biomolecules

Article
Investigation of the Repairing Effect and Mechanism of Oral
Degraded Sericin on Liver Injury in Type II Diabetic Rats
Zhen-Zhen Wei, Yu-Jie Weng and Yu-Qing Zhang *

School of Biology and Basic Medical Sciences, Medical College, Soochow University,
RM702-2303, No. 199, Renai Road, Industrial Park, Suzhou 215123, China;
20204021006@stu.suda.edu.cn (Z.-Z.W.); 20184021002@stu.suda.edu.cn (Y.-J.W.)
* Correspondence: sericult@suda.edu.cn

Abstract: In the sericulture and silk production industry, sericin is discharged in the degumming
wastewater, resulting in a large amount of wasted natural protein and environmental pollution.
This study investigated the effect of degraded sericin recovered by the Ca(OH)2 –ultrasound degum-
ming method (a green process) on liver injury in T2D rats. After 4 weeks of dietary sericin supple-
mentation, the liver masses and organ coefficients of the T2D rats improved compared with those of
the model rats that were not fed sericin. Oral sericin activated the damaged PI3K/AKT/AMPK path-
way to enhance glycogen synthesis, accelerate glycolysis, and inhibit gluconeogenesis. The protein
expression levels of the inflammatory factors NF-κB, IL-6, and TNF-α in the T2D model group were
up to two times higher than in the normal group. However, all three T2D groups that received oral
sericin showed significant decreases in these factors to the level found in the normal group, indicating
that inflammation in the body was significantly reduced. These results show that the sericin protein
might improve glycogen synthesis, accelerate glycolysis, and inhibit gluconeogenesis by enhancing
the anti-oxidation capability and reducing inflammatory reactions. Therefore, sericin recovered by





 Ca(OH)2 degradation has potential use in the development of functional health foods that can lower
Citation: Wei, Z.-Z.; Weng, Y.-J.; blood sugar.
Zhang, Y.-Q. Investigation of the
Repairing Effect and Mechanism of Keywords: sericin; oral administration; protein expression; signaling pathway; inflammatory factors
Oral Degraded Sericin on Liver
Injury in Type II Diabetic Rats.
Biomolecules 2022, 12, 444. https://
doi.org/10.3390/biom12030444 1. Introduction
Academic Editors: Undurti N. Das The classic symptom of diabetes is insulin resistance, a primary cause of oxidative
and Natalia Osna stress [1,2], which is reflected in increased levels of reactive oxygen species (ROS) in the
Received: 25 January 2022
liver. The liver is the most important target organ of insulin [3]. In the liver, insulin binds to
Accepted: 10 March 2022
the insulin receptor on the cell membrane to activate the insulin receptor substrate, which
Published: 13 March 2022
in turn binds to the regulatory subunit p85 of PI3K (phosphatidylinositide3-kinases) and
activates catalytic subunit p110 of PI3K. Activated PI3K generates a second messenger
Publisher’s Note: MDPI stays neutral
and promotes the activation of AKT (protein kinase B). Therefore, the insulin/PI3K/AKT
with regard to jurisdictional claims in
pathway plays an important role in regulating liver carbohydrate metabolism. Studies
published maps and institutional affil-
have found that it can be an important part of the treatment of diabetes, cancer, neurode-
iations.
generative diseases, and stroke targeting; AMPK (AMP-dependent protein kinase) is an
important sensor molecule that regulates bioenergy homeostasis, which can control the
synthesis and decomposition pathways of energy metabolism. AMPK activation can reg-
Copyright: © 2022 by the authors.
ulate the metabolism and vascular function of adipocytes, promoting glucose transport
Licensee MDPI, Basel, Switzerland. and fatty acid oxidation. The liver is also the main organ that regulates blood sugar levels.
This article is an open access article When blood sugar levels rise after meals, the liver uses blood sugar to synthesize glycogen.
distributed under the terms and Excessive sugar is converted into fat in the liver and accelerates the pentose phosphate
conditions of the Creative Commons cycle, thereby reducing blood sugar and maintaining a constant blood sugar concentration.
Attribution (CC BY) license (https:// Conversely, when the blood sugar concentration decreases, the hepatic glycogenolysis and
creativecommons.org/licenses/by/ gluconeogenesis are strengthened, and the generated glucose is sent into the blood to adjust
4.0/). the blood sugar concentration so as not to be too low.

Biomolecules 2022, 12, 444. https://doi.org/10.3390/biom12030444 https://www.mdpi.com/journal/biomolecules

