was
obviously reduced, which is almost back to the normal level.
Compound Reduced the Production of ROS in Micro-
vascular Endothelial Cells. The previous results indicated
the compound could reduce the production of the inflammatory
cytokines. Next, we further wanted to explore the relationship
between the compound and the ROS production in the cells.
After the construction of the oxygen-glucose deprivation model
Reaction conditions: aMe3SiCN (1.0 mmol), aldehyde (0.5
mmol), 1a of 0.1 mol%, at room temperature, under an atmos-
phere of N2. Determined via GC on the basis of the carbonyl
b
substrate.
activation
O
Me SiO CN
3
R
H
R
H
TMSCN
TMSNC
(
OGD) in hCMEC/D3 microvascular endothelial cells of
human brain, the compound was exposure to cells at a specified
concentration for one day. ROS related gene expression was
determined via RT-PCR. According to the results of Figure 4.
The oxygen-glucose deprivation model could lead to a signifi-
cant increase of the ROS level in the cells, that is significantly
distinct with matched group (p < 0.005). After treating with
compound, the up-regulated level of ROS was reduced, almost
reaching control group level. The result revealed that the
compound could inhibit the inflammatory condition of the
human brain microvascular endothelial cells. In addition, com-
pared with the MOFs reported previously, the synthetic MOF
in this study showed stronger induction activity on the ROS
O
O
R
R
C
N
Si
C N Si
H
H
Scheme 1. The mechanism of 1a carbonyl compound
catalytic cyanation is proposed.
has also been acquired, which reveals a good match with that of
the freshly prepared 1a, showing its framework stability in the
catalytic process (Figure 2a).
2
4
production.
Compound Reduced the Relative Expression of the ROS
Related Genes. In the above research, we have demonstrated
that this compound could inhibit production of ROS in human
brain microvascular endothelial hCMEC/D3 cells. However,
the detailed mechanism was still unknown, thus, in this experi-
ment, we further wanted to explore the relative expression of
gp91 and gp22, which is reported to have a strong relationship
with the production of the ROS in human brain microvas-
cular endothelial. According to the results of Figure 5, we can
observe that there was a significant increased level of the gp91
On the basis of the experimental results and reports in the
literature, a rational reaction mechanism was put forward to
explain the process of cyanation catalyzed by 1a. The unstable
water molecules within the 1a channels were dislodged via
heating to lay bare the metal centres of unsaturation. With
coordinated K centres of unsaturation to active aldehydes,
reaction with TMSCN occurred (Scheme 1). Using aldehydes
to displace products, the catalysts activate continually alde-
hydes for the next cycle of catalysis.
46 | Bull. Chem. Soc. Jpn. 2020, 93, 43–49 | doi:10.1246/bcsj.20190260
© 2020 The Chemical Society of Japan

Table 3. Neurological deficit scores in animals before and
after treatment.
Pre-treatment Post-treatment
Score
difference
Group
score
score
Control group
21.78 « 6.75
21.65 « 6.42 0.21 « 0.63
(n = 30)
Treatment group 22.03 « 6.86
6.12 « 2.75 16.92 « 4.35
(n = 30)
t
0.5021
6.7612
6.1249
P
>0.05
<0.05
<0.05
hetero-donor organic ligand 5-(1H-tetrazol-5-yl)isophthalic
acid (H TZIA) solvothermal condition in water and DMF
3
+
mixed solvent. X-ray single crystal study shows that K ions
were immobilized on the surface of pores via trinuclear Mn
2
+
­
tetrazole coordination motif. The resulting activated 1a has
been explored in detail by BET analysis to probe its porosity.
The removal of water molecules in order to generate 1a makes
+
Figure 4. ROS production within hCMEC/D3 cells de-
creased after combined treatment. OGD was established in
the hCMEC/D3 cells, followed by the compound treat-
ment. The ROS level in the human brain microvascular
endothelial cells was detected by ROS detection.
it possible for K ions to be applied as an efficient and size-
selective multiphase catalyst for solvent-free cyanosilylation of
acetaldehyde under mild conditions. The ELISA assay of IL-18
along with IL-1β indicated that compound could reduce the
inflammatory level in human brain microvascular endothelial
hCMEC/D3 cells. Besides, the ROS detection and RT-PCR
results revealed the compound could also inhibit ROS produc-
tion via reducing the ROS related gene expression. Finally, the
protective effect of the compound in an animal model was
evaluated, which suggested the excellent prevention activity of
compound in vivo.