Biomolecules 2022, 12, 444 2 of 12

Metformin is one of the most commonly used oral drugs clinically used to lower
blood glucose levels in diabetic patients. After issuing warnings for decades about the
risks of lactic acidosis in patients with chronic nephropathy, most evidence from the
recent literature has demonstrated that the risk of lactic acidosis is low and acceptable [4].
Acarbose can cause gastrointestinal reactions [5]. Thiazolidinediones may cause water and
sodium retention, bone looseness, etc. [6]. Therefore, natural medicines with a reliable anti-
hyperglycemic effect that are less toxic and have fewer side effects have received increasing
attention [7]. Silk sericin protein from Bombyx mori cocoons contains 18 essential amino
acids. Sericin is biologically active [8] and has strong physiological and pharmacological
effects such as anti-oxidation [9,10], antitumor [11–13], anti-apoptosis [14], and blood sugar
reducing activities [15,16]. Sericin has also been shown to reduce serum cholesterol in
rats [17,18] and enhance cognition in Alzheimer’s disease patients [19,20]. Our group has
focused on the green processing and recovery of sericin from silkworm cocoons and its anti-
hyperglycemic effects, shown in vitro and in vivo. It has recently been reported that sericin
added to the diet can effectively reduce blood sugar levels in a streptozotocin-induced
type II diabetic (T2D) mouse model. It has also been found to reduce intestinal cholesterol,
triglycerides, and other lipids and improve glucose and insulin tolerance in mice [21].
We recently published the results of the first half of the present study, which seeks to
better understand the blood-sugar-lowering function of sericin and its regulatory mecha-
nism. Degraded sericin was shown to regulate blood glucose levels and improve impaired
liver function in T2D rats by reducing oxidative stress [22]. At the same time, some studies
have also found that mulberry leaf polysaccharides can reduce inflammatory factors in
T2D rats, improve mitochondrial function and relieve oxidative stress damage, and have
an anti-diabetic effect [23]. The present article reports the second half of the study, which
investigated the effect of sericin administration on T2D rats. We explored the ability of
sericin to repair liver damage and its mechanism of action in diabetic rats in terms of
glucose and lipid metabolism, the PI3K/AKT signaling pathway, and the AMPK/ACC
signaling pathway.

2. Materials and Methods

2.1. Materials
Western blot antibody, TNF-α (tumor necrosis factor-α), IL-6 (interleukin- 6), and NF-
κB (nuclear factor kappa-B) enzyme-linked immunosorbent assay (ELISA) kits, Jiangsu KGI
Biotechnology Co., Ltd., Nanjingn China; bicinchoninic acid assay (BCA) determination kit,
Shanghai Biyuntian Institute of Biotechnology, Shanghai, China; multifunctional sample
homogenizer, QIAGEN Biotech, Hilden, Germany; MIKro120 low-speed centrifuge, Ger-
many Hettich Scientific Instruments Co., Ltd., Kirchlengern, Germany; desktop high-speed
refrigerated microcentrifuge, SCILOGEX, Czech Republic, USA. The rest of the reagents
are analytical pure.

2.2. Preparation of Sericin Peptides

We used an improved degumming method developed by our laboratory to obtain
the sericin peptides [24]. First, the silk underwent degumming by boiling in 0.025% (w/v)
calcium hydroxide for 30 min; this boiling step was repeated once to increase the recovery
rate. This was followed by rotary evaporation of the obtained degumming liquid to
concentrate it. The concentrated solution was neutralized with 6M dilute sulfuric acid
and centrifuged at 10,000 rpm/min to remove the precipitate. Finally, the supernatant
underwent vacuum freeze-drying to obtain the low-molecular-weight sericin (referred to
hereafter as LS). In this study, LS samples containing 1%, 2.5%, and 5% were defined as
LLS, MLS, and HLS, respectively, for the subsequent feeding of T2D rats.

2.3. Rat Feed

Sprague–Dawley (SD) rats are often used as a diabetic animal model due to their
higher sensitivity to streptozotocin (STZ) and their easily attainable and inexpensive char-
Biomolecules 2022, 12, 444 3 of 12

acteristics. This can help further elucidation of the underlying mechanisms of diabetes
and its complications [25–28]. The procedures for the rat breeding and handling, model
construction, blood glucose measurement, and anatomical measurement in the experiment
were carried out according to our previous report [22]. Purchased male SD rats were raised
to approximately 200 g in a controlled environment at 20–25 ◦ C and 50%–80% humidity.
In this study, rats fed with metformin were used as the positive control group. We randomly
tested several STZ injection doses for T2D modeling, and the 90 mg/kg dose was identified
as the optimal dose for use in the study. The rats fasted overnight before the first STZ dose,
and they received one dose per day for 3 days. Two days after the final injection, blood
was collected from the tail vein on an empty stomach to test the fasting blood glucose
level. The model successfully established if the fasting blood glucose was higher than
11.1 mmol/L. The rats were randomly divided into six groups (n = 6). The normal group
and T2D model group were fed normal irradiated feed. Three sample groups of T2D rats
were fed irradiated chow containing LLS, MLS, and HLS samples. The positive control (PC)
group was fed irradiated feed containing 0.5% (w/w) metformin. The entire test process
was completed in 7 weeks. All animal experiments were conducted in accordance with
the relevant regulations required by the International Animal Welfare Committee and the
Animal Experiment Operation and Ethics Committee of Soochow University.

2.4. Liver Weight and Organ Body Coefficients

After the rats were sacrificed by dislocation, the liver was removed and weighed.
The organ coefficient was calculated as liver mass/rat weight × 100%.

2.5. Extraction of Total Protein from Liver Samples

First, 10 µL of phosphatase inhibitor, 1 µL of protease inhibitor, and 5 µL of 100 mmol/mL
PMSF were added to 1 mL of prechilled lysis buffer and mixed well. The samples were
kept on ice for a few minutes until use. Each 100 mg liver was cut into 3 mm pieces.
One milliliter of chilled lysis buffer containing inhibitors was added to each 3 mm piece,
and the sample was homogenized at 50 r/min. All operations were carried out at low
temperature. After homogenization, the sample was centrifuged for 5 min at 10,000 r/min
and 4 ◦ C. The supernatant containing the whole protein extract was divided into two
precooled centrifuge tubes and stored at −70 ◦ C to avoid repeated freezing and thawing.
The protein concentration of the obtained supernatant was determined using a BCA protein
concentration kit.

2.6. Western Blotting

Total protein content in liver tissue was extracted and separated by SDS-PAGE elec-
trophoresis. The separated proteins were transferred to NC membranes. Protein-transferred
membranes were then blocked with 5% nonfat milk for 2 h at room temperature and in-
cubated with dilution-related antibodies overnight at 4 ◦ C. Membranes were washed and
then mixed with Rabbit Anti-AKT (1:10,000), Rabbit Anti-p -ACC (1:5000), Rabbit Anti-P-
ULK1 (1:500), and other corresponding antibodies diluted 1:1000 and incubated at room
temperature for 1.5 h. After imaged using G:BOX chemiXR5., the results were analyzed in
grayscale using Gel-Pro32 software.

2.7. Determination of Related Inflammatory Factors

According to the instructions of each ELISA kit, the contents of TNF-α, IL-6, and
NF-κB in the supernatant of the liver homogenate of each group of rats were detected.

2.8. Data Processing

The data obtained from the experiment were processed using Prism 8 statistical
software. The results are presented as means ± standard deviation. One-way analysis of
variance was conducted, and p < 0.05 indicates statistical significance.
Biomolecules 2022, 12, 444 4 of 12

3. Results
3.1. Liver Organ Quality and Organ Coefficient
The liver is the most important organ in nutrient metabolism. Diabetes can disrupt
fat metabolism, affect liver function, and cause organ enlargement. The liver masses and
organ coefficients of the rat groups were compared, and the results are shown in Table 1.
The normal group had the largest average liver mass (16.38 ± 1.70 g). The liver mass of rats
in the T2D model group was greatly reduced (10.86 ± 1.43 g; p < 0.01). The liver quality of
the PC group was significantly increased compared with the T2D model group (p < 0.01)
and was very close to that of the normal group. The liver mass of the HLS group was also
increased to a certain extent, but the other sericin test groups did not have significantly
different liver masses. The organ coefficient refers to the ratio of the organ to the body
weight. If the coefficient increases, the organ may show signs of hyperemia, edema, or
hyperplasia. The organ coefficient of rats in the normal group was 3.25 ± 0.34%, while that
of rats in the model group was 4.34 ± 0.54%, which was significantly greater (p < 0.01).
The organ coefficients of the PC group and each sericin group were increased compared
with the normal group, but the increases were not significant. The coefficient of the HLS
group was significantly decreased compared with the model group (p < 0.05). These results
indicate that HLS can improve liver hyperplasia and hypertrophy caused by diabetes.

Table 1. Effect of LS oral administration on liver mass and liver coefficient of T2D rats.

Group Liver Mass (g) Coefficient (%)

Normal 16.38 ± 1.70 3.25 ± 0.34
Model 10.86 ± 1.43 ** 4.35 ± 0.54 **
PC 15.73 ± 2.92 ## 4.23 ± 0.79
LLS 10.43 ± 1.20 4.11 ± 0.49
MLS 10.49 ± 1.33 4.23 ± 0.54
HLS 11.90 ± 1.34 # 4.03 ± 0.73 #
Normal: normal group; Model: diabetic model group; PC: positive control group (0.5% metformin); LLS, MLS,
and HLS: 1%, 2.5%, or 5% LS, respectively. n = 3, ** p < 0.01 versus normal group. # p < 0.05, ## p < 0.01 versus
model group.

3.2. Carbohydrate Metabolism Pathway

The unbalanced blood glucose levels in T2D rats were largely due to disordered
gluconeogenesis and glycolysis metabolism. In a high-glucose environment, Glucose-6-
phosphatase (G6pase), phosphoenolpyruvate carboxylase (PCK), and glucokinase (GLK)
are involved in the conversion and storage of glucose. Glycolysis is the process in which
sugars are broken down to produce lactic acid and energy. The main rate-limiting en-
zymes of glycolysis are 6-phosphofructokinase-1 (PFK1) and M2 pyruvate kinase (PKM2).
This study investigated these key enzymes of sugar metabolism. As can be seen from
Figure 1, the levels of G6pase and PCK in the model group and PC group were higher than
those in the normal group, and the protein level in the HLS group basically recovered to
the level of the normal group, but the expression levels of G6pase in the LLS and MLS
groups were not significantly up-regulated, indicating that HLS-fed rats can accelerate
the conversion and storage of glucose; the expression levels of GLK, PFK1, and PKM2
in each experimental group were higher than those in the normal group and the model
group, and the indicators in the HLS group were close to or even higher than those in the
PC group. Compared with the PC group, the expression levels of LLS and MLS were also
increased, indicating that feeding LS could accelerate the glucose conversion and weaken
the glycolytic activity. In general, these results indicate that feeding with a higher amount
of sericin can evidently reduce the blood sugar levels by promoting gluconeogenesis and
weakening glycolysis.
 group. Compared with the PC group, the expression levels of LLS and MLS were also
increased, indicating that feeding LS could accelerate the glucose conversion and weaken
the glycolytic activity. In general, these results indicate that feeding with a higher amount
Biomolecules 2022, 12, 444 5 of 12
of sericin can evidently reduce the blood sugar levels by promoting gluconeogenesis and
weakening glycolysis.