4
. Experimental
Chemicals and Measurements.
Solvents, reagents as
Figure 5. Reduced gp91 and gp22 expression level in
human brain microvascular endothelial cells after com-
pound treatment. OGD was established in the cells of
hCMEC/D3, followed via the compound treatment. The
expression level of the gp91 and gp22 in the cells of
hCMEC/D3 was determined via RT-PCR.
well as the raw materials were all purchased from commercial
sources and could be utilized with no further purification. With
a Nicolet Avatar 360 FT-IR spectrophotometer we determine
FT-IR spectra. The thermogravimetric analysis was performed
under nitrogen flow with a Q50 TGA (TA) thermal analyzer,
¹1
with a heating rate of 5 °C¢min . Powder X-ray diffraction
patterns (PXRD) of samples of bulk were measured at room
temperature on a MiniFlex (Cu Kα, λ = 1.5418 ¡). GC analy-
sis was carried out on an Agilent 7890B GC analyzer. The gas
and gp22 expression level in the hCMEC/D3 cells, which also
explains the high level of ROS in the cells. After the treatment
via compound with the indicator concentration, gp91 and gp22
expression level was obviously reduced, compared with the
model group.
adsorption isotherms (N ) at low pressure (up to 1 bar) were
2
detected by porosity analyzer along with ASAP 2020 specific
surface area.
Compound Prevents Neurological Deficit.
After the
Preparation and Characterization of {(Me NH )[Mn K -
2
2
3
2
previous in vitro study, we have revealed the protective effect
of the compound in human brain microvascular endothelial
cells by reducing the production of ROS. Subsequently, the
treatment activity and prevention ability of the compound in
the rat model was evaluated with the neurological deficit score.
In Table 3, we can see that the neurological deficit score was
reduced to 6.12 « 2.75, which is significantly lower than the
score in control groups (p < 0.05). The results showed that the
compound had a good protective effect within the rat model.
(TZIA)3(H2O)3]¢(DMF)4}n (1). We synthesized complex 1
via MnCl ¢4H O using 23.79 mg and 0.10 mmol, KF¢2H O of
2
2
2
14.10 mg and 0.15 mmol as well as H TZIA and 23.40 mg and
3
0.10 mmol solvothermal reaction in the mixed solvent of 2 mL
H O, N and N-dimethylformamide (7 mL, DMF) for three days
2
at 140 °C. We acquired light yellow crystals with block-shape.
After filtration and separation from the mother solvent, we
washed the crystals with anhydrous DMF three times, then
dried it in air. The yield was approximately 62% on the basis of
H TZIA. Anal. Calcd for C H K Mn N O (1): C, 37.06;
3
41 51
2
3
17 19
3
. Conclusion
H, 3.87; N, 17.92%; found: C, 37.26; H, 3.99; N, 17.94%.
With an Oxford Xcalibur E diffractometer we acquired com-
pound 1 X-ray data. CrysAlisPro software was used to analyze
In conclusion, we have generated a novel porous metal-
organic framework by corresponding K(I)and Mn(II) salts and
Bull. Chem. Soc. Jpn. 2020, 93, 43–49 | doi:10.1246/bcsj.20190260
© 2020 The Chemical Society of Japan | 47

Table 4. Compound 1 parameters of crystallography and
refinement details.
experiment was carried out three or more times, the results were
showed as mean « SD.
ROS Detection. After treated with the compound at the
indicated concentration, the ROS level in the cells was detect-
ed by ROS detection kit according to the instruction of the
protocols. In brief, human brain microvascular endothelial cells
hCMEC/D3 were inoculated into a 96 well plate and then the
oxygen-glucose deprivation model (OGD) was constructed.
The compound was added for treatment for the indicated time,
followed by 20 ¯M DCFH-DA solution addition for 30 min cell
incubation at 37 °C in the dark. Then, PBS was used to wash
cells three times and then the cells were harvested. An ROS
detection kit was used for the ROS measurement, the absor-
bance in each group was detected by flow cytometry (BD Via,
New Jersey, USA) at 488/530 nm and analyzed with FlowJo7.6
software.