1.4
Normal
##
Model ##
1.2 ## ##
PC ## ** ##

Protein Relative Expression

LLS ##
Normal Model PC LLS MLS HLS ##
1.0 MLS ## ##
## ## ##
HLS ##
G6pase **
##
0.8 **
GLK **

PCK 0.6
##
PFK1 ** #
0.4
PKM2 ##

GAPDH 0.2

0.0
G6pase GLK PCK PFK1 PFK2
Key Proteins
Figure
Figure1.1.Effect
Effectof
oforal
oralsericin
sericinon
onthe
theexpression
expressionof
ofkey
keyhepatic
hepaticglucose
glucosemetabolism
metabolismproteins
proteinsininT2D
T2D
rats. Normal: normal group; Model: diabetic model group; PC: 0.5% melbin; LLS, MLS, and HLS:
rats. Normal: normal group; Model: diabetic model group; PC: 0.5% melbin; LLS, MLS, and HLS: 1%,
1%, 2.5%, and 5% LS, respectively. n = 3, ** p < 0.01 versus normal group.# # p < 0.05,##
## p < 0.01 versus
2.5%, and 5% LS, respectively. n = 3, ** p < 0.01 versus normal group. p < 0.05, p < 0.01 versus
model group.
model group.
3.3.
3.3.Impact
Impacton onPI3K/AKT
PI3K/AKTPathwayPathway
Insulin is the main regulatory
Insulin is the main regulatory hormone hormoneininthe theprocess
process ofof glucose
glucose andand lipid
lipid metabo-
metabolism,
lism, and insulin
and insulin mainly mainly regulates
regulates glucoseglucose
uptake uptake and transport
and transport throughthrough the PI3K/AKT
the PI3K/AKT path-
pathway.
way. PI3K, PI3K,
AKT, AKT, fructose-2,6-bisphosphatase
fructose-2,6-bisphosphatase cardiac
cardiac enzyme
enzyme (PFKFβ2)
(PFKFβ2) andand forkhead
forkhead box
box protein O1(FoxO1) in the liver are the key regulatory enzymes.
protein O1(FoxO1) in the liver are the key regulatory enzymes. After PI3K activation, After PI3K activation,
phosphate
phosphategroupsgroupscan canbe berecruited
recruitedto tothe
theplasma
plasmamembrane
membraneof ofAKT
AKTprotein
proteinto topartially
partially
phosphorylate
phosphorylate and activate, and p-AKT will further activate the downstream PFKFß2and
and activate, and p-AKT will further activate the downstream PFKFß2 and
FOXO1.
FOXO1.As Asshown
shown ininFigure
Figure 2, 2,
compared
compared with thethe
with model
modelgroup, withwith
group, the increase
the increasein thein
sericin concentration,
the sericin concentration, PI3K,PI3K,
p-PI3K, p-AKT,
p-PI3K, PFKFß2,
p-AKT, p-PFKFß2,
PFKFß2, and p-FOXO1
p-PFKFß2, and p-FOXO1 in the three
in the
dose
threegroups
dose groupswere gradually
were gradually increased, whilewhile
increased, the indexes
the indexesin theinHLS group
the HLS almost
group re-
almost
turned
returned to to
normal
normal levels;
levels;FoxO1
FoxO1 ininthe
thesericin
sericintreatment
treatmentgroup groupdecreased,
decreased,and andthetheHLS
HLS
group
group decreased
decreased by by about
about50%.50%.Compared
Comparedwith withthethe
PCPC group,
group, thethe levels
levels of p-PI3K,
of p-PI3K, p-AKT,p-
AKT,
PFKFß2,PFKFß2,
p-PFKFß2,p-PFKFß2, and p-FOXO1
and p-FOXO1 in the
in the three dosethree dosewere
groups groups were increased,
gradually gradually and in-
creased,
the HLSand group thebasically
HLS group basically
reached reached
the same theassame
level level as group;
the normal the normal group;
for PI3K andfor PI3K
AKT in
the MLS
and AKT group,
in the MLS the protein
group,content was content
the protein almost the wassame
almost as that of theas
the same PCthatgroup, which
of the PC
exceeded
group, whichtheexceeded
level of the thenormal
level ofgroup;
the normalthe FOXO1
group; theprotein
FOXO1 level of the level
protein HLS of group was
the HLS
almostwas
group thealmost
same as thethat
sameof asthethat
PCof group,
the PCand it was
group, andreduced to the level
it was reduced to the of level
the normal
of the
group; for
normal PI3K,for
group; p-PI3K,
PI3K, and p-AKT,
p-PI3K, andthe levels the
p-AKT, of PFKFß2
levels of and p-FOXO1
PFKFß2 andwere
p-FOXO1significantly
were
lower than those in the normal group, there was almost
significantly lower than those in the normal group, there was almost no differenceno difference in the amount
in theof
total AKT
amount and AKT
of total p-PFKFß2, the level the
and p-PFKFß2, of FoxO1
level ofwasFoxO1significantly increased,
was significantly and various
increased, and
phosphorylated
various proteinsproteins
phosphorylated were highlywereexpressed in the HLS
highly expressed in thegroup,
HLSindicating that the that
group, indicating HLS
group
the HLSthat activated
group the PI3K/AKT
that activated pathway
the PI3K/AKT has thehas
pathway strongest effect. The
the strongest effect.above
The results
above
indicate
results that adding
indicate sericinsericin
that adding can inhibit hepatichepatic
can inhibit gluconeogenesis
gluconeogenesisand sugar
andproduction
sugar produc- and
increase the synthesis and storage of hepatic glycogen, thereby
tion and increase the synthesis and storage of hepatic glycogen, thereby stabilizing the stabilizing the blood sugar
level of
blood the body.
sugar level of the body.
Biomolecules 2022, 11, x 6 of 12
Biomolecules 2022, 12, 444 6 of 12

## Normal
1.0 # Model
PC

Protein Relative Expression

Normal Model PC LLS MLS HLS
LLS
PI3K 0.8 MLS
##
p-PI3K HLS
AKT
0.6 ##
#
p-AKT # ##

##
PFKFß2

p-PFKFß2
0.4 ## ## #

FOXO1 #
## ## ##
#
p-FOXO1 0.2 # #
## ##
GAPDH ##
##

0.0
PI3K p-PI3K AKT p-AKT PFKFβ2 p-PFKFβ2 FOXO1 p-F0X01
Key Proteins

Figure 2. Effect of oral sericin on the expression of PI3K/AKT

PI3K/AKT pathway
pathway proteins
proteins in T2D
T2D rats.
rats. Normal:
Normal:
normal group;
normal group; Model:
Model: diabetic
diabeticmodel
modelgroup;
group;PC:
PC:0.5%
0.5%melbin;
melbin;LLS,
LLS,MLS,
MLS,and
andHLS:
HLS:1%,
1%,2.5%,
2.5%,and
and 5
5%
% LS.
LS. n =n3,= #3,p <p 0.05,
# < 0.05,
## ##p p<<0.01
0.01versus
versusmodel
modelgroup.
group.

3.4. Impact on AMPK/ACC Pathway

AMPK is a key protein in energy metabolism and is of great significance to the study
of
of metabolic diseases
metabolic diseases in in diabetes. Glucose transporter
diabetes. Glucose transporter 44 (GLUT4)
(GLUT4) exists
exists only
only inin insulin-
insulin-
sensitive tissues such as hepatocyte membranes and has a high affinity
sensitive tissues such as hepatocyte membranes and has a high affinity for glucose, for glucose, soso
it is
it
involved
is involved in in
insulin-stimulated
insulin-stimulated glucose
glucosetransport.
transport. Although
Although GLUT2
GLUT2 hashasa high
a highspecificity
specific-
for glucose,
ity for it has
glucose, a high
it has affinity
a high for for
affinity glucosamine,
glucosamine, so this study
so this investigated
study investigated the the
expression
expres-
of GLUT4 in the liver of rats in each group. GLUT4 accelerates cellular
sion of GLUT4 in the liver of rats in each group. GLUT4 accelerates cellular uptake of uptake of glucose,
which
glucose, in which
turn accelerates hepatic glycogen
in turn accelerates synthesis,
hepatic glycogen ultimately
synthesis, loweringlowering
ultimately blood glucose
blood
levels. Excessive blood glucose concentrations stimulate insulin
glucose levels. Excessive blood glucose concentrations stimulate insulin secretion, secretion, resulting in a
result-
dramatic increase in GLUT4 expression on the membrane, accelerating
ing in a dramatic increase in GLUT4 expression on the membrane, accelerating glucose glucose conversion
and storageand
conversion [29].storage
When the [29].intracellular AMP/ATP ratio
When the intracellular increases,
AMP/ATP ratioactivated
increases, AMPK can
activated
enhance glucose uptake, improve insulin sensitivity, promote fatty
AMPK can enhance glucose uptake, improve insulin sensitivity, promote fatty acid oxi- acid oxidation, and
reduce
dation, sugar and lipid
and reduce production.
sugar and lipid Liver kinase Liver
production. B1 (LKB1)
kinase is B1
the(LKB1)
major upstream
is the major kinase
up-
of AMPK and is closely related to intracellular energy metabolism, muscle contraction,
stream kinase of AMPK and is closely related to intracellular energy metabolism, muscle
and ATP regeneration. Acetyl-CoA carboxylase (ACC) is an important substrate of AMPK
contraction, and ATP regeneration. Acetyl-CoA carboxylase (ACC) is an important sub-
and plays an important role in regulating fatty acid synthesis, which can not only inhibit
strate of AMPK and plays an important role in regulating fatty acid synthesis, which can
the production of cholesterol and fat, but also enhance fatty acid oxidation. In Figure 3,
not only inhibit the production of cholesterol and fat, but also enhance fatty acid oxida-
the LKB1, p-LKB1, p-AMPK, p-GLUT4, and GLUT4 in the model group were significantly
tion. In Figure 3, the LKB1, p-LKB1, p-AMPK, p-GLUT4, and GLUT4 in the model group
lower than those in the normal group (p < 0.01). The levels of AMPK and ACC in the
were significantly lower than those in the normal group (p < 0.01). The levels of AMPK
model group were higher than those in the normal group (p < 0.05). The expression levels
and ACC in the model group were higher than those in the normal group (p < 0.05). The
of the three experimental groups showed a downward trend, which was similar to the
expression levels of the three experimental groups showed a downward trend, which was
PC group but did not reach the level of the normal group, indicating that AMPK/ACC
similar to the PC group but did not reach the level of the normal group, indicating that
was activated in T2D rats after feeding with sericin; the expressions of p-AMPK, p-LKB1,
AMPK/ACC was activated in T2D rats after feeding with sericin; the expressions of p-
p-GLUT4, GLUT4, ACC, and p-ACC were up-regulated. It almost recovered to the level
AMPK,
of the PC p-LKB1,
group p-GLUT4, GLUT4, ACC,
and even partially reachedandthe
p-ACC
level were
of theup-regulated.
normal group, It almost
in whichrecov-
the
ered to the level of the PC group and even partially reached the level
high expression of GLUT4 can more directly show that the glucose level in T2D rats is of the normal group,
in which the high
accelerating. expression
Increased enzyme of expression
GLUT4 canlevelsmore accelerate
directly show that theactivated
glycolysis; glucose level
AMPK in
T2Dalso
can ratsinhibit
is accelerating. Increased
ACC production, enzyme
thereby expression
inhibiting levels accelerate glycolysis; acti-
fat synthesis.
vated AMPK can also inhibit ACC production, thereby inhibiting fat synthesis.
 Biomolecules 2022, 11, x 7 of 12
Biomolecules 2022, 12, 444 7 of 12