Empirical formula
Formula weight
Temperature/K
Crystal system
C27H15K2Mn3N12O15
990.51
386.3
hexagonal
P63/m
19.336(2)
19.336(2)
9.989(4)
Space group
a/¡
b/¡
c/¡
α/°
β/°
γ/°
90
90
120
3234.2(14)
2
1.017
0.755
1735/6/103
1.254
R1 = 0.0987, ωR2 = 0.3033
R1 = 0.1119, ωR2 = 0.3127
1.15/¹0.68
Volume/¡3
Z
3
ρcalcg/cm
μ/mm
¹
1
RT-PCR of gp91 and gp22. Human brain microvascular
endothelial cells HCMEC/D3 were inoculated into a 96 well
plate and then the oxygen-glucose deprivation model (OGD)
was constructed before the compound treatment. Subsequently,
the relative expression of the gp91 and gp22 in cells were
examined by RT-PCR according to the protocols. Briefly, total
RNA in cells was extracted with TRIzol and quantified with
the detection kit. Next, a One-Step RT-PCR kit was utilized for
the RNA reverse-transcribing into cDNA, and the RT-PCR was
Data/restraints/parameters
Goodness-of-fit on F
Final R indexes [I ½ 2σ(I)]
Final R indexes [all data]
Largest diff. peak/hole/e ¡
CCDC
2
¹3
1952188
μ
the strength data, then convert strength data into hkl files.
Based on the direct method, a primitive structure model was
established using the SHELXS program, and modified via
SHELXL-2014 based on the least square approach. All non-H
atoms were mixed with the anisotropic parameters, and all the
H atoms were geometrically fixed on their linked C atoms via
utilizing the AFIX commands. Table 4 exhibits the parameters
of crystallography along with the information of the numerical
values of compound 1 after recombination.
Cell culture and MOF Treatment. Human brain micro-
vascular HCMEC/D3 endothelial cells were purchase from
American Type Culture Collection, and then cultured in ATCC-
formulated RPMI-1640 culture medium. 2%L-glutamine, 100
U/ml penicillin-streptomycin solutions (Gibco, Life Tech-
nologies) as well as 10% (v/v) heat-inactivated fetal bovine
serum (FBS) were added to the medium. The cells were cul-
carried out utilizing SYBR Premix Ex Taq· kit (Takara).
Finally, we calculated relative expression quantification of
¹
¦¦CT
gp91 and gp22 with 2
of gapdh mRNA.
approach after the standardization
Neurological Deficit Score.
The experimental animals
were randomly divided into a control group and a treatment
group. The neurological function of the experimental animals
was evaluated before and after the compound treatment. The
greater the difference between the two groups, the better the
treatment effect and the better recovery of nerve function.
According to the physiological functions of the experimental
animals, the evaluation of the therapeutic effect of the com-
pound evaluation was divided into: Complete recovery, no
obvious sequelae, neurological function score reduced by
>90%; Improved, with slight sequelae, neurological function
score decreased >65%; Ineffective, with no recovery or
improvement.
tured in an incubator at 37 °C along with 5% CO . The culture
2
medium was replaced when the cells reached 80% confluence.
The MOF was dissolved in DMSO to the concentration of
Statistical Analysis. All the results in this research were
collected from the independent experiments of three or more
repeats. All data were analyzed by Graphpad Prism 5.0 soft-
ware and expressed as average value («) standard deviation
(SD). Two-way t-test was used for comparing differences of the
two groups. One-way ANOVA was applied to compare more
than three groups. When p < 0.05, it is estimated that there is
statistical difference.
1
mg/mL for storage, before experiment the MOF solution was
thoroughly dissolved in PBS to the indicated experimental
concentration.
ELISA Assay. To detect the pro-inflammatory cytokines
in hCMEC/D3 cell lines after complex treatment, IL-18 and
IL-1β ELISA detection assay was carried out. We performed
the experiment under the guidance of the manufacturer’s proto-
cols with some modifications. In brief, hCMEC/D3 endothelial
cells were inoculated into a 96 well plate and then the oxygen-
glucose deprivation model (OGD) was constructed. The com-
pound was used for 24 h treatment. After that, we collected the
supernatant of cell and the level of pro-inflammatory IL-18 and
IL-1β was acquired by ELISA detection. All the plates were
washed and added to a biotin-conjugated antibody for 2 h, then
the absorbance of each well was measured at 450 nm. This
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