Normal Model PC LLS MLS HLS

AMPK

p-AMPK

LKB1

p-LKB1

GLUT4

p-GLUT4

ACC

p-ACC

GAPDH

1.8 Normal
Model
1.6 PC
Protein Relative Expression

LLS
1.4
MLS
#
HLS
1.2

##
1.0 #
#
#
0.8 # # #
## ##
##

0.6
## ##

##
0.4
#### ##
# # ##
0.2

0.0
AMPK p-AMPK LKB1 p-LKB1 GLUT4 p-GLUT4 ACC p-ACC
Key Proteins
Figure 3. Effect of sericin on the expression of key AMPK/ACC proteins in T2D rats. Normal: normal
Figure 3. Effect of sericin on the expression of key AMPK/ACC proteins in T2D rats. Normal: normal
group; Model: diabetic model group; PC: 0.5% melbin; LLS, MLS, and HLS: 1%, 2.5%, and 5 % LS.
group; #Model: diabetic model group; PC: 0.5% melbin; LLS, MLS, and HLS: 1%, 2.5%, and 5 % LS.
n = 3, p < 0.05, ## p < 0.01 versus model group.
# ##
n = 3, p < 0.05, p < 0.01 versus model group.
3.5. Impact on Liver Damage
3.5. Impact on Liver Damage
Oxidative stress in the liver of T2D mice triggers a cascade of reactions, resulting in
Oxidative stress in the liver of T2D mice triggers a cascade of reactions, resulting in
inflammation. NF-κB is an important immune-related transcription factor that is involved
inflammation. NF-κB is an important immune-related transcription factor that is involved
in the transcriptional regulation of a variety of cytokines. A large amount of IL-6 in a high-
in the transcriptional regulation of a variety of cytokines. A large amount of IL-6 in a high-
glucose environment can reduce insulin sensitivity [30], and excessive TNF-α will cause
glucose environment can reduce insulin sensitivity [30], and excessive TNF-α will cause an
an increase in the free fatty acid content, which will eventually lead to insulin resistance
increase in the free fatty acid content, which will eventually lead to insulin resistance [31].
[31]. Figure 4 shows that the levels of NF-κB, IL-6, and TNF-α in the liver of the T2D model
Figure
group 4were
shows that the levels
significantly higherof NF-κB,
than thoseIL-6,
of theand TNF-α
normal groupin the
(p < liver
0.01) of
by the T2D 1.3-
2.1-fold, model
group were significantly higher than those of the normal group (p < 0.01)
fold, and 1.2-fold, respectively. These results show that severe inflammation occurred in by 2.1-fold,
1.3-fold,
the liversand
of 1.2-fold, respectively.
the diabetic rats. After These
4 weeksresults showwith
of feeding that sericin,
severe inflammation
the NF-κB, IL-6,occurred
and
inTNF-α
the livers of the diabetic rats. After 4 weeks of feeding with sericin, the NF-κB,
levels in the three dose groups were all downregulated to normal levels in a dose- IL-6,
and TNF-α levels in the three dose groups were all downregulated to normal levels in a
dose-dependent manner (p < 0.05, p < 0.01). In all three oral sericin groups, the anti-TNF
inflammatory factor was strong and almost the same as in the PC group. These results
Biomolecules 2022, 11, x 8 of 12
Biomolecules 2022, 12, 444 8 of 12

dependent manner (p < 0.05, p < 0.01). In all three oral sericin groups, the anti-TNF inflam-
show
matorythat adding
factor sericinand
was strong effectively reduces
almost the samethe inflammatory
as in the PC group.response in theshow
These results livers of
that
diabetic rats. effectively reduces the inflammatory response in the livers of diabetic rats.
adding sericin
45 9
TNF IL-6
40 ** 8
**
35 7
TNF (ng/mg protein)

IL-6 (pg/mg protein)

30 6
##
25 5 ##
##
##
20 ##
4
##
## ##
15 3

10 2

5 1

0 0
Normal Model PC LLS MLS HLS Normal Model PC LLS MLS HLS
Groups Groups

(a) (b)
35
NF-kB
**
30
NF-kB (ng/mg protein)

25 ## ##
##

##
20

15

10

0
Normal Model PC LLS MLS HLS
Groups

(c)
Figure 4. Effect of oral sericin on TNF-α, IL-6, and NF-κB levels in livers of T2D rats. (a–c) are TNF-
Figure 4. Effect of oral sericin on TNF-α, IL-6, and NF-κB levels in livers of T2D rats. (a–c) are TNF-α,
α, IL-6, and NF-κB levels, respectively. Normal: normal group; Model: diabetic model group; PC:
IL-6, and NF-κB levels, respectively. Normal: normal group; Model: diabetic model group; PC: 0.5%
0.5% melbin administration; LLS, MLS, and HLS: 1%, 2.5%, and 5% L. n = 3, ** p < 0.01 versus normal
melbin ##administration; LLS, MLS, and HLS: 1%, 2.5%, and 5% L. n = 3, ** p < 0.01 versus normal
group. p < 0.01 versus model group.
group. ## p < 0.01 versus model group.
4. Discussion
4. Discussion
Typical symptoms
Typical symptoms of of diabetes
diabetes include
include insulin
insulin resistance
resistance (IR)
(IR) and
and accumulation
accumulation of of
triglycerides in the liver, which not only aggravate the progression
triglycerides in the liver, which not only aggravate the progression of diabetes, but may of diabetes, but may
also lead
also lead toto nonalcoholic
nonalcoholic fatty fatty liver
liver disease
disease (NAFLD).
(NAFLD). NAFLD NAFLD affects
affects more than 20%
more than 20% of of
adults [32,33].
adults [32,33]. NAFLD
NAFLD also also has
has the
the potential
potential to to develop
develop into into diseases
diseases such such asas cirrhosis,
cirrhosis,
liver failure or hepatocellular carcinoma. The liver is the most critical target organ of
liver failure or hepatocellular carcinoma. The liver is the most critical target organ of in-
in-
sulin [9,34], and insulin is the only hormone that can lower blood sugar.
sulin [9,34], and insulin is the only hormone that can lower blood sugar. Insulin mainly reg- Insulin mainly
regulates
ulates glucose
glucose absorption,
absorption, glycogen
glycogen synthesis,
synthesis, and and degradation
degradation throughthrough insulin
insulin regu-
regulation
lation through the PI3K/AKT signaling pathway [16,35]. In the liver,
through the PI3K/AKT signaling pathway [16,35]. In the liver, insulin binds to the insulin insulin binds to the
insulin receptor
receptor IR on the IRcell
on membrane,
the cell membrane, then activates
then activates the IR substrate
the IR substrate (IRS) and(IRS)then and then
activates
activates
PI3K PI3K toa generate
to generate a second that
second messenger messenger
promotes thatthepromotes
activationthe activation
of AKT. p-AKT ofregulates
AKT. p-
AKT
the regulates themetabolism
carbohydrate carbohydrate metabolism
process processthrough
of hepatocytes of hepatocytes through
the following the following
pathways. First,
AKT activation promotes the transfer of GLUT4 to the cell membrane and promotesand
pathways. First, AKT activation promotes the transfer of GLUT4 to the cell membrane the
promotes
inward the inward
transport transport ofglucose
of extracellular extracellular glucosep-AKT
[36]. Second, [36]. Second,
mediates p-AKT mediates gly-
glycogen-forming
cogen-forming
enzyme kinase enzyme
3 (GSK3)kinase 3 (GSK3)
[37]. Third, [37].inhibits
p-AKT Third, p-AKT
hepatocyteinhibits hepatocyte gluconeo-
gluconeogenesis through
genesis through
peroxisome peroxisome
receptor-γ receptor-γ
coactivator-α coactivator-α
[38]. Therefore, [38]. Therefore,pathway
the insulin-PI3K/AKT the insulin-
plays
an important
PI3K/AKT role in regulating
pathway the hepatic
plays an important carbohydrate
role in regulating metabolism.
the hepatic Studies have shown
carbohydrate me-
that adding
tabolism. sericinhave
Studies to the daily that
shown diet adding
can not sericin
only effectively
to the daily reduce
diet the
can blood
not onlysugar level in
effectively
Biomolecules 2022, 12, 444 9 of 12

the STZ-induced T2D rat model [39,40], but also reduce the intestinal cholesterol, triglyc-
eride, and other lipid intakes while improving the body’s tolerance to glucose [41]. AMPK
is an energy sensor in cells. Activated AMPK can promote GLUT4 transport and increase
glucose uptake [42]. Sericin supplementation resulted in up-regulation of LKB1, AMPK,
and GLUT4 and down-regulation of ACC protein expression. This suggests that sericin
may accelerate glycolysis and reduce the body’s absorption of fat by activating AMPK.
Sericin is a glue that binds silk fibroin fibers together to strengthen silkworm cocoons.
In the industrial processing of raw silk, it is often discharged together with degumming
agent as alkaline waste, which not only wastes biological resources, but also pollutes
the environment. Therefore, its green recycling and efficient utilization has always been
the focus of research and development in the silk industry [43,44]. The utilization of
sericin mainly involves two aspects: industrial materials and biological actives. The larger
molecular weight is suitable for the former application [8], while degraded sericin and even
hydrolysate are suitable for the latter application [45]. In the past ten years, our team has
been engaged in the green recovery of sericin, the preparation of degraded sericin, and
its application in hypoglycemic functional foods, etc. [46,47]. Recently, our group newly
developed a green technology for degumming silk fiber with Ca(OH)2 aqueous solution [24].
It was found that this degumming method can recover the low molecular weight of
degraded sericin peptide and its hydrolysate, which not only have strong antioxidant
activity, but also inhibition activity on glycosidase and tyrosinase in vitro [48]. Colleagues
from Chengde Medical University (Chengde, Hebei 067000, P.R. China) also investigated
the hypoglycemic effect of sericin water extract on rats and its mechanism. The sericin
water extract could enhance the insulin-PI3K/AKT signaling pathway in the liver of a
rat model of T2D [49]. It also reduced hippocampal neuronal apoptosis in a model rat
by activating the AKT signal transduction pathway [50] and has a protective effect on
the nervous system in diabetic rats [33,51]. However, it should be noted here that the
sericin sample used in that experiment was an aqueous extract of sericin obtained by
boiling water from the shell layer of a colored silkworm cocoon, which contains a lot of
β-carotene with anti-oxidation. The above anti-diabetic effects are probably the result
of the combined action of high-molecular-weight sericin and β-carotene. In our recently
published paper [22], orally administered sericin samples were degraded sericin peptides
and their hydrolysates obtained by the Ca(OH)2 method from the shells of white silkworm
cocoons that are now commercially available. After the oral administration of sericin to
diabetic model rats, the pathological slices showed that the rat liver cells in the model
group were severely damaged, with inflammatory infiltration and edema. Furthermore,
the serum levels of ALT, AST, and ALP were significantly increased, which also indicated
liver damage. These results show that adding sericin to the diet alleviated diabetic liver
damage. The present paper is an in-depth investigation of the aforementioned research.
As shown in Figure 5, after oral administration of sericin, the first noticeable effect was
an increase in the antioxidant capacity of the T2D rats, followed by a significant decrease
in the level of inflammatory factors in the body. This decrease resulted in changes in
the expression of genes and proteins related to insulin synthesis and secretion, glucose
synthesis and decomposition, glycogen synthesis and storage, and even the synthesis and
metabolism of fat. These changes might ultimately lead to a gradual improvement in insulin
resistance in diabetic rats and the normalization of blood sugar levels. The present results
are mostly consistent with our previous results obtained with T2D mice. In the future, we
will continue to investigate orally administered sericin and the ability of functional foods to
lower blood sugar. This new use of sericin provides a reason for utilizing this waste product
of silk processing and will support the sustainable development of the silk industry.
Biomolecules 2022, 11, x 10
Biomolecules 2022, 12, 444 10 of 12

Figure 5.5.Possible
Figure Possiblepathways through
pathways which which
through oral sericin
oralmight affect
sericin T2D rats.
might affect↓& ↑, means
T2D ↓ &↑ , means u
rats. upstream
and downstream of the pathway and feedback regulation, respectively.
stream and downstream of the pathway and feedback regulation, respectively.
5. Conclusions
5. Conclusions
Sericin is a natural macromolecular protein with various biological activities, such
as antioxidation
Sericin is aand hypoglycemia.
natural In this paper,
macromolecular the hypoglycemic
protein with variousmechanism
biological of low-
activities, suc
molecular-weight sericin (LS) prepared by the ultrasonic degumming method
antioxidation and hypoglycemia. In this paper, the hypoglycemic mechanism of low- and Ca(OH) 2 -
ultrasonic degumming method after feeding T2D rats was investigated. The results show
lecular-weight sericin (LS) prepared by the ultrasonic degumming method and Ca(O
that all the concentrations of LS can improve the liver hyperplasia and hypertrophy caused
ultrasonic
by diabetes; degumming method
at the same time, after feeding
it was found T2Dconcentrations
that different rats was investigated. The results sh
of LS can regulate
that all GLK,
G6pase, the concentrations
and PCK relatedof to LS
the can
glucoseimprove the liver
metabolism hyperplasia
pathway, andthe
and regulate hypertro
caused
PI3K/AKT byand
diabetes;
AMPK/ACCat thepathways
same time, it wasglycogen
to enhance found that different
synthesis, concentrations
accelerate glycolysis, of LS
and inhibit
regulate gluconeogenesis.
G6pase, GLK, andItPCK can also reduce
related the glucose
to the expression of inflammatory
metabolism factors
pathway, and regu
TNF-α, IL-6, and NF-κB, and significantly relieve liver oxidative stress.
the PI3K/AKT and AMPK/ACC pathways to enhance glycogen synthesis, accelerate
colysis, and inhibit gluconeogenesis. It can also reduce the expression of inflamma
Author Contributions: All authors conducted experiments. Z.-Z.W.: literature search, data analysis,
factors
writing, editing andIL-6,
TNF-α, and Y.-J.W.:
reversion; NF-κB, and
data significantly
analysis, relieve
writing original liver
draft oxidative
and editing; stress.
All authors
have read and agreed to the published version of the manuscript. Y.-Q.Z.: conceiving the idea,
refining the
Author manuscript, reviewing,
Contributions: and providing
All authors conducted funds.
experiments. Z.-Z.W.: literature search, data anal
writing, editing and reversion; Y.-J.W.: data analysis, writingSystem
Funding: This study was funded by the China Agriculture Research original draft and editing;
(CARS-18-ZJ0502) and All aut
have read Academic
the Priority and agreed to theDevelopment
Program published version
of JiangsuofHigher
the manuscript. Y.-Q.Z.: conceiving
Education Institutions (PAPD). the idea
fining the manuscript, reviewing, and providing funds.
Institutional Review Board Statement: The Institutional Review Board Statement and approval
number forThis
Funding: studies involving
study humansby
was funded orthe
animals.
ChinaApproval Code:Research
Agriculture 201911A063, Approval
System Date:
(CARS-18-ZJ0502)
5 November 2019.
the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD
Informed Consent Statement: Not applicable.
Institutional Review Board Statement: The Institutional Review Board Statement and appr
Data Availability
number Statement:
for studies Code humans
involving and material;
or The datasetsApproval
animals. used and/or analyzed
Code: during the current
201911A063, Approval Da
study as well as analysis scripts are available from the corresponding author on reasonable request.
November 2019
Conflicts of Interest: All authors declare that they have no known competing financial interests
Informed
or personal Consent that couldNot
Statement:
relationships haveapplicable.
appeared to influence the work reported in this paper.
The manuscript has been read and approved by all named authors, and that there are no other
Data Availability Statement: Code and material; The datasets used and/or analyzed during
persons who satisfied the criteria for authorship but are not listed.
current study as well as analysis scripts are available from the corresponding author on reason
request.
Conflicts of Interest: All authors declare that they have no known competing financial interes
personal relationships that could have appeared to influence the work reported in this paper.
manuscript has been read and approved by all named authors, and that there are no other per
who satisfied the criteria for authorship but are not listed.
Biomolecules 2022, 12, 444 11 of 12

